(ACS Symposium Volume 207) Harvey W. Blanch, E. Terry Papoutsakis, and Gregory Stephanopoulos (Eds.)-Foundations of Biochemical Engineering. Kinetics and Thermodynamics in Biological Systems-American .pdf
(ACS Symposium Volume 207) Harvey W. Blanch, E. Terry Papoutsakis, and Gregory Stephanopoulos (Eds.)-Foundations of Biochemical Engineering. Kinetics and Thermodynamics in Biological Systems-American .pdf
(ACS Symposium Volume 207) Harvey W. Blanch, E. Terry Papoutsakis, and Gregory Stephanopoulos (Eds.)-Foundations of Biochemical Engineering. Kinetics and Thermodynamics in Biological Systems-American .pdf
Biochemical Engineering
Boulder, Colorado,
W A S H I N G T O N , D.C. 1983
Copyright © 1983
American Chemical Society
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Advisory Board
ix
HARVEY W. BLANCH
University of California
Berkeley, CA
E. TERRY PAPOUTSAKIS
Rice University
Houston, TX
GREGORY STEPHANOPOULOS
California Institute of Technology
Pasadena, CA
June 1, 1982
Financial assistance for the 1982 Winter symposium was received from
National Science Foundation
and
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MICHAEL A. SAVAGEAU
University of Michigan, Department of Microbiology and Immunology,
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© 1983 American Chemical Society
Power-law Formalism
E a r l y e f f o r t s t o a n a l y z e b i o l o g i c a l systems were b l i n d e d by
the success t h a t engineers have had in analyzing complex
t e c h n o l o g i c a l ( o f t e n e l e c t r i c a l and mechanical) systems and by the
power and elegance o f the mathematical methods a t t h e i r d i s p o s a l .
Over a p e r i o d o f years the e f f o r t t o make b i o l o g i c a l phenomena f i t
i n t o the same mold became i n c r e a s i n g l y f r u s t r a t e d . Biological
phenomena a r e h i g h l y n o n l i n e a r and cannot be made t o wear the
l i n e a r s t r a i g h t j a c k e t t h a t t e c h n o l o g i c a l systems o f t e n s u b m i t t o
willingly. F u r t h e r m o r e , t h e t y p e s o f n o n l i n e a r i t i e s t h a t are
(1)
i = 1, 2 , ···
P r i n c i p a l Advantages. T h e r e a r e a number o f d i f f i c u l t i e s
a s s o c i a t e d with the m o d e l i n g and a n a l y s i s o f complex n o n l i n e a r
systems, ( a ) The f u n c t i o n a l form o f the n o n l i n e a r i t i e s i s o f t e n
unknown, as are the numbers o f i n t e r a c t i o n s and p a r a m e t e r s t h a t
must be s p e c i f i e d , ( b ) Once one h a s assumed a f u n c t i o n a l form
there i s s t i l l d i f f i c u l t y i n e x t r a c t i n g s t a t i s t i c a l e s t i m a t e s o f
the p a r a m e t e r v a l u e s from experimental data, (c) The amount o f
experimental data i t s e l f t h a t i s r e q u i r e d t o c h a r a c t e r i z e many
n o n l i n e a r mechanisms i n c r e a s e s e x p o n e n t i a l l y with the dimensions
of the problem, (d) Genera
system o f n o n l i n e a r equation
Linear analysis largely overcomes these difficulties.
However, there i s a c o n s i d e r a b l e c o s t . I t i s v a l i d f o r only small
v a r i a t i o n s i n t h e c o n c e n t r a t i o n v a r i a b l e s and t h i s i s u s u a l l y
u n r e a l i s t i c f o r most b i o l o g i c a l systems o f i n t e r e s t .
The power-law formalism provides a j u d i c i o u s compromise that
has many o f the advantages o f l i n e a r a n a l y s i s w i t h o u t i t s s e v e r e
limitations. ( a ) The f u n c t i o n a l form i s known and the
i n t e r a c t i o n s and p a r a m e t e r s a r e r e a d i l y s p e c i f i e d , ( b ) From
experimental data one can e x t r a c t parameter values f o r the
i n d i v i d u a l r a t e laws by l i n e a r r e g r e s s i o n i n a l o g a r i t h m i c s p a c e ,
(c) The d a t a n e c e s s a r y f o r such an a n a l y s i s has a p o l y n o m i a l
( r a t h e r than e x p o n e n t i a l ) i n c r e a s e w i t h t h e d i m e n s i o n s o f t h e
problem, (d) General methods f o r o b t a i n i n g s t e a d y - s t a t e s o l u t i o n s
a r e a v a i l a b l e by t r a n s f o r m i n g t h e p r o b l e m i n t o l i n e a r t e r m s .
General purpose computer algorithms have been developed f o r
dynamic s o l u t i o n s , and parameter e s t i m a t i o n from i n t e g r a t e d system
behavior i s also possible. (For more d e t a i l e d d i s c u s s i o n o f the
advantages and disadvantages o f t h i s and o t h e r a p p r o a c h e s t o t h e
modeling and a n a l y s i s o f n o n l i n e a r systems, see Ref. 3·)
T h i s concludes my b r i e f review o f the k i n e t i c methods t h a t we
have developed f o r d e a l i n g w i t h complex b i o l o g i c a l systems. These
methods a l r e a d y have been a p p l i e d t o a v a r i e t y o f biochemical and
g e n e t i c systems (e.g., see Réf. 3)· However, f o r the remainder o f
t h i s paper I s h a l l t r e a t one p a r t i c u l a r a p p l i c a t i o n i n some
detail·
Environment + Physiology
Demand f o r Gene E x p r e s s i o n
REPRESSIBLE SYSTEMS
corepressor^.
REPRESSOR
Figure 1. Repressor control of transcription initiation. A regulatory gene, R, codes for a repressor
protein. Structural genes, SG, are preceded by a control region in the DNA consisting of a promoter
site, P, and a modulator site, O.
INDUCIBLE SYSTEMS
inducer
ACTIVATOR
SGi SG 2 SGi SG 2
3C I I
OPERON- 1 RNA tronscript~*>
REPRESSIBLE SYSTEMS
compressor^.
ACTIVATOR
Ο
Ο
Figure 2. Activator control of transcription initiation. A regulatory gene, R, codes for an activator m
protein. Structural genes, SG, are preceded by a control region in the DNA consisting of a promoter
• LEADER-
LT SG,
REPRESSOR SYSTEMS
corepressor
ANTITERMINATOR
«—LEADER
<_>
SGi SG2 SG. SG 2
RNA tronscript
te SG, i m {
=>
REPRESSOR SYSTEMS
corepressor »...
PROTERMINATOR
1-
SGi
Table I . Terminology
Positive Activato
Regulatory mechanism
Demand
Positive Negative
Type o f C o n d i t i o n corresponding t o
system high demand f o r expression
R e p r e s s i b l e drug g f r e q u e n t l y presen
sensitivity i n high concentrations
Experimental Implications
R e p r e s s i b l e B i o s y n t h e t i c Systems. In e n t e r i c b a c t e r i a the
r e p r e s s i b l e s y s t e m s f o r h i s t i d i n e and t r y p t o p h a n b i o s y n t h e s i s
provide examples o f systems c o n t r o l l e d by p o s i t i v e and n e g a t i v e
elements, r e s p e c t i v e l y (see Table I V ) . Since the l e v e l o f
h i s t i d i n e i n the colon i s among t h e l o w e s t o f a l l amino a c i d s ,
e x p r e s s i o n o f t h e h i s t i d i n e b i o s y n t h e t i c operon i n the bacterium
w i l l be i n high demand (Table I I I ) . Thus, demand theory p r e d i c t s
that the h i s t i d i n e b i o s y n t h e t i c operon w i l l be p o s i t i v e l y
r e g u l a t e d (Table I I ) . I n f a c t , t h e r e i s good e v i d e n c e t o show
t h a t t h i s o p e r o n i s c o n t r o l l e d p r i m a r i l y by an a n t i t e r m i n a t o r
mechanism (26.27). On the other hand, the l e v e l o f t r y p t o p h a n i n
t h e c o l o n i s among the h i g h e s t o f the amino a c i d s and, t h e r e f o r e ,
expression o f the tryptophan b i o s y n t h e t i c operon i n the b a c t e r i u m
Nature o f Demand f o r
System" " 1
regulator expression
R e p r e s s i b l e b i o s y n t h e t i c systems:
Histidine
Nature o f Demand f o r
System" "1
regulator expression
Tryptophanase • High
Positive
I n d u c i b l e g e n e t i c movement:
Prophage i n d u c t i o n
λ Negative Low
P1 Negative Low
P2 Negative Low
Transposition
Conjugation
F Negative Low
Inducible resistance/
d e t o x i f i c a t i o n systems:
Nature o f Demand f o r
System" " 1
regulator expression
Toluene (P. p u t i d a )
Inducible r e p a i r systems:
Adaptive response t o
* Low
Negative
a l k y l a t i n g agents
Iron « High
Positive
Sulfate Positive High*
C o l i c i n e E1 Negative Low
D i p t h e r i a (C. d i p h t h e r i a e ) Negative «
Low
Nature o f Demand f o r
System" "1
regulator expression
+
Enteric bacteria unless i n d i c a t e d otherwise
ft
Predicted
b a c t e r i a l p l a s m i d becomes i n c o r p o r a t e d i n t o t h e p l a n t genome.
T h i s s p e c i f i c DNA a l s o codes f o r a b i o s y n t h e t i c system that causes
t h e p l a n t t o produce a r a r e amino a c i d , octopine i n t h i s example.
Octopine i n t u r n a c t s as a s p e c i f i c inducer upon the b a c t e r i a t h a t
h a r b o r t h e t u m o r - i n d u c i n g p l a s m i d t o c a u s e e x p r e s s i o n o f an
o c t o p i n e c a t a b o l i c pathway and e x p r e s s i o n o f a s p e c i f i c system of
b a c t e r i a l c o n j u g a t i o n f o r plasmid t r a n s f e r (see R e f s . 39,40).
S i n c e o c t o p i n e i s o n l y r a r e l y found i n nature, e x p r e s s i o n o f the
octopine c a t a b o l i c system and t h e b a c t e r i a l c o n j u g a t i o n s y s t e m
w i l l be i n low demand. Demand theory p r e d i c t s t h a t these systems
w i l l be under t h e c o n t r o l o f a n e g a t i v e - a c t i n g e l e m e n t . The
e x p e r i m e n t a l e v i d e n c e showing t h a t t h e s e systems are under the
c o n t r o l o f a r e p r e s s o r p r o t e i n (40) i s e n t i r e l y c o n s i s t e n t w i t h
these e x p e c t a t i o n s .
Discussion
In summary, the r e s u l t s p r e s e n t e d i n t h i s p a p e r i l l u s t r a t e
the use o f k i n e t i c methods t o achieve fundamental understanding o f
gene f u n c t i o n . T h e r e a l s o a r e p r a c t i c a l i m p l i c a t i o n s that have
yet t o be f u l l y e x p l o r e d . C l e a r l y , i f one w i s h e s t o o p t i m a l l y
e x p l o i t t h e p r o d u c t o f a p a r t i c u l a r gene, then one must know the
physiology and n a t u r a l ecosystem o f i t s h o s t organism as
i n t i m a t e l y as i t s molecular g e n e t i c s .
Acknowledgments
Literature cited
DAVID F. OLLIS
University of California, Chemical Engineering Department, Davis, CA 95616
1. Soluble substrates
1
The Henri form f o r r e a c t i o n v e l o c i t y , v, of an enzyme
c a t a l y z e d r e a c t i o n was proposed i n 1902 as eqn (1.1):
v ( 1 υ
- W ï f s ï ·
0097-6156/83/0207-0027$07.50/0
© 1983 American Chemical Society
S + Ε «- ES (1.2a)
k 2
where
Michaelis-Menten: Κ Ξ
ν = k E3 where Ε = t o t a l (1.3b)
max ο ο
enzyme c o n c e n t r a t i o n
(E + ES)
3
The subsequent Briggs-Haldane treatment recognized that i n
general the r a t e constants ( k i , k ) and k could be of a r b i t r a r y
2 3
Briggs-Haldane: Κ = (1.4a)
= k E ν 3
3 (1.4b)
max ο
Thus, the constant Κ i s no longer a simple e q u i l i b r i u m constant^
Extensions of form (1.1) a r e apparently endless i n number.
For example, f o r a common case of a (two substrate)-(one enzyme)
system g i v e n below,
Ε + S i / J ESi Ki (1.5a)
Ε + S 2 J ES 2 K 2 (1.5b)
ES S £ Ρ + E (1.5e)
kE S 2
ν = — - — (1.6a)
max Ki2 + S 2
Κ 2 1 l K l 2
substrate Κ = ^ + * (1.6b)
S2 + K i 2
Thus, i f S2 i s approximately constant as expected f o r S2 = s o l
vent or S2 = b u f f e r e d Η or OH , the Henri form of constant
parameters ν and Κ w i l l appear to f i t the data f o r S i .
a
S i m i l a r l y , i¥ ?he order of s u b s t r a t e b i n d i n g r e a c t i o n s (5a) and
(5b) i s o b l i g a t o r y , form (1.1) i s again obtained (e.g., by
e l i m i n a t i o n of eqn (1.5d)).
When the second bindin e n t i t y S2 i t s u b s t r a t e but
instead an enzyme a c t i v a t o
i s again regained with
X
i n h i b i t o r l e v e l as i n d i c a t e d 6y eqns (1.6a,b).
The extension of the network i n eqns (1.5 a-e) to m u l t i p l e
intermediates i s important i n d e v i s i n g p l a u s i b l e multi-enzyme
r a t e forms f o r m i c r o b i a l metabolism. The Henri form obtained i n
eqns (1.5a-e) i n v o l v e s three d i f f e r e n t enzyme complexes, a l l
e q u i l i b r a t e d with each other "upstream" to the k i n e t i c a l l y slow
step (eqn (1.3)). The n e g l i g i b l e f r e e energy changes evident
f o r many^of the steps i n the Embden-Meyerhof g l y c o l y t i c
sequence (Figure 1) i n d i c a t e that the three r e a c t i o n sequences
(1.7a,b,c),
f r u c t o s e - d i - P J · · · J 2(phosphoenolpyruvate) (1.7b)
and
pyruvate j l a c t a t e (1.7c)
50
40
<
CD
CD
30
A ( e x t r a c e l l u l a r glucose) membrane
0
permease ( E i )
B(fructoee-6-phoephate)(Si) - phosphofructokinase
ki k
G + E J EXJE + F (1.8)
k-i k- 2
κ ] G + 9 K ( L E 9 C )
κ = [ ΐ ς ^ Π ν ν * ** *ρ G
and K* = g l u c o s e / f r u c t o s e e q u i l i b r i u m constant
Kj, = ( k e l +k /k
2 - 2 and K Q = (k^+k^/^
G = glucose c o n c e n t r a t i o n a t e q u i l i b r i u m .
ν = ν · bfzr]; ν
L J>
= kS (2.1)
max K+E max ο
where Ε = f r e e (uncomplexed) enzyme c o n c e n t r a t i o n and ν , Κ =
constants as f o r eqn. (1.1). In c o n t r a s t to s o l u b l e s u B s i r a t e s
where a s i n g l e r e a c t i o n product i s t y p i c a l l y i d e n t i f i a b l e ,
m u l t i p l e r e a c t i o n products r o u t i n e l y occur i n p a r t i c u l a t e degra
d a t i o n and/or enzymatic depolymerizations. A p a r a l l e l / s e q u e n t i a l
conversion network appears to be provided f o r t r i g l y c e r i d e s by
9
the data o f Constantin et a l . (Figure 3) which a r e c o n s i s t e n t
with the f o l l o w i n g network (2.2):
ι—ι—τ- ι ι—ι 1
— Ι 0.1 0.3 0.5 0.7
- Soluble ι Insoluble
S 4 - i /
-
1JA
I I 1
0.5S LOS 1.5S 2.0S 3.5S 0 0.5S LOS 1.5S 2.0S
(0.328 M) (0.153 M)
Saturation Saturation
Hydrolysis, %
,triglyceride^
glycerol'
κ
«
Ε + S ± J (ES^j j = 0,1,1 1 1 i + i (2.3a)
(ES ).
± - C
Aj + S ( i - j ) j = 1, . . . , i (2.3b)
k
V
[(ES.). - A, + H 0 ? (2.3c)]
k.
1
3
Α..(+Η 0) V Ε + S
2 Jj
(2.3d)
*Τ
±A + S^ Ε + S ± + j (2.3e)
1
Here, the (i+2)-mer S^, i s complexed at the j * * s i t e ,
numbered 0 to i+1, but only b i n d i n g at s i t e s 1< j < i can r e s u l t
i n r e a c t i o n , as a s i t e at an end i s u n r e a c t i v e . The model f i t
11
k i n e t i c data from e x p e r i m e n t s s t a r t i n g with (uncleavable) dimer
only. Other measures of r e a c t i o n r a t e f o r c e l l l y s i s are t y p i
c a l l y more convenient: o p t i c a l d e n s i t y of p a r t i a l l y l y s e d c e l l s
N
τ = K(^) (2.4)
XT - . x l pseudoplastic
Ν = power law inde
r
=
As s t a r c h pastes were hydrolyzed by α-amylase , the para
meters Κ and Ν were observed to change with r e a c t i o n progress i n
a coupled manner according to the equation (2.5):
i n v o l v e s a c e l l o b i o s e noncompetitive b i n d i n g to the c e l l u l a s e ;
t h i s i n h i b i t i o n i s r e l i e v e d by c e l l o b i o s e conversion to glucose
15
by β-glucosidase :
k
i
k
E [G]
1 -> 3
Ε χ + G 2 (2.6b)
E + G E G c e l l o b i o s e
l 2 * 1 2 ( binding) (2.6c)
E (B-glucosidase) + G
2 £ 2G + E £ (2.6d)
The form of the i n h i b i t i o
b i o s e , thus Κ i s unaffecte
same K) but the maximal v e l o c i t y i s diminished according t o eqn
(2.7):
k
i 3
1 + (
K^ k
N
max = k
3 o E [
l+î/Κ^ 1 ( 2
' 7 )
C e l l u l o s i c residues c h a r a c t e r i s t i c a l l y c o n s i s t of a mixture
of c e l l u l o s e , h e m i c e l l u l o s e , and l i g n i n . Even the i n d i v i d u a l
components are heterogeneous: the i n s o l u b l e c e l l u l o s e i t s e l f con
s i s t s of a s t r u c t u r e d m a t e r i a l of v a r y i n g degrees of c r y s t a l -
linity. An i l l u s t r a t i v e s t r u c t u r e d substrate d e s c r i p t i o n
i n c l u d i n g a k i n e t i c model and supporting data was provided by
16
Ryu, Lee, T a s s i n a r i and Macy. These i n v e s t i g a t o r s modelled the
substrate heterogeneity by assuming that the c e l l u l o s e c o n s i s t e d
p r i m a r i l y of two phases: "an impermeable, denser, and h i g h l y
ordered p a r a c r y s t a l l i n e or amorphous phase." Correspondingly,
each phase possesses a d i f f e r e n t r e a c t i v i t y . With S (amor
phous), S ( c r y s t a l l i n e ) and S ( r e s i d u a l i n e r t , i n c l u d i n g
l i g n i n ) making up the t o t a l ceïlulose mass, S , the k i n e t i c
model i n c l u d i n g binding of s o l u b l e product Ρ So the enzyme i s the
system of r e a c t i o n s given by eqns (2.8a-3):
k
(enzyme ad
Ε E* (2.8a)
adsorption)
des
k k
la M „ 3
(amorphous E* + S J E*S ·> E*+P (2.8b)
conversion) k 0
Za
(crystalline
E* +S E*+P (2.8c)
conversion)
(inert
E* + S (2.8d)
binding)
( s o l u b l e pro
duct b i n d i n g
Ε + Ρ EP (2.8e)
to s o l u b l e
enzyme)
These authors determine
x-ray d i f f r a c t i o n data; molecular a c c e s s i b i l i t y and surrace area
of v a r i o u s l y m i l l e d samples were obtained from i o d i n e adsorption
and BET measurements. Degree of c e l l u l o s e p o l y m e r i z a t i o n was
determined from viscometry of cadoxen-dissolved s o l u t i o n s , with
the s p e c i f i c v i s c o s i t y extrapolated to zero c o n c e n t r a t i o n to
obtained the i n t r i n s i c v i s c o s i t y , [η], from which i n turn the
v i s c o s i t y average molecular weight M was estimated from the Mark-
Houwink equation:
-5 0.76
W
[η] = 38.5 χ 10 J
M (2.9)
Immobilized Enzymes
The attachment of c a t a l y t i c a l l y a c t i v e s i t e s to m a t e r i a l s
which may be e a s i l y recovered from a r e a c t i o n mixture has been
the s i n e qua non of most u s e f u l examples of c a t a l y s i s o u t s i d e of
enzymology, and t h i s l a t t e r area has begun to f o l l o w s u i t (see
17
f o r example, the s i x Enzyme Engineering C o n f e r e n c e s , as w e l l
as s e v e r a l summary t e x t s ). In a p l e a s a n t l y exhaustive review
1 9
of the k i n e t i c s of immobilized enzyme systems, G o l d s t e i n
s e v e r a l years ago assigned "the e f f e c t s of i m m o b i l i z a t i o n on the
k i n e t i c behavior of an enzyme" to four s i t u a t i o n s :
where ze = s u b s t r a t e charge, ψ = e l e c t r o s t a t i c p o t e n t i a l of
8;^
>—
E S
MOLA
ENZYME LOADING) χ Ι Ο - 2
1.0
>- I ^
α- -Chymotrysin
>
ι
ο
± 0.1
V \
χ
\ MAcylated)
ο.
Lysozyme^ \
L( Diazotized)
°\
^ Lipase
3 0.01 ο
Κ ο ο \ >
Figure 4. Top, relative specific activity of immobilized enzyme versus the molar
ratio (ratio of immobilized surface coupling groups to enzyme immobilized). Key:
· , diazotized lysozyme; X, diazotized lipase; Δ, acylated a-chymotrypsin. Bottom,
relative specific activity of modified soluble enzymes versus the molar ratio (ratio of
soluble coupling reagent to enzyme). Key: O, diazobenzenesulfonic acid lysozyme;
X, diazobenzenesulfonic acid-lipase; | , diazobenzenesulfonic acid-chymotrypsin;
Δ, acetic anhydride-chymotrypsin. Reproduced, with permission, from Ref. 20.
ι — ι — ι — ι — ι — ι — ι — Γ
f
K = Kexp(zei|;/kT) (3.1b)
D
ν ν S
max i _ max ο ,~ , ν
V
K+S ± Kexp(zei|;/k T) + S B q U-ic;
f , l$
K = γΚ[1 - K Zm /KI] c (3.2)
i n n e r b u l
where the a c t i v i t y c o e f f i c i e n t r a t i o i s γ = y /y k^ Z m
2 2
Table l
ΙΟ" 2
12000 13.3 (^8.3) -36mV
icf 1
12000 22.6 (^8.3) -37.3mV
* ( f i r s t numbers a r e experimental r e s u l t s , second ( i n parentheses)
are values p r e d i c t e d by p a r t i c l e c o l l e c t o r theory, i n c l u d i n g i n
fluence of i n t e r c e p t i o n ( p a r t i c l e s i z e ) and Brownian motion on
mass t r a n s f e r of p a r t i c l e s to immobilized enzyme surface.)
a
r e p u l s i v e (ψ = ¥feï to 83.5mV) as determined from e l e c t r o -
p h o r e t i c m o b i l i t y measurements. A M i c h a e l i s constant (K) modi-
f i c a t i o n may a l s o be involved (eqn 3.2 above). The agreement
between observed r a t e and the c a l c u l a t e d mass t r a n s f e r l i m i t e d
r a t e ( l a s t column i n parenthesis) means that the f l u i d mechanical
and c o l l o i d a l f o r c e s which a c t to a t t r a c t / r e p e l enzyme and c e l l
p a r t i c l e s are, a t the highest i o n i c strengths, dominating the
slow step k i n e t i c s , i . e . , that every c e l l which encountered the
22
immobilized enzyme surface was subsequently h y d r o l y z e d . Simi-
l a r l y , the d e c l i n e i n r a t e f o r s o l u b l e enzyme i s to be a s s o c i a -
ted with a more d i f f i c u l t binding o r lessened substrate a f f i n i t y
occasioned i n part by the s h i f t from a t t r a c t i v e to r e p u l s i v e
c o l l o i d a l f o r c e betwee
The mass t r a n s f e r enzym p a r t i
c l e brings a r e l a t i v e l y modest number of hydrolyzable bonds per
c e l l p a r t i c l e to the c a t a l y s t . The above lysozyme data i n d i c a t e
that the r e a c t i o n r a t e i s e s s e n t i a l l y mass t r a n s f e r c o n t r o l l e d ,
i . e . , the enzyme can cleave the small number of c e l l w a l l bonds
per u n i t volume of c e l l rather r a p i d l y . S o l i d and emulsion
p a r t i c l e s of s i m i l a r s i z e , such as c e l l u l a s e p a r t i c l e s or l i p i d
emulsion d r o p l e t s , b r i n g a much l a r g e r number of c l e a v a b l e bonds
per p a r t i c l e to the immobilized enzyme. Immobilized p a n c r e a t i c
2 3
lipase shows an observed r e a c t i o n r a t e which i s 10 to 100
times l e s s than that p r e d i c t e d from c o l l o i d a l p a r t i c l e t r a n s -
port (Figure 6 ) . The p a r t i c l e c o l l e c t i o n e f f i c i e n c y u t i l i z e d i n
c a l c u l a t i o n of the p a r t i c l e mass t r a n s f e r c o e f f i c i e n t i s composed
of only two important terms, i n t e r c e p t i o n and Brownian motion
( g r a v i t a t i o n a l s e t t l i n g and i n e r t i a l impactions a r e n e g l i g i -
2 3
ble). Thus, the p a r t i c l e mass e f f i c i e n c y of c o l l e c t i o n , η, i s
23 2
the simple sum of two terms: * **
η +
overall ^Brownian ^interception
9( ) 2 / 3 + x 5 ( 2 (3 3
• °· ΐ Τ Τ Ί Γ - d^> ' >
e enz ο enz
where d , d ^ e are substrate and immobilized enzyme p a r t i c l e d i a
n
M
^ ^ 2 0 0 - 2 5 0 Mesh 3
s ι , - —325 Mesh
CD
i 10 •
# —50-100 Mesh^
/ r 325 Mesh 3
£
1 ^ « *
50-100 Mesh
250 mesh; and Δ, 325 mesh. Reproduced, 43 0 0.4 0.8 1.2 1.6 2.0 2.4 2.8
with permission, from Ref. 23. Copyright
1975, John Wiley and Sons, Inc. Substrate Concentration (Volume %)
s t r u c t u r e : Micrococcus l y s o d e i k t i c u s ( p r o c a r y o t i c ) and t r i b u t y -
r i n , r e s p e c t i v e l y . More complex p a r t i c u l a t e s t r u c t u r e s provide
a v a r i e t y of hydrolysable bonds per p a r t i c l e , l e a d i n g to a
greater d i f f i c u l t y i n reaching a d e s c r i p t i o n of enzyme h y d r o l y t i c
kinetics. Examples here i n c l u d e e u c a r y o t i c c e l l w a l l s (e.g.,
yeast, with c r o s s l i n k e d mannan and glucan components), and the
c e l l u l o s i c substrates discussed i n the e a r l i e r s e c t i o n .
Enzyme immobilization leads always to the need to consider
d i f f u s i o n a l l i m i t a t i o n s , as the l a t t e r can give r i s e to r a t e
l i m i t a t i o n s and d i s g u i s e s . The general k i n e t i c r e s u l t s are w e l l
e s t a b l i s h e d , and we touch on only a few i l l u s t r a t i v e examples i n
t h i s short paper; d i f f u s i o n a l l i m i t a t i o n s , enzyme e l e c t r o d e
design, and polystep conversions.
Internal d i f f u s i o n a l r e s i s t a n c e i s widely appreciated now,
and t e s t s f o r freedom fro
used i n k i n e t i c s t u d i e s
i n t r u s i o n s i s i l l u s t r a t e d with c o n s i d e r a t i o n of an immobilized
dual system: glucose oxidase (E^) and c a t a l a s e (E«) a r e used to
o x i d i z e glucose (G) to o x i d i z e d glucose ( G ^ ) , and to decompose v
hydrogen peroxide ( H 0 ) : ox
2 2
G + 0 G + H ( 3 4 a )
2 Î ox 2°2 -
E
2 1
( 3 , 4 b )
H
2°2 t H
2° +
2°2
ν
- V
l• Γ E
ox / ( 1 + K
G / C
G +
V a>
c ( 3
- 5 a )
= V - k Ε C (3.5b)
z ca ca D
Oxidase d e a c t i v a t i o n :
dE
O X
- - k 0 C E_ B (3.5c)
dt 3 Β ox
Catalase activity:
dE
= -k.C^E (3.5d)
dt 4 Β ca
dP = ^dG
(3.5e)
dt dt
In commercial l a r g e s c a l e c a t a l y t i c conversions, a c t i v i t y
27
maintenance i s important. C a l c u l a t i o n s by C h a n g i n d i c a t i n g the
glucose oxidase a c t i v i t y p r o f i l e s vs. d i s t a n c e i n the c a t a l y s t
p e l l e t are shown i n Figure 7 f o r v a r i o u s c a t a l a s e enzyme loadings
l 9
20
0 = / R k
Ε /D
ca ca A
i n F i g s . 7a (t=0), 7b (t=100), 7d(t=150), 73(t=20C0, and
7f(t=300). Lack of any c a t a l a s e (φ = 0, F i g s . 7a-3, curve a)
2
i n "planar" membrane:
d i f f u s i o n + r e a c t i o n of urea s u b s t r a t e :
American Chemical
Society Library
1155 16th St., N-W.
In Foundations of Biochemical Engineering; Blanch, H., et al.;
Washington,
ACS Symposium Series; D.C. 20036
American Chemical Society: Washington, DC, 1983.
48 BIOCHEMICAL ENGINEERING
d 2 g k kE S3 Q
P = ( 3 6 a )
s ^ 2 " (1+KS) (1+K- [NH +] 4 ° '
v; +
( n«)(i«'[HH^i) = 0 ( 3
· 6 b )
d[NH+] d s
—j = 0 =— at ζ = L (ammonium i o n (3.6c)
d z d z
electrode)
V ~ = L( ) ζ (externa (3.6d)
s αζ Z D - ν
surface)
. dNHT
V 4 -d^ N H4 = k
^ " îlb> A
( [ N H l M H
· ( 3 6 β )
2 9
Using publisheddata of L a i d l e r et a l . , the c a l c u l a t e d ammonium
ion concentration p r o f i l e i s presented i n Figure 8 f o r v a r i o u s
urease loadings i n the t h i n "planar" membrane g e l surrounding the
t i p of the ammonium i o n e l e c t r o d e . These r e s u l t s c o n t a i n u s e f u l
design information. I f ^e wish to use the e l e c t r o d e to measure
substrate (urea), the NH^ s i g n a l a t the ammonium e l e c t r o d e sur
face (z/L = 1.0, F i g . 8) should be independent of Ε , thus we
should produce an e l e c t r o d e with Ε > 20-30 mg urease per ml
of g e l . I f we wish t o detect an i n h i b i t o r , then we should load
the enzyme membrane e l e c t r o d e very l i g h t l y (e.g., Ε = 2 mg/ml)
so that a 50% a c t i v i t y inhibition,corresponding to E^ = Ε /2,
produces a considerable drop i n NH^ s i g n a l . The experimental
voltage response o f an urease enzyme e l e c t r o d e with v a r i o u s
30
urease loadings was determined by G u i l b a u l t and M o n t a i v o ;
F i g . 9 i n d i c a t e s that the i n i t i a l l o a d i n g of 20-30 mg c a l c u l a t e d
above i s i n reasonable agreemen£ with the experimental r e s u l t s
(note that c a l c u l a t i o n gives NH^ (Z=0) while measurement i s
reported as ammonium i o n e l e c t r o d e voltage.) Many other sub
stances have been analyzed experimentally with s i m i l a r enzyme
3 2
e l e c t r o d e probes ( G u i b a u l t ) , i n d i c a t i n g major p o t e n t i a l f o r
analogs of eqns (3.6a-e).
Summary
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
DIMENSIONLESS THICKNESS
Figure 8. Influence of enzyme loading (mg enzyme/cc gel) on product profiles for
50μ membrane; bulk urea concentration, 0.0833 M; bulk ammonium ion, negligible
1
(10 M). Reproduced, with permission, from Ref. 28. Copyright 1972, Plenum
Publishing Corp.
τ 1 1 1 1 Γ
UREASE (mg//tgel)
Literature Cited
1. Henri, V. Comptes Rendus Acad. Sci., Paris, 1902, 135, 916.
2. Michaelis, L; Menten, M. L., Biochem. Ζ., 1913, 333, 49.
3. Briggs, G. E.; Haldane, J. B. S. Biochem. J., 1925, 19, 338.
4. Dixon, M.; Webb, E. C. "Enzymes", Academic Press, 1964;
Chapter IV).
5. Gileadi, E. (ed), "Electrosorption", Plenum Press, New York,
1967.
6. Lehninger, A. L. "Biochemistry", Worth Publishers, New York,
1970, p. 327.
7. Sarda, L.; Desnuelle, P. Biochim. Biophys. Acta, 1958, 30,
513.
8. Wills, E. D. Adv. in Lipid Research, 1965, 3, 219.
9. Constantin, M. J.; Pasero L.; Desnuelle P Biochim
Biophys. Acta, 1960
10. Chipman, D. M. Biochemistry, , ,
11. Chipman, D. M.; Pollock, J . J.; Sharon, N. J. Biol. Chem.,
1968, 243, 481.
12. Cruz, Α., Russel; W. B.; Ollis, D. F. AIChE J., 1976, 22,
832.
13. Whitaker, J . "Enzymology of Foods", John Wiley and Sons,
New York.
14. Tuczinski, W.; Scot Blair, G. W. Nature, 167, 216, 367.
15. Howell, J . Α.; Stuck, J . D. Biotech. Bioeng., 1975, 17, 873.
16. Ryu, D. D. Y.; Lee, S. F . ; Tassinari, T.; Macy, C. "Effect
of Compression Million on Cellulose Structure and on Enzyma
tic Hydrolysis Kinetics", private communication, 1981.
17. "Enzyme Engineering I", L. B. Wingard (ed.), Plenum Press,
1972; II, Pye Ε. K. and L. B. Wingard (eds), Plenum Press,
New York, 1974; III, Pye Ε. K. and Weetall, H. H. (eds),
Plenum Press, New York, 1977; IV, Brown, G. and Mannecke, G.
(eds), Plenum Press, New York; V, Plenum Press, New York,
1980; VI, 1982 (in press).
18. Zaborsky, O. "Immobilized Enzymes", CRC Press, 1973; Chibata,
(ed), "Immobilized Enzymes: Research and Development",
Kodansha Press, Tokyo, 1978.
19. Goldstein, L. "Adv. In Enzymology, XLIV, (K. Mosbach (ed.)),
Academic Press, New York, 1976, pp. 397-443.
20. Datta, R.; Ollis, D. F., "Immobilized Biochemicals and
Affinity Chromatography, (R.B. Dunlap (ed.)), Plenum Ρress,
New York, 1974, p. 293; "Adv. in Enzymol.", XLIV, pp. 444-
450 (K. Mosbach (ed.)).
21. Wharton, C. M.; Crook, Ε. M.; Brocklehurst, K. Eur. J.
Biochem., 1968, 6, 572.
22. Datta, R., PhD Thesis, Princeton Univ., 1974.
23. Lieberman, R.; Ollis, D. F . , Biotech. Bioengig, XVII,
1401 (1975).
24. Bailey, J . B.; Ollis, D. F. "Biochemical Engineering Funda
mentals", McGraw H i l l , 1977, p. 469.
Prolonged productio
by in vivo stabilizatio
As a model system we have focused on the gramicidin S
synthetase complex in Bacillus brevis. Our group
previously reported an O -dependent loss of total bio-
2
0097-6156/83/0207-0053$06.00/0
© 1983 American Chemical Society
c o n t r o l l i n g the i n i t i a t i o n of a n t i b i o t i c s y n t h e s i s , such as r e -
p r e s s i o n and i n h i b i t i o n of t h e i r synthetases. For example, carbon
c a t a b o l i t e r e g u l a t i o n i s r e l i e v e d through a d d i t i o n of a slowly
u t i l i z e d carbon source at the beginning of a batch fermentation or
through the slow a d d i t i o n of the otherwise r e a d i l y consumed carbon
source i n various fed-batch type o p e r a t i o n s . S i m i l a r l y , n i t r o g e n
and phosphate r e g u l a t i o n are bypassed e i t h e r through r e s t r i c t i o n
of the r e s p e c t i v e N- or P- source or through a d d i t i o n of a slowly
metabolized a l t e r n a t i v e source of those n u t r i e n t s . Feedback
i n h i b i t i o n of a n t i b i o t i c synthetases has been minimized by appro-
p r i a t e b i o r e a c t o r design, e.g., a d i a l y s i s c u l t u r e i n which the
end product i s continuously removed; (c) f i n a l l y , the s p e c i f i c
growth r a t e of the a n t i b i o t i c - p r o d u c i n g microorganism i s optimized
during the idiophase (e.g., through l i m i t a t i o n of a key n u t r i e n t ,
pH c o n t r o l , etc.) s i n c e i n i t i a t i o n and maintenance of i d i o l i t e
production i s favored a
Genetic s o l u t i o n s t problem regu
l a t o r y mechanisms have a l s o been a p p l i e d s u c c e s s f u l l y f o r the
e f f i c i e n t production of secondary m e t a b o l i t e s . Mutation and
s e l e c t i o n f o r s u p e r i o r producing mutants can account f o r much of
the success of the modern a n t i b i o t i c i n d u s t r y .
However, even with s u p e r i o r producer s t r a i n s and r a t i o n a l
environmental s t r a t e g i e s f o r adequate expression and a c t i v i t y of
the synthetases, these b i o s y n t h e t i c enzymes u s u a l l y decay q u i c k l y
during the idiophase. Thus, the i n v i v o i n a c t i v a t i o n of
b i o s y n t h e t i c enzymes i s widespread and appears to be a l i m i t i n g
f a c t o r i n the prolonged production of a n t i b i o t i c s and r e l a t e d
secondary m e t a b o l i t e s . Despite i t s importance, i n v i v o enzyme
i n a c t i v a t i o n of secondary metabolism has not received any a t t e n -
t i o n as a d i s t i n c t b i o l o g i c a l phenomenon, although a fundamental
understanding of the process(s) could hold the key to i t s success-
f u l circumvention as i s the case with other types of c e l l u l a r
enzyme r e g u l a t i o n .
We have reasoned that a novel approach to prolong the produc-
t i o n phase of secondary metabolites should be based on an attempt
to prevent or r e t a r d the process of in v i v o i n a c t i v a t i o n of the
enzymes (synthetases) c a t a l y z i n g t h e i r formation i n fermentations.
Such an attempt would r e q u i r e an understanding of the chemical
nature of the i n a c t i v a t i o n . T h i s knowledge could be subsequently
t r a n s l a t e d i n t o process development and c o n t r o l i n a c t u a l fermen-
t a t i o n s , which, f o r the most p a r t , are c a r r i e d out i n batch
r e a c t o r s . If the object of the fermentation i s the recovery of
the enzymes f o r f u r t h e r use i n a c e l l - f r e e system or the a c q u i s i -
t i o n of a c t i v e whole c e l l s f o r repeated use i n a f i x e d bed-type
b i o r e a c t o r , the prevention or r e t a r d a t i o n of the i n a c t i v a t i o n
process would ensure both an adequate margin of time for primary
h a r v e s t i n g and a longer h a l f - l i f e of the a c t i v i t y f o r the b i o -
c a t a l y s t s i n the c e l l s .
The purpose of t h i s communication i s to i l l u s t r a t e the
p o t e n t i a l of the above-mentioned approach by f o c u s i n g on our
GROWTH (Klett)
4 5 6 7 8 9
Table I.
Table I I .
Effect o
Stabilit
O v e r a l l synthetase
Atmosphere Additive specific activity Activity
Γ "g GS 1 (%)
Lmg-hr J
a
I n c u b a t i o n with shaking f o r 90 min at 250 rpm, 37°C.
Table I I I .
F a i l u r e of Carbon Source to R e - a c t i v a t e
i n a c t i v a t e d GS Synthetases
O v e r a l l Synthetase
Atmosphere Additive specific activity
Γ Ug GSΤ
L mg'hr J
a
I n c u b a t i o n with shaking f o r 90 min at 250 rpm, 37°C.
0 1 2 3 4 5
time [ hr ]
Figure 3. Time course of specific activity of G S synthetase under air in the presence
of various levels of utilizable carbon source (glycerol). Shown also are the corre
sponding levels of dissolved oxygen (DO). Standard conditions for inactivation:
agitation at 250 rpm, 3TC. Glycerol levels: M, 4%; A, 2%; Δ, 1%; O, 0%.
S SH
Enz'i + GSH ^ Enz' -h GSSG
S ^ ^SH
GSH reductase
n a d p* nad ρ H
Stabilizer compound —> —> —> —> Τ
Conclusions
0 4 8 12 16 20 24
time [ hr J
the presence and absence of an organic thiol (DTT). Standard conditions: agitation
at 250 rpm, 37°C. Key: O, N present; Δ, N + 15 mM DTT present.
t a
%
S.a.
mm DTT at 37°C for 45 min. Key: O, air only; Δ, air, then N, + 15 mM DTT.
Acknowledgements
Literature Cited
H. E. UMBARGER
Purdue University, Department of Biological Sciences, West Lafayette, IN 47907
0097-6156/ 83/0207-0071 $ 0 6 . 5 0 / 0
© 1983 American Chemical Society
Table I
Table I I
General mechanisms:
Energy charge Retardation of asparto- (
kinase I I I a c t i v i t y at
low energy charge
Reducing I n h i b i t i o n of pyruvate (
potential dehydrogenase a t low
NAuVNADH r a t i o s
Substrate None known
availability
Table I I I
P h y s i o l o g i c a l Patterns o f Regulation of P r o t e i n Amount
E f f e c t on Enzyme D e s t r u c t i o n
Specific:
S t a b i l i z a t i o n by l i g a n d s The s t a b i l i z a t i o n of (24)
glutamine phospho-
ribosylpyrophosphate
amidotransferase of
B. subtilis by
substrates
General:
Starvation of c e l l s f o r Increased p r o t e i n turn (25)
carbon or n i t r o g e n over with onset o f
starvation
I n h i b i t i o n of protease Serine protease i n h i b i (26)
activity ±. υ·
t o r of B. subtilis Ό * S ^ ^ »
Table IV
Transcriptional Control
C o n t r o l of T r a n s c r i p t i o n I n i t i a t i o n
Specific:
Negative C o n t r o l Elements
A c t i v a t e d by endproducts Repression of the trp (28)
I n a c t i v a t e d by substrat
or precursor operon by a l l o l a c t o s e
P o s i t i v e C o n t r o l Elements
A c t i v a t e d by s u b s t r a t e Induction of acetohydroxy (30)
or precursor isomeroreductase by
acetohydroxy acids
( I n a c t i v a t i o n by None known
endproduct)
General:
P o s i t i v e C o n t r o l Elements
A c t i v a t e d by cAMP Dependence of lac (31)
operon i n d u c t i o n on
cAMP-CRP i n t e r a c t i o n
A c t i v a t e d by temperature Dependence of s e v e r a l (32)
heat-induced p r o t e i n s
on the htp gene product
σ-Like p r o t e i n s S h i f t o f RNA polymerase (33)
s p e c i f i c i t y by B a c i l l i
during s p o r u l a t i o n
[ppGpp-mediated I n h i b i t i o n of RNA (34)
regulation] polymerase b i n d i n g to
rrn promoters.
Enhancement of polymerase (35)
pausing i n rrn opérons
[Nitrogen-mediated
regulation] The glnG-dependent a c t i v a - (36)
t i o n of genes i n v o l v e d i n
catabolism of n i t r o g e n -
c o n t a i n i n g compounds
General:
p-Dependent termination trpt* terminator a t end (37)
of trp operon
t R 1 terminator of λ (38)
p-Independent termination Termination o f t r a n - (39)
s c r i p t i o n at the end
of leader sequence
f o r amino a c i d
b i o s y n t h e t i c operon
Transient terminâtion
polymerase pausing i n rmB operon
Specific:
A n t i t e r m i n a t i o n by λ Ν gene-dependent (41)
s p e c i f i c proteins t r a n s c r i p t i o n beyond
the tp2 terminator
A n t i t e r m i n a t i o n by Attenuation o f ilv, trp, (39)
ribosome h a l t at his, and other amino
s p e c i f i c codon a c i d b i o s y n t h e t i c codons
Control of T r a n s l a t i o n
Specific-
Masking of ribosomal Binding o f small r i b o - (42)
b i n d i n g s i t e s by p r o t e i n somal p r o t e i n s a t r i b o
binding some b i n d i n g s i t e f o r
the rbsL (str) operon
By p e r t u r b a t i o n of The masking o f erm (43)
secondary s t r u c t u r e (erythromycin r e s i s t a n c e )
of message ribosome b i n d i n g s i t e by
retarded t r a n s l a t i o n o f
the erm leader t r a n s c r i p t
General:
(Amino a c i d supply) None known
(Inhibition) None known
Pyruvote
Ο Ο Ο Ο NH 3
IR OH TrB
CH -CH-C-COO
3
CH -C-C-COO"
3 CH -C-CH-COO"
3 CH -CH-C-COO"
3 CH -CH-CH-COO"
3
AHSU
a-Ketobutyrote CH 2 CH 2 ÇH 2 CH 2
ι ι
CH 3 CH 3
CH 3
CH 3
a - Aceto - a - α,β-Dihydroxy- a-Keio-β-
hydroxybutyrate /3-methylvolerote methy I valerate L- Isoleucine
TD-*--
NH 3
CH -CH - CH-COO"
3
L-Threonjne
H I Β C Y A D E G G G Gp
a e
I—; 1 q
—ι—-pi ι—:—ι—;—ι—;—ι ; ι—;
I I ι
—ι—I, t i *ι "i
AHSm AHSI ι TD DH TrB AHSU
IR
Figure 1. Biosynthesis of isoleucine and valine. The enzymes catalyzing the indicated steps are
abbreviated, and the corresponding structural genes (where known) are indicated in parentheses
as follows.
meso-diaminopimelate
cJ/ysA
i ! L-Lysine
AECrl74
N-ll
uth
KNr31
ilvA uth
uth ilvA
?(Met-leaky)
ilvA r ? (Met-leaky)
lysC (AK ) r
?(Met-leaky) lysC (AK )
0.3 g T h r / 1
T-570
HNr21 T-693
uth uth uth
ilvA ilvA ilvA
r r
r
thrA (AK HD ) r thrA (AK HD )
?(Itet-leaky)
11 g T h r / 1
ileS lysC (AK ) r
8 g Thr/1
lyeCo ?
r r
thrA (AK HD )
île S
25 g T h r / 1
HNr59 E-60
uth uth
ilvA ilvA
r
thrA (HD ) thrA (HD ) r
T-803
ileS ileS
5 g Thr/1 thrB/C uth
r
ilvA (TD )
GIHVL-r6426 ilvGa ?
r
ilvA (TD ) ?(Met-leaky)
r
ilvGa ? lysC (AK )
Mu-910 3.5 g I l e / 1 lysCo ?
r r
Literature Cited
1. Umbarger, H. E. Scienc
2. Kotlarz, D.; Buc, H. Biochim. Biophys. Acta 1977, 484, 35-48.
3. Anderson, P. M.; Marvin, S. V. Biochemistry 1969, 9, 171-8.
4. Cedar, H.; Schwartz, J . H. J. Biol. Chem. 1969, 244, 4112-21.
5. Hoffler, J . G.; Burns, R. O. J . Biol. Chem. 1978, 253, 1245-
51.
6. Ginsburg, A.; Stadtman, E. R. "The Enzymes of Glutamine
Metabolism"; Prusinev, S.; Stadtman, E. R., Ed.; Academic:
New York, 1973; p. 9.
7. Crawford, I. P.; Yanofsky, C. Proc. Natl. Acad. Sci. USA
1958, 44, 1161-70.
8. Klungsøyr, L.; Hagemen, J . H . ; Fall, L . ; Atkinson, D. E.
Biochemistry 1968, 7, 4035-40.
9. Shen, L. C.; Atkinson, D. E. J . Biol. Chem. 1970, 245, 5974-
78.
10. Pardee, A. B.; Potter, V. R. J . Biol. Chem. 1948, 176, 1085-94.
11. Koch, A. L. J. Theoret. Biol. 1967, 15, 75-102.
12. Monod, J.; Jacob, F. Cold Spr. Harbor Symp. Quant. Biol.
1961, 26, 389-401.
13. Krebs, E. G.; Beavo, J . A. Annu. Rev. Biochem. 1979, 48,
923-59.
14. Garnak, M.; Reeves, H. C. J. Biol. Chem. 1979, 254, 7915-20.
15. Pennincky, M.; Simon, J . P.; Wiame, J . M. Eur. J. Biochem.
1974, 49, 429-42.
16. Atkinson, D. E. Biochemistry 1968, 7, 4030-34.
17. Chapman, A. G.; Fall, L . ; Atkinson, D. E. J . Bacteriol.
1971, 108, 1072-86.
18. Vogel, R. H.; McLellen, W. L.; Hirvonen, A. P.; Vogel, H. J .
"Metabolic Pathways," 3rd ed.; Vogel, H. J., Ed.; Academic:
New York, 1971, vol. 5, p. 464.
19. Pardee, A. B.; Jacob, F . ; Monod, J. J . Mol. Biol. 1959, 1,
165-78.
20. Nakada, D.; Magasanik, B. J . Mol. Biol. 1964, 8, 105-27.
Why S i n g l e - C e l l Models?
0 0 9 7 - 6 1 5 6 / 8 3 / 0 2 0 7 - 0 0 9 3 $ l 1.50/0
© 1983 American Chemical Society
Examples of S i n g l e - C e l l Models
ers (40, 41, 42)· They have made extensive measurements of RNA
metabolism and p r o t e i n s y n t h e s i s i n IS. c o l i and have c o r r e l a t e d
t h e i r r e s u l t s with phenomenological expressions. These expres-
sions coupled with s i m i l a r r e l a t i o n s h i p s suggested by other
workers (e.g. Cooper and Helmstetter (43)) allow r a t h e r accurate
c a l c u l a t i o n of c e l l s i z e and composition f o r u n r e s t r i c t e d growth
i n media of v a r i o u s compositions. These expressions appear to be
u n s a t i s f a c t o r y f o r growth r e s t r i c t e d by a l i m i t i n g n u t r i e n t as
glucose (41). The above expressions do not e x p l i c i t l y depend on
the e x t e r n a l environment; the growth r a t e which a media w i l l
support must be known beforehand. These expressions are r e a l l y
c o r r e l a t i o n s r a t h e r than a t r u e model. They are l i m i t e d to a
narrow range of growth c o n d i t i o n s and are not p o t e n t i a l l y as
general as the other models d i s c u s s e d .
Another type of s i n g l e - c e l
Nishimura and B a i l e y (24)
c e l l mass and DNA content are e x p l i c i t l y recognized. Rules con
cerning DNA r e p l i c a t i o n and the timing of i n i t i a t i o n DNA s y n t h e s i s
are imposed on the c e l l ; the r u l e s are d e r i v e d from the observa-
t i o n s of Cooper and Helmstetter (43) and Donachie (44). The
s i n g l e - c e l l model was e s s e n t i a l l y the b a s i s f o r the c o n s t r u c t i o n
of a p o p u l a t i o n model - a formulation among the most general
c u r r e n t l y a v a i l a b l e . The main l i m i t a t i o n of the model i s i t s
i n a b i l i t y to permit an e x p l i c i t c a l c u l a t i o n of each c e l l ' s growth
r a t e i n terms of the e x t e r n a l environment and the c e l l ' s p h y s i o -
logical state.
Ho and Shuler (45) proposed a mathematical model f o r the
growth of an i n d i v i d u a l bacterium i n c o r p o r a t i n g feedback c o n t r o l
of n u t r i e n t uptake. T h i s simple model could p r e d i c t the growth
p a t t e r n f o r a c e l l of a given shape (filamentous, b a c i l l u s , or
s p h e r i c a l ) . The model c e l l contained four components (ammonium
ion, glucose, p r e c u r s o r s , and macromolecules). An a n a l y t i c a l
s o l u t i o n was p o s s i b l e f o r filamentous c e l l s , but numerical s o l u -
t i o n s were r e q u i r e d f o r other c e l l shapes.t
More r e c e n t l y Shuler, Leung, and Dick (46) have presented a
more complete model f o r the growth of a s i n g l e - c e l l of E s c h e r i c h i a
c o l i B/r A. The model contained 14 components. A l l of the c e l l ' s
components were included i n one of the model components. The
model c e l l r e l i e d on chemical concentrations as c o n t r o l s i g n a l s so
that the growth p a t t e r n , timing of DNA s y n t h e s i s , c e l l shape, c e l l
s i z e , c e l l composition, and c e l l d i v i s i o n could be considered
n a t u r a l responses to e x p l i c i t changes (e.g. glucose concentration)
i n the e x t e r n a l environment. An attempt was made to evaluate
k i n e t i c parameters from independent measurements on e x p o n e n t i a l l y
growing c e l l s . Four parameters, having to do with c e l l envelope
The C o r n e l l S i n g l e - C e l l Model
The prototype model has been revamped. The base model has
20 components; the a d d i t i o n a l components are necessary to allow
an accurate d e s c r i p t i o n of the systems a l l o w i n g the i n c o r p o r a t i o n
of ammonium i o n i n t o amino a c i d s , to allow more accurate estimates
of c e l l u l a r energy expenditures, and to allow a more complete
s i m u l a t i o n of systems c o n t r o l l i n g t r a n s c r i p t i o n and t r a n s l a t i o n .
Some of the parameters i n the prototype model (46) were c a l c u
l a t e d based on c e l l dimensions obtained f o r c e l l s f i x e d i n osmium
t e t r o x i d e ; these have been r e c a l c u l a t e d u s i n g s i z e parameters
obtained from g l u c o s e - l i m i t e d chemostat c u l t u r e with g l u t e r
aldehyde-fixed c e l l s .
F i g u r e 1 and Tables I to IV d e s c r i b e the current model.
J u s t i f i c a t i o n of parameter values have been given elsewhere (46-
50). Almost a l l parameters were estimated from independent
measurements on e x p o n e n t i a l l y growing c e l l s or c e l l - f r e e systems.
Values f o r η2 and η (parameters a s s o c i a t e d with the r a t i o of
3
1
at μ = 0.95 hr match experimental measurements. Values f o r
K P Z a n < K r e ( u i r e <
iIF* * * * ZMi» l * experimental growth data f o r μ <
1
0.95 hr but were evaluated independently of the model's p r e
d i c t i o n s . A l l parameters were set based on g l u c o s e - l i m i t e d
growth o n l y . No f u r t h e r adjustments were made f o r p r e d i c t i o n of
ammonium i o n - l i m i t e d growth.
The model makes reasonable p r e d i c t i o n s of the dependence of
c e l l s i z e , c e l l shape, c e l l composition, growth r a t e , and the
timing of c e l l u l a r events on e x t e r n a l concentrations of glucose.
Results f o r g l u c o s e - l i m i t e d growth are given elsewhere (47, 50).
The c u r r e n t model makes use of recent observations of
F r a l i c k (51), Messer, et a l . (52) , and Fayet & Louarn (53) to
construct a mechanism f o r the c o n t r o l of the i n i t i a t i o n of DNA
s y n t h e s i s . The scheme i s i l l u s t r a t e d i n Figure 2. The dnaA gene
which i s l o c a t e d near the o r i g i n makes a gene product (RP) which
represses the t r a n s c r i p t i o n of the 0-RNA gene. I n i t i a t i o n r e
q u i r e s 0-RNA as a primer. An a n t i - r e p r e s s o r (ARP) i s made which
i n a c t i v a t e s RP. Experimental evidence f o r ARP e x i s t s (51), and
there are i n d i c a t i o n s that ARP production i s r e l a t e d to c e l l
envelope formation (54). In the model we have made the r a t e of
Transport *Couple
(1) Αχ* t r a n s p o r t s i n & becomes Αχ ω. Αι + ω ^ 0 2 •> ω
χ
Η2Ο Α Α
Αι Αχ Α χ
Precursor Formation
(3) otiAi + 6 ι Α + ... •+ P i
2
+ ... δ ΑΤΡ
ρ ι
-V δ (ΑΟΡ
ρι
+
(4) ε Ρ ι + 32A2 + . . .
2 •> P 2 + ... δ Α Τ Ρ -> δ
ρ 2 ρ (ADP + ν
(5) ε Ρ2 + ω
3 Ό
r
Χ + ... + Ρ 3 + .. . δ Α Τ Ρ -> δ ( Α 0 Ρ
ρ 3 ρ 3 +
3
(6) ει»Ρι + $ι*Α + ... -> Ρι» 2 + ... δ ρΐ( ΑΤΡ -> δ ρ (ADP + p
i>
Macromolecule Formation
->
(7) ΎιΡι
J
Μι + ... V ATP
y (ADp +
(8) Ύ2?2 £ Μ 2 + ... δ Μ ΑΤΡ ->-
V ( A D P
+
(9) Ύ3Ρ3 J Μ 3 + ... ΑΤΡ -+ δ (ADP + p
i>
Μ Μ3
3
(10) Ύ^Ρ* δ Τ Ρ
-> δ (ADP + p
i>
Μ/ Mit
A d d i t i o n a l D i r e c t Reductant Use
Transport of ions + ω W H 2
W ΐ Ο Ν *>* - I0N °
+
Membrane recharge to o f f s e t H
leakage
Table I , continued.
Additional Biosyntheti
=
SO" -> S ω 8 0 ι + Χ + SO -> [ S ]
Mass use ω Β Ι ( ) Χ + M J * - M^* D
A d d i t i o n a l ATP Coupling
PG formation ό ATP -> 6 „ ( A D P + P.)
PG PG i
*
Coupled to e i t h e r phosphate bond energy or o x i d a t i v e r e a c t i o n s .
Products of o x i d a t i o n i n c l u d e pmf and reducing e q u i v a l e n t s .
**
M = u n r e c u c e ( a n c
0X* **RED * l * reduced c e l l mass, r e s p e c t i v e l y .
it " V s
- α ι [ (
ΐΓ> 5
(
- -dT> D
] a (
- i,GLN ^r> s
( 1 )
NET
,dM . 5
(2)
Table I I , continued.
.dATP ,dP!
K ;
dt
rf d M 2
s
ν
i RTI
dt
[1 »
,dPG
l d t >
s
(3)
GLN ^ t -
g +
W~dt> s
s + < ω + ω
f - "a!'V ΐΟΝ Ο
dP 3
δ + ω (4)
ν Ρ <1ΠΓ>
3
(dATP/dt). + dX/dt(P/0)
(dA /dt),2
S max •180 (5)
4 + (12 - 4·Ζ)(Ρ/0)
max
/ (dMWdt) s
Ζ =μ (6)
^<d*/dt) g + K z M i >
dA 2
(7)
A L»
GLN/V A /V
2
(8a)
+ A l / V
SxGUI + G L N / V
«ΡχΑ,
dt " "A 2
(8b)
,dGLN.
s
( d t )
ι · r.
N E T
dp
ει
t T h i s term i s equivalent t o : *(~ΠΓ*)
S
dMi
(12)
dM 2
RTI P /V iPG
= P2
2
dt P + PG/
^ P ^ * ^ 1 P G 7
\LIF
•GD -
rr dMi/dtv Mi l S ι
K + [ (
iIF Μ
rdM 2
RTI RTM
k
dt (13)
TM 2 (dMi/dt) s | M 2
RTI
RTI1
RTI
dM 2
RTM RTM
(14)
dt RTM <RTI T M 2 R U + A 2 / V
RTM
dM 2
"*M /dM \ . X
(15)
ίγ ' μ 2
Μ ^ - ν δ Μ * Μ 2
Μ
dM 3
= „ / Pa/V V A 2 / V
(16)
dt
(17)
dM 5
(18)
dt
(19)
C
A* __Aj7y__ A /V 2
Αχ Ai K. . + A /V 2
K
A L
+ C
A J ' K
A X + A L / V AiA 2
Κ
Ai A /V2 ^
V +
(21)
^K c
Ul iAiK + A i / v
A A K
I A 2
+
A2/V
J
/ c* 2 \/ K i A 2 \
(22)
*A 2
dEi dMi
niN (23)
dt ο dt
dE dMi
2
n (24)
dt 2
dt
dE 2
TI3M1
dt (25)
dEij_
ni»
dt (26)
Μι»
V =
(27)
cyto
2
S • nW + πΝ·α,+ 2πW·SL (29)
SEPF
SL (31)
2ÏÏW
SEPFι = SEPF, + _ E _ - dS 3
(32)
|tH-At 11 E + E 2 3
(33)
Table I I , continued.
At (34b)
^IRP
RP (35)
burst
/dM l / d t\ («i/^) "
8
K +
iRP
6
K * = 3.6 χ 10"
Αχ cc
2.056 χ 1 0 - 7
JES- κ - 1.0 χ 10~ 5
Β»
ce h r Αι ce
Κ Α
1
= 1.0 χ Ι Ο " 7
&* Κ Α = 1.0 χ ΙΟ" 5
Β&.
Αι ce Αχ ce
Α2
κ 2
hr cm Α ce
~iA 2
cc'
gms
amino a c i d s k i = 0.390
hr cc *P I A i -· *2 5 1 0 - 5
S
IL, = 5.0 χ ΙΟ" -SB 2
Τι ce
-5 gm
= 0.025 h r " 1
= 1.1 χ 10
^Pi
β
q 3
k '' β 0.39 J = S - 3 3 1 0 1 2
hr cc ^GLN = · * "
m
ribonucleotides k 2 - 0.19 , S
hr cc ce
3
K- = 9.0 χ 10" S E K
= 2.5 χ Î O " -
«
r 2 CC P A 2 2 c c
V = 0-03 h r " 1
^ f - l . l x l O - « a
CC
7 2 1 0 5
VA, * 7
· 2 x 1 0
" 5
f? NM 1 ° ° · 0 2 5 H R _ 1
K 1 x 1 0 - 5 = 0 2 5h r _ 1
V • f! ν °·
"te "
3
K i p G - 2.0 χ 1 0 - i ^ K i I F = 0.009 ( ^ )
1 1
μ 2 = 14 h r " k =0.07 h r "
RTM ™2 R T M
τ> ·» Λ οο gm RNA
Ρ 3 μ 2 3 2
" Μ " °· gmProtein
5 1
Κ™ - 1.1 χ ΙΟ" « k = 21.0 h r "
2 C C 2
™ RTM ™ M
1S 8 6
DNA u = 6.0 x 10" . S ? .
3 1C, _ = 4.0 χ 10" f §
hr fork M P 3 3 CC
s
IL, . = 2.0 x 10"
M A 3 2 CC
VA, " · 1 8 x 10
~* f ? Si, " °' 2 3h r _ 1
1 8X 1 0
W a = · "S
2 Uh r _ 1
glycogen μ - 2.0 χ 10"
5 ^TMs " ° '
2 x10 3
VA. =·° " S ^ 1
- -° * ' f ! l 0 3
Table I I I , continued* 3
enzymes = 1.0 χ 10 Π ι η 2 == 3Q. 2ο χ 1iθn" 3 v
2 1
η13 - 1.6 χ 10" h r "
3
2
10
IL, = 2 . 0 χ ΙΟ" (SE)' n i f = 0.01
£ι|» ce
β 2 k 0 5 h r X
* TE, - ° · '
P p G - 6 . 4 χ ΙΟ" - J g L - 3
k ± p i - 1 2 . 7 χ ΙΟ"» £
k 1 2 5 h r X k 7 2 X 1 0 5
TPG « ' TPGA = · 2 ' S
6
c e l l shape ρ = 0 . 2 5 8 *E* f = 259 χ 1 0 —
cyto ce s gm
ο = 0.553 BS.
env cc
3
DNA i n i t i a t i o n k = 2.6 χ 1 θ " - ^ r - IT = 0.22 - S * —
ARP gm Mi* iRP cc-hr
3 6 5 x 1 0 - 1 7
*** · · S r
Stoichiometric Coefficients:
Table I I I , continued*
2 - 1
glycogen y = 2.0 χ 1 0 "
5 = .14 h r
0
5
K
M A 5 2
= 2
-° x 1 0
" f 3 K
T M « 1-0 x 10"
5
3
g
3 3
enzymes n i = 1.0 χ 1 0 " n = 3.2 χ 1 0 "
2
2 1
n = 1.6 x 1 0 " h r "
3
%= 2 1
k TEit = 0.05 h r '
k T p G = 125hr- 1
K TpGft2 - 7.2 χ 1(Γ« g
oxidation Pi
^| =1.5
I
max
6
c e l l shape . = 0.258 = 5 9 χ 10 ^
P /
cyto ce f
s 2
gm
env = 0.553 c3 c
P p n v 2i
Ύ = 1 Λ 0
β 2 = -0.456 "
3H. 1.28 Ύ5 = 1 . U
A , NH^+,
x transport 0.028
3
2 χ 10" mole
Ion Transport
gm c e l l
3 moles
PG Formation
mole
0.0633 moles
Uncoupled ATP Use
3
cm
3
1.21 χ 1Q- mole
SO^ S
gm
2
1.39 χ I P " mole
Biosynthesis Reduction
gm
+
(2) α-Ketoglutarate + L-glutamine + NADPH -> 2 glutamate + NADP
+ +
(3) α-Ketoglutarate + NHi» + NADPH -> L-glutamate + H 0 2 + NADP
y C* _ D* ^
Snodel ^model expt. expt
1
hr" min. min. min. min.
0.95 41 21 42 22
0.68 47 28 42 22
0.51 59 35 56 28
0.39 75 42 73 39
0.29 99
0.24 120 60 ^115 -57
0.16 189 81 -178 -89
t/τ *
t / T
Model expt.
1
hr"
0.8 h
Figure 4. Variation of cell volume with growth rate. For glucose-limited cells:
O, data from Cornell; data from Ref. 64. For ammonium-limited cells: Δ,
data from Cornell. Solid line, the model's prediction for glucose-limited cells;
dashed line, model's prediction for ammonium-limited cells.
l.2r
1.0
UJ
0.8
<
tr
UJ 0.6
u 0.4
0.2
0.0
4 6 8 10 12
GENERATIONS
The s i n g l e - c e l l model i s l i m i t e d i n i t s u s e f u l n e s s f o r
engineering c a l c u l a t i o n s unless a p o p u l a t i o n model can be con-
s t r u c t e d from the information i n the s i n g l e - c e l l model. I t i s
p o s s i b l e to b u i l d a p o p u l a t i o n model u s i n g an ensemble of s i n g l e -
c e l l models to mimic the response of a l a r g e p o p u l a t i o n of c e l l s
(24, 46, 68). Since computer c a p a c i t y i s f i n i t e , the question i s
r e a l l y , "how few c e l l models can be included i n a p o p u l a t i o n and
s t i l l allow reasonable p r e d i c t i o n s of the behavior of a n a t u r a l
p o p u l a t i o n of c e l l s ? "
F i g u r e 9 presents a sketch of an " i d e a l " s i z e d i s t r i b u t i o n
and a " t y p i c a l " s i z e d i s t r i b u t i o n . The " i d e a l " d i s t r i b u t i o n
would occur i f there were no v a r i a t i o n producing processes w i t h i n
the c e l l ( i . e . a l l c e l l s have i d e n t i c a l c e l l c y c l e s ) . In the
i d e a l d i s t r i b u t i o n c e l l s a u t o m a t i c a l l y d i v i d e at twice the b i r t h
volume, and there are two times as many c e l l s of b i r t h s i z e as
d i v i s i o n s i z e . The frequency f u n c t i o n of c e l l s of intermediate
s i z e would decrease approximately e x p o n e n t i a l l y from b i r t h to
fission-ready c e l l s .
However, v a r i a t i o n causing processes e x i s t . H y p o t h e t i c a l
p r o b a b i l i t y f u n c t i o n s r e l a t i n g b i r t h and d i v i s i o n to c e l l s i z e
are shown. Such f u n c t i o n s w i l l r e s u l t i n a " t y p i c a l " s i z e
8r
I4r
0.2l 1 1 1 1 L
0 2 4 6 8 10 12
GENERATIONS
Figure 8. Transient response of the computer cell to a step-change in external
ammonium-ion concentration: O, response to step-decrease (2.5 mg/L to 1.4
mg/L); Δ, response to step-increase (lΛ mg/L to 2.5 mg/L). The relative rate
corresponds to the average growth rate during that cell generation.
Figure 9. Sketches of ideal size distribution ( - ' - · - ) and typical size distribu
tion ( ). The typical distribution results from random processes that cause the
cell to have a significant probability of fission over a range of sizes.
Ο
c
q>
3
σ-
ο
c
3
σ
0)
>% >*
ο ο
c c
0) α>
3 3
σ
α) σ
α)
Summary
Acknowledgment
φ
3
σ
α)
Figure 12. Computer simulation of a population using 240 structured cells (the
Cornell single-cell model). There were 16 size classes and 15 cells per class in the
simulation. The steady-state size distributions display a level of stability acceptable
for most applications.
Literature Cited
1. Tsuchiya, Η.Μ.; Fredrickson, A.G.; Aris, R. Adv. Chem. Eng.
1966, 6, 125-205.
2. Painter, P.R.; Marr, A.G. Ann. Rev. Microbiol. 1968, 22,
519-548.
3. Van Uden, N. Ann. Rev. Microbiol. 1969, 23, 473-486.
4. Garfinkel, D.; Garfinkel, L.; Pring, M.; Green, S.B.;
Chance, B. Ann. Rev. Biochem. 1970, 39, 473-498.
5. Fredrickson, A.G.; Megee, R.D., III; Tsuchiya, Η.Μ. Adv.
Appl. Microbiol. 1970, 13, 419-465.
6. Nyiri, L.K. Adv. Biochemical Eng. 1972, 2, 49-95.
7. Boyle, W.C.; Berthouex, P.M. Biotechnol. Bioeng. 1974, 16
1139-1159.
8. Fredrickson, A.G
9. Bailey, J . E . Chem
10. Fredrickson, A.G.; Ramkrisha, D.; Tsuchiya, Η.Μ. Chem. Eng.
Symp. Series 1971, 67(108), 53-59.
11. Campbell, A. Bacteriol. Rev. 1957, 21, 263-272.
12. Williams, F.M. J . Theo. Biol. 1967, 15, 190-207.
13. Williams, F.M. "Systems Analysis and Simulation in Ecology";
Patten, B.C., Ed.; Academic Press: New York, 1971, Vol. 1.
14. Ramkrishna, D.; Fredrickson, A.G.; Tsuchiya, Η.Μ.
Biotechnol. Bioeng. 1967, 9, 129-170.
15. Van Foerster, Η. "The Kinetics of Cellular Proliferation";
Stohlman, F . , Ed.; Grune & Stratton: New York, 1959, p. 382.
16. Trucco, E. Bull. Math. Biophys. 1965, 27, 285-304, 449-471.
17. Yakovlev, A.Y.; Zorin, A.V.; Isanin, N.A. J . Theor. Biol.
1977, 64, 1-25.
18. Fredrickson, A.G.; Tsuchiya, Η.Μ. AIChE J. 1963, 9, 459-468.
19. Kozesnik, J . "Continuous Cultivation of Microorganisms";
Malek, I.; Beran, K.; Hospodka, J., Eds.; Academic Press:
New York, 1964, p. 59.
20. Lebowitz, J . L . ; Rubinow, S.I. J . Math. Biology 1974, 1,
17-36.
21. Koch, A.L.; Schaechter, M. J . Gen. Microbiol. 1962, 29,
435-454.
22. Eakman, J.M.; Fredrickson, A.G.; Tsuchiya, Η.Μ. Chem. Eng.
Prog. Symp. Series 1966, 62(69), 37-49.
23. Bailey, J . E . ; Fazel-Madjlessi, J.; McQuitty, D.N.; Lee, L . Y . ;
Oro, J.A. AIChE J. 1978, 24, 570-576.
24. Nishimura, Y . ; Bailey, J . E . Math. Biosciences 1980, 51,
305-328.
25. Nishimura, Y . ; Bailey, J . E . AIChE J. 1981, 27, 73-81.
26. Pickett, A.M.; Bazin, M.J.; Topiwala, Η.Η. Biotechnol.
Bioeng. 1980, 22, 1213-1224.
27. Pickett, A.M.; Bazin, M.J.; Topiwala, Η.Η. Biotechnol.
Bioeng. 1979, 21, 1043-1055.
28. Tanner, R.D. Biotechnol. Bioeng. 1970, 12, 831-843.
29. Bischoff, K.B. Can. J . Chem. Eng. 1966, 44, 281-284.
JAMES E. BAILEY
California Institute of Technology, Department of Chemical Engineering,
Pasadena, CA 91125
0097-6156/83/0207-0135$07.50/0
© 1983 American Chemical Society
^ [ r ( p , s ) f ( p , s ) ] = D[4<K2p,s)-<f>(p,s)-f(p,s)]
f (1.1)
f(0,s) = 0 (1.2)
STAIN PROTEIN
0
WITH
FLUORESCENT SU
COMPOUND
STAINED
SAMPLE FLOW
SAMPLE
PHOTOMULTIPLIER
TUBE
ANALYZE
PULSES
<
60 20 20-0
J I I I L
60 50 40 30 20 10 0
30 20
<
ζ *ΞΕ=
ο
>
<
UJ DOUBLING TIME = 50 MIN
or 50 40 30 20 10 0
TIME UNTIL
DIVISION (MIN)
Figure 2. DNA synthesis rates and chromosome configurations according to the
Cooper-Helmstetter (11) model for E . coli growing at different rates. (The circular
chromosome is shown here in linear form for schematic convenience.)
CELL
ENVIRONMENT
μ (t)
CELL
GROWTH
ι DONACHIE
INITIATION OF
DNA
ONA
COOPER- ^ CONFIGURATION
REPLICATION HELMSTETTER AND UtLL
m DIVISION
"m =
M m = 2 m" TIMING
DAUGHTER
t BINARY FISSION
CELL MASS
Figure 3. Summary of a model for coordinated mass and DNA synthesis and cell
division for individual cells of E . coli. Reproduced, with permission, from Ref. 14.
Copyright 1980, Elsevier North Holland, Inc.
1 i near
mass i n -
crease ,
constant
volume
1 i n e a r mass
increase , exp-
onential volume
increase
Cell size
CS - c e l l separation ; ND - n u c l e a r division ;
CP - c e l l plate formation
Figure 6. Schematic diagram of the nuclear and cell division cycles of fission
yeast S. pombe.
r^(p,s) = k ( s ) ,
Q zero order (2.1)
r (p»s) = k ( s ) p ,
f 1 f i r s t order (2.2)
0.06
^ 0.02
ΰ 0 J \ L . I I I I \—ι I I I I
0 0.06
£0.04 " I 0.132 - Λ 0.088
LU 0.02
F ο λ V-M I I I I /| ι ι ι ι
<0.06
- Λ 0.131 0.088
ω 0.04
λ
Ο,
Ο 6
C E L L DNA C O N T E N T
(CHANNEL N U M B E R )
Figure 7. Frequency functions for cellular DNA content for S. pombe populations
propagated in a chemostat at different dilution rates (specific growth rates shown in
4
each frame). Channel number, one division = 6.8 X 10' pg DNA/cell. Repro
duced, with permission, from Ref. 9. Copyright 1981, John Wiley & Sons, Inc.
0.03
0.249 0.176
Ζ 0.01
r^i
3 ο
ι J ι ι ι 1 / ι ι \ - ι ι
Ο 0.03
- ι^ν 0.132 - Λ 0.088
uj 0.01
Ê ο ι J\ ι j ι Il 1 N-i- ι ι ι
< 0.03
- /V 0.131 - 0.088
UJ 0.02
0.01
. 1/1 J*. 1
6 4 128 192 0 6 4 128 192 2 5 6
C E L L PROTEIN CONTENT
(CHANNEL NUMBER)
American Chemical
Society Library
1155 16th St., N.W.
In Foundations of Biochemical Engineering; Blanch, H., et al.;
ACS Symposium Series; Washington, U.C.
American Chemical 20036
Society: Washington, DC, 1983.
148 BIOCHEMICAL ENGINEERING
r
f(p»s) = k ( ) Q
s +
k^(s)p, two term (2.3)
2
r (p,s) = k (s) + k (s)p + k (s)p ,
f Q 1 2 three term (2.4)
> 0.03
ZERO ORDER FIRST ORDER
S 0.02
0 1
S °·
Ê Ο
TWO TERM THREE TERM
LU
> 0.02
LU —ι ι^^^^ J ι J^^^^^NS^ 1
64 128 192 0 64 128 192 256
C E L L PROTEIN CONTENT
π r
0 64 128 192 256
CELL PROTEIN CONTENT
(CHANNEL NUMBER)
second,
first DNA gap (62) 61
gap (G|) synthesis - and ~
nuclear
(S) division (M)
I NUCLEAR CYCLE
Figure 11. Schematic diagram of the nuclear and cell division cycles for the
budding yeast S. cerevisiae. Reproduced, with permission, from Ref. 35. Copyright
1980, Pergamon Press Ltd.
Discussion
Acknowledgements
LITERATURE CITED
A C y b e r n e t i c P e r s p e c t i v e of Microbial Growth
DORAISWAMI RAMKRISHNA
Purdue University, School of Chemical Engineering, West Lafayette, IN 47907
0097-6156/83/0207-0161 $06.00/0
© 1983 American Chemical Society
THE CYBERNETI
s t a n t s , R i s a f i x e d r a t e of t o t a l resource a v a i l a b i l i t y the
f r a c t i o n of which a l l o c a t e d f o r enzyme E i i s given by U i . The
#
J = - b E Y . ^ (3)
dt i ι dt
The biomass, whose concentration i s represented by b, i s viewed
as comprising key enzymes and the r e s t of the biomass. For the
sake of s i m p l i c i t y no m a t e r i a l resources have been e x p l i c i t l y i n
cluded i n the biomass f o r the present.
D h u r j a t i [10] based the optimal a l l o c a t i o n of resources on
minimizing the time r e q u i r e d f o r r e a l i z i n g a l l of the biomass
from the d i f f e r e n t s u b s t r a t e s . Thus the c o n t r o l o b j e c t i v e i s
given by
Mi
l 1
i î
where t f i s the time required f o r the substrate l e v e l s to drop to
some preassigned values (since a r b i t r a r i l y small values would
lead to a r b i t r a r i l y l a r g e times mathematically).
1
This completes D h u r j a t i s model. The mathematical techniaue
of computing the optimal a l l o c a t i o n r a t e s to accomplish the objec-
t i v e (4) i s contained i n the well-known minimum p r i n c i p l e of
Pontryagin [11] which w i l l not be discussed here. The l i n e a r
f u n c t i o n a l i t y of the c o n t r o l v a r i a b l e s u^ leads to what i s r e f e r -
red to as a "bang-bang" p o l i c y which i n the present context im-
p l i e s e x c l u s i v e u t i l i z a t i o n of one of the s u b s t r a t e s . ( D h u r j a t i
[10] has i n v e s t i g a t e d the p o s s i b i l i t y of s i n g u l a r c o n t r o l which
could accommodate simultaneous consumption of d i f f e r e n t sub-
s t r a t e s and f i n d s the c o n s t r a i n t s to be i m p l a u s i b l e ) . The
d i a u x i c growth s i t u a t i o n i s thus described by t h i s model.
D h u r j a t i was able to adapt model constants to d e s c r i b e growth
data obtained by him on K l e b s i e l l a pneumoniae grown on a mixture
of glucose and xylose. The preference f o r glucose with a higher
growth r a t e on i t i s c o n s i s t e n t with the i n t e r p r e t a t i o n s of the
model. The r e s u l t i s d i s p l a y e d i n F i g u r e 1. The question a r i s e s
as to whether the organism i n f a c t consumes both glucose and
xylose a f t e r the former has dropped to s u f f i c i e n t l y low l e v e l s .
While t h i s i s not r e a d i l y v e r i f i a b l e because of the d i f f i c u l t i e s
inherent i n measuring small d i f f e r e n c e s i n small substrate con-
1
c e n t r a t i o n s some a s s e r t i o n s can be made i n regard to D h u r j a t i s
model. The l i n e a r i t y i n the c o n t r o l v a r i a b l e w i l l permit only
e x c l u s i v e u t i l i z a t i o n of one substrate or the other. I f f o r ex-
ample 'more p r e f e r r e d * s u b s t r a t e , say drops to s u f f i c i e n t l y
low l e v e l s , the preference would switch to the 'less p r e f e r r e d '
substrate ( S ) a f t e r which e x c l u s i v e u t i l i z a t i o n of S w i l l
2 2
Figure 1. The diauxic growth curve predicted by Dhurjati's model for Klebsiella
pneumoniae grown on a glucose-xylose mixture. Dashed lines, model; solid line,
experimental.
TIME (HOURS)
Figure 2. Dissolved oxygen curve for Klebsiella pneumoniae grown on xylose
with periodic addition of glucose. G, 250 mg/mL glucose; X, 250 mg/mL xylose
(t = 0, 50 mLX).
dT - φ τ γ ι " 6
i i e ( 5 )
j = { i : m a x
(K'Vs.) 2 } ( 6 )
β
where ^Λ ^9 u r
i ) i s the r a t e of consumption of resources f o r
the synthesis of i enzyme E^; i t i s r e l a t e d to the r a t e of
t n
f ( s U r ) = 6 ( 1 0 )
i i ' i ί(Κ| + S i ) (κ. + u.r)
with Eq.(5) modified by
de. a. S i u r
±
- 6!e. (11)
dt (iq + S i ) (κ ± + u.r) M
i ic
Concluding Remarks
Ackrawledgnents
Nomenclature
Β Biomass
b Concentration of biomass
Ε Key enzyme
e Concentration of key enzyme
f Rate of comsumption of resources
for s y n t h e s i s of key enzyme.
Κ M i c h a e l i s constant f o r growth
Κ» M i c h a e l i s constant of enzyme syn
thesis
R T o t a l resource a b a i l a b i l i t y r a t e
r Concentration of l i m i t i n g resource
S Substrate
s Concentration of s u b s t r a t e
t Time
u Fractional allocation
V Rate constant i n growth
Yield coefficient
Greek symbols
α,a1
Rate constants i n enzyme s y n t h e s i s
β Rate constant f o r enzyme breakdown
Ύ S t o i c h i o m e t r i c constant f o r r e
plenishment of resources by growth
S t o i c h i o m e t r i c constant f o r con
sumption of resources f o r enzyme
synthesis
Kroenecker d e l t a equals u n i t y when
i=j and vanishes otherwise
M i c h a e l i s constant f o r consumption
of resources f o r enzyme s y n t h e s i s
Subscripts
Refers to i substrate
Refers to f i n a l s t a t e of growth
when s u b s t r a t e s drop to p r e a s s i g n -
ed l e v e l s
Literature Cited
CHARLES L. COONEY
Massachusetts Institute of Technology, Department of Nutrition and Food Science,
Biochemical Engineering Laboratory, Cambridge, MA 02139
0097-6156/83/0207-0179$06.00/0
© 1983 American Chemical Society
Production o f C e l l Mass
Overall
Initial Final Fermentation cell y i e l d Volumetric
cell cone cell cone time (g cells/ productivity
( g/liter) (g/liter) (hr) g sugar) (g/liter-hr)
Source: Ref. 3.
S i n g l e - C e l l P r o t e i n . T h e concept of single-cell p r o t e i n ,
while not new i n that it was utilized b y both the A z t e c s a n d v a r -
ious c u l t u r e s i n c e n t r a l A f r i c a , has only r e c e n t l y (since 1960)
been p r o p o s e d as a means for p r o d u c i n g l a r g e - s c a l e , h i g h quality
protein to supplement traditional p r o t e i n resources ( 8 ) . T h e man-
u f a c t u r i n g cost for S C P i s dominated b y the raw material cost, as
well as capital investment ( 1 ) . A s a consequence, the objective
i n p r o c e s s development i s to maximize the c o n v e r s i o n y i e l d a n d
the volumetric p r o d u c t i v i t y . In this way, the o p e r a t i n g a n d cap-
ital investment cost c a n be minimized. Continuous c u l t u r e o f f e r s
a u s e f u l a p p r o a c h i n which cells c a n be grown u n d e r c a r b o n
source to p r o t e i n u n d e
ity.
Two strategies fo optimizatio o productio are sugges
ted i n F i g u r e 3. F i g u r e 3A shows a traditional continuous c u l t u r e
isotherm. T h e maximum p r o d u c t i v i t y o c c u r s at a value D x ( 9 ) . m a
does not take into account the need to operate the fermentor at
i t s maximum p r o d u c t i v i t y , the value o f which i s determined b y the
o x y g e n t r a n s f e r rate (or i n the case o f v e r y large r e a c t o r s , the
heat t r a n s f e r r a t e ) . T h i s i s i l l u s t r a t e d i n Equation 1:
m a x
W m = DX m = Y n R (1)
m m 0 2 0 2
Dilution Rate
that for fixed oxygen transfer rate, OTR (curves show the maximum cell concen-
tration for a given OTR and cell yield).
Enzyme Production
Maltase P r o d u c t i o n
Table II
Maltase (units/g-cell)
Carbon Source Wild T y p e Mutant 1-4
Sucrose no growth
Maltose 870 770
Glycerol 10 1100
Acetate 10 770
Fructose 10 380
Glucose 10 320
TIME (HOURS)
Table III
Specific
Activity Productivity
(units/ ( unit s7
Process g-cell) g-cell-hr) (units/1-hr)
Batch
(1-4)
Continuous 2 9 Q 3 4 0 0
(1-4)
Batch
890 70 470
(wild type)
Heparinase Production
CE
Lu
I
O 10 20 30
HOURS
Metabolite Production
T h e p r i m a r y objective f u n c t i o n s f o r p r o d u c t i o n o f metabolites
are to maximize the concentration (to minimize r e c o v e r y costs)
while maintaining h i g h c o n v e r s i o n y i e l d s o f costly raw materials to
the p r o d u c t , u n d e r conditions o f h i g h volumetric p r o d u c t i v i t y (to
r e d u c e capital c o s t ) . T h e u s u a l a p p r o a c h i s to r a p i d l y p r o d u c e a
h i g h cell concentration u n d e r conditions that maximize the c o n v e r -
sion rate o f raw materials to the d e s i r e d p r o d u c t .
The example c o n s i d e r e d here i s penicillin p r o d u c t i o n b y Péni-
cillium c h r y s o g e n u m . Penicillin i s u n d e r control of c a r b o n catab-
olite r e p r e s s i o n a n d is t y p i c a l l formed secondar product
after p r i m a r y growth i
oped i n i n d u s t r y f o r thi generally
fed-batch fermentation which r e s t r i c t s the flow o f c a r b o n to the
c u l t u r e to slow down growth, t h u s p r e v e n t i n g c a r b o n catabolite
r e p r e s s i o n . Mou a n d Cooney (16) initiated a p r o g r a m to examine
the application of on-line computer control as a means f o r achiev-
i n g improved c a r b o n source management f o r penicillin p r o d u c t i o n .
The rationale was to utilize on-line monitoring o f growth to assess
the demand o f the c u l t u r e for the c a r b o n source a n d i n t h i s way
develop a means to control the specific growth rate i n the fermen-
tation. It was reasoned that on-line computer c o n t r o l would p r o v e
to be more flexible i n allowing one to balance s u p p l y a n d demand
of the c a r b o n source d u r i n g this fermentation.
A series o f studies were conducted i n which a computer con-
t r o l system was utilized to control both growth phase a n d p r o d u c -
tion phase growth rates a n d to a s k the question, "what i s the ef-
fect o f changes i n these growth rates on the specific p r o d u c t i v i t y
of p e n i c i l l i n ? " . In F i g u r e 8 are shown r e s u l t s i n which the p r o -
1
duction phase growth rate was manipulated from 0 to 0.015 h "
while the initial growth rate was manipulated constant at i t s maxi-
mum value o f 0.107. T h e specific rates of penicillin p r o d u c t i o n
are shown i n F i g u r e 9. It i s clear that control o f the p r o d u c t i o n
phase growth rate i s important to maintaining the ability of the
cells to make p e n i c i l l i n . While it i s not clear from these r e s u l t s
what the optimum value i s , this a p p r o a c h does allow one to b e g i n
to design a set of experiments to answer that q u e s t i o n . In a
second set o f experiments, two growth phase growth rates were
compared. In the first case, the growth was controlled at 0.11
1
h " a n d i n the second case at 40% o f this value. T h e r e s u l t s ,
shown i n F i g u r e 10, indicate that cells grown more slowly d u r i n g
the initial growth phase have a h i g h e r specific rate of penicillin
p r o d u c t i o n . A g a i n , it i s not clear what the optimum values f o r
growth phase growth rate are; i t i s clear, however, that c a r b o n
source management d u r i n g both the growth a n d p r o d u c t i o n phase
is important to maximize the specific rate of penicillin p r o d u c t i o n .
600Γ
HOURS
6Γ
HOURS
HOURS
Conclusions
Literature Cited
183-197.
2. Cooney, C.L.; Rha, C . K . ; Tannenbaum, S.R. Adv. in
Food Res., 1980, 26, 1-52.
3. Wang, H . Y . ; Cooney, C . L . ; Wang, D.I.C. Biotech. Bio-
engr., 1979, 21, 975-995.
4. Pirt, S . J . "Principles of Microbe and Cell Cultivation",
1975, Blackwell Scientific Publications, London.
5. Aiba, S.; Nagai, S.; Nishizawa, Y. Biotechnol. Bioengr.,
1976, 19, 1001.
6. Cooney, C . L . ; Wang, H . ; Wang, D.I.C. Biotech. Bioengr.,
1977, 19, 55-66.
A. G. FREDRICKSON
University of Minnesota, Department of Chemical Engineering and
Materials Science, Minneapolis, MN 55455
0097-6156/83/0207-0201 $ 0 7 . 7 5 / 0
© 1983 American Chemical Society
C l a s s i f i c a t i o n of population interactions
Effect Effect
of presence of presence
of Β on of A on
growth rate growth rate Name of
of A of Β Qualifying remarks interaction
-
—
—
—
Ο
—
Negative effects caused by
removal of resources
Negative effects caused by
COMPETITION
ANTAGONISM
— Ο production of toxins or inhibitors AMENSALISM
Negative effects caused by
- + production of lytic agents;
positive effects caused by
solubilization of biomass
ECCRINOLYSIS
FEEDING -
- + Β feeds on A
INCLUDES
PREDATION AND
SUSPENSION-
FEEDING
The parasite (B) penetrates
- +
the body of its host (A) and
therein converts the host's
biomaterial or activities
PARASITISM
D i s c u s s i o n of the i n t e r a c t i o n
A
Β I Figure 2. An association of two popu-
lations that interact by commensalism
E c c r i n o l y s i s . C o n s t r u c t i o n of t h e o r i e s of t h i s i n t e r a c t i o n i s
faced with d i f f i c u l t i e s s i m i l a r to those f a c i n g c o n s t r u c t i o n of
t h e o r i e s of protocooperation, but the d i f f i c u l t i e s are even more
severe. In order to construct such a theory, we would need know-
ledge of the k i n e t i c s of formation of exoenzymes by the one popu-
l a t i o n and of the k i n e t i c s of a c t i o n of those enzymes on the other
population of the p a i r . The k i n e t i c s of both processes are complex,
i f we may judge from some recent attempts at modeling exoenzyme
production and a c t i o n presented by Van Dedem and Moo Young (48),
and thus, i t i s not s u r p r i s i n g that attempts to make models of sys-
tems e x h i b i t i n g e c c r i n o l y s i s seem not to have been made. The
i n t e r a c t i o n i s probably of importance i n some n a t u r a l systems, f o r
i t must be involved i n the c y c l i n g of minerals. Therefore, attempts
to study i t q u a n t i t a t i v e l y should be made.
We turn now to d i r e c t i n t e r a c t i o n s between p a i r s of m i c r o b i a l
populations.
i n o c u l a t e d a t high d e n s i t y i n t o a c u l t u r e of non-growing b a c t e r i a ,
even though i n o c u l a t i o n of T_. p y r i f o r m i s at low d e n s i t y i n t o a
c u l t u r e of non-growing b a c t e r i a d i d r e s u l t i n c o l l e c t i o n (the
i n i t i a l b a c t e r i a l d e n s i t i e s were the same i n both experiments). I
do not think that the observed f a i l u r e to s t a r t feeding i n the high
d e n s i t y case i s an a r t i f a c t to be a t t r i b u t e d to the procedures used
to prepare the protozoan inoculum, although that does need to be
checked out.
An a l t e r n a t e working hypothesis which i s suggested by the
foregoing d i s c u s s i o n i s that T. p y r i f o r m i s c e l l s possess sensing
and c o n t r o l mechanisms which lead to c e s s a t i o n of t h e i r feeding
a c t i v i t y under c e r t a i n c o n d i t i o n s of environmental s t a t e . A
c o r o l l a r y of t h i s hypothesis i s that T. p y r i f o r m i s c e l l s which do
not eat b a c t e r i a l c e l l s under one set of c o n d i t i o n s can eat these
c e l l s — t h e same o n e s — u n d e different t f conditions Testin
of these d i f f e r e n t hypothese
at the present time.
A d d i t i o n a l adaptations which make m i c r o b i a l feeders "prudent"
or which allow m i c r o b i a l "predators" to c o e x i s t with t h e i r "prey"
are discussed i n a recent review by Alexander (67).
C e s s a t i o n of feeding, whether i t be caused by encystment o r
by some process that i s not accompanied by a morphological t r a n s -
formation of the feeders, implies that there i s some threshold
d e n s i t y of food. The threshold d e n s i t y i s such that feeding w i l l
stop (or f a i l to s t a r t ) i f the density f a l l s (or i s ) below the
threshold d e n s i t y . One would expect that the threshold density of
food would depend on the i d e n t i t i e s of the feeding and fed-upon
populations as w e l l as on such things as the composition of the
medium. However, there i s some evidence that i n a d d i t i o n to these
f a c t o r s the threshold d e n s i t y of food changes with the density of
the feeders themselves. F o r example, i n the experiment of Habte
and Alexander c i t e d above (64) , Tetrahymena c e l l s i n o c u l a t e d at
high d e n s i t y f a i l e d to s t a r t feeding whereas Tetrahymena c e l l s
i n o c u l a t e d at a low density d i d s t a r t , and t h i s i s evidence that
the threshold density of b a c t e r i a i s r a i s e d by increase of the
protozoan d e n s i t y . Bader £t a l . (59) reached s i m i l a r conclusions
about thresholds f o r encystment of Colpoda s t e i n i i , and a d d i t i o n a l
evidence from the l i t e r a t u r e could be c i t e d .
Results such as these suggest that tht threshold r e l a t i o n
between the two d e n s i t i e s might be as shown i n F i g u r e 3. Herein,
feeding w i l l occur i f the combination of d e n s i t i e s l i e s above and
to the l e f t of the curve but feeding w i l l not occur i f the combi-
n a t i o n l i e s below and to the r i g h t of the curve.
The curve shown i s drawn with a h o r i z o n t a l asymptote because,
i f the d e n s i t y of the feeding p o p u l a t i o n i s s u f f i c i e n t l y low, there
can be no e f f e c t s o f crowding i n t h i s population. Under such con-
d i t i o n s , the threshold d e n s i t y of the fed-upon p o p u l a t i o n — i f there
i s such a threshold d e n s i t y — m u s t be independent of the density of
the feeding population. The curve i s drawn with a v e r t i c a l
asymptote, a l s o . This i s because one expects t h a t , under very
° c
> ο
W CO
I! Feeding
ο,ο.
ο
tr α No Feeding
b < b t
Φα) = (1)
φ (b - b.)
m t b > b_
L + b
* ( b )
" (L, Λ(L 2 + b) ( 2 )
(3)
(73), but more data of the same kind are needed before one can
conclude that food preference by suspension-feeders i s based
s o l e l y on d i f f e r e n c e s of hydromechanically s i g n i f i c a n t p r o p e r t i e s
of food organisms.
P a r a s i t i s m . In t h i s d i r e c t i n t e r a c t i o n a p a r a s i t e c e l l or
p a r t i c l e attaches i t s e l f to a host c e l l and makes a p a r t i a l or
t o t a l p e n e t r a t i o n i n t o i t , where i t then uses the host's biomass
or metabolic a c t i v i t i e s to grow and reproduce i t s e l f . Examples
are provided by the p a r a s i t i s m of v i r u s e s on b a c t e r i a and other
microorganisms, by the p a r a s i t i s m of the very small bacterium
B d e l l o v i b r i o on other b a c t e r i a , and by the p a r a s i t i s m of b a c t e r i a
on protozoa (30,74_,75) . P a r a s i t i s m i s c h a r a c t e r i z e d by a v a r i a b l e
but always high degree of host s p e c i f i c i t y . That i s , a given
p a r a s i t e i s able to i n f e c t onl l i m i t e d numbe f hosts d i
some cases, the number ma
A number of mathematica host-parasit
have been published. For references to l i t e r a t u r e appearing
before 1977, see F r e d r i c k s o n (2). A more recent model has been
given by L e v i n ^ t a l . (76).
P a r a s i t i s m can serve to r e g u l a t e competition between d i f f e r e n t
host populations. An example i s provided by the work of L e v i n
et a l . (76). They found that p a r a s i t i s m by the v i r u l e n t b a c t e r i o
phage T2 on two s t r a i n s of E s c h e r i c h i a c o l i s t a b i l i z e d the compe
t i t i o n of the s t r a i n s f o r sugar, and allowed the competitors to
c o e x i s t i n a chemostat. One of the s t r a i n s was s u s c e p t i b l e to
i n f e c t i o n by T2 but the other was not.
A most i n t e r e s t i n g aspect of the v i r u s - b a c t e r i a h o s t - p a r a s i t e
i n t e r a c t i o n i s the tendency f o r genetic changes of the populations
to keep a l t e r i n g the dynamics of the i n t e r a c t i o n . T h i s i s w e l l
i l l u s t r a t e d by some a d d i t i o n a l work of Chao et a l . (77). A host
bacterium B Q (a s t r a i n of E. c o l i ) and a bacteriophage T were Q
T .
0 Mutation of B produced a new s t r a i n of b a c t e r i a , B^, which
Q
Summary and d i s c u s s i o n
Acknowledgement
Literature Cited
1. Odum, P. "Fundamentals of Ecology", 3rd. ed., Saunders:
Philadelphia, 1971, pp. 211-213.
2. Fredrickson, A. G. Annu. Rev. Microbiol. 1977, 31, 63.
3. Kaiser, D.; Manoil, C . ; Dworkin, M. Annu. Rev. Microbiol.
1979, 33, 595.
4. Fredrickson, A. G.; Stephanopoulos, G. N. Science 1981, 213,
972.
5. Stephanopoulos, G. N.; Fredrickson, A. G. Biotechnol. Bioeng.
1979, 21, 1491.
6. Stewart, F. M.; Levin, B. R. Am. Nat. 1973, 107, 171.
7. Grenney, W. J.; Bella, D. Α.; Curl, H. C., Jr. Am. Nat. 1973,
107, 405.
8. Chisholm, S. W.; Nobbs P A "Modeling Biochemical Processes
in Aquatic Ecosystems"
Ann Arbor, Michigan, , pp
9. Stephanopoulos, G. N.; Fredrickson, A. G.; Aris, R. AIChE J.
1979, 25, 863.
10. Hsu, S. B. J. Math. Biol. 1980, 9, 115.
11. Smith, H. L. SIAM J. Appl. Math. 1981, 40, 498.
12. Hsu, S. B.; Hubbell, S.; Waltman, P. SIAM J. Appl. Math.
1977, 32, 366.
13. Hsu, S. B. SIAM J. Appl. Math. 1978, 34, 760.
14. Koch, A. L. J. Theor. Biol. 1974, 44, 387.
15. Hsu, S. B.; Hubbell, S. P.; Waltman, P. SIAM J. Appl. Math.
1978, 35, 617.
16. Hus, S. B.; Hubbell, S. P.; Waltman, P. Ecol. Monogr. 1978,
48, 337.
17. Jost, J . L.; Drake, J . F . ; Fredrickson, A. G.; Tsuchiya,
H. M. J . Bacteriol. 1973, 113, 834.
18. León, J . Α.; Tumpson, D. B. J. Theor. Biol. 1975, 50, 185.
19. Peterson, R. Am. Nat. 1975, 109, 35.
20. Taylor, P. Α.; Williams, P. J . LeB. Can. J. Microbiol. 1975
21, 90.
21. Yoon, H.; Blanch, H. W. J. Appl. Chem. Biotechnol. 1977, 27,
260.
22. Yoon, H.; Klingzing, G.; Blanch, H. W. Biotechnol. Bioeng.
1977, 19, 1193.
23. Stephanopoulos. G. N.; Schuelke, L. M.; Stephanopoulos, G.
Theor. Pop. Biol. 1979, 16, 126.
24. Smouse, P. E. Theor. Pop. Biol. 1980, 17, 16.
25. Hsu, S. B.; Cheng, K-S.; Hubbell, S. P. SIAM J. Appl. Math.
1981, 41, 422.
26. Gottschal, J. C.; de Vries, S.; Kuenen, J . G. Arch. Micro
biol. 1979, 121, 241.
27. Laanbroek, H. J.; Smit, A. J.; Nulend, G. M.; Veldkamp, H.
Arch. Microbiol. 1979, 120, 61.
28. DeFreitas, M.; Fredrickson, A. G. J . Gen. Microbiol. 1978,
106, 307.
The Role of S p e c i a l i s t s a n d G e n e r a l i s t s
in Microbial Population Interactions
J. G. KUENEN
Delft University of Technology, Laboratory of Microbiology,
Julianalaan 67a, 2628 BC Delft, The Netherlands
0097-6156/83/0207-0229$06.75/0
© 1983 American Chemical Society
Specialist
Table I
11
Types of metabolism of "model b a c t e r i a used f o r the study of
competition between " s p e c i a l i s t s " and " g e n e r a l i s t s " .
GENERALIST:
Facultative chemolitho(auto)troph ("mixotroph") (Thiobacillus A2)
= °2
S 0 2 3 (or S ) > S0 4
U
2
(many) organic compound(s) > CO^
CO2 — * cellmaterial
Carbon source: and/or
(many) organic compound(s) — ^ c e l l m a t e r i a l
SPECIALIST I
O b l i g a t e chemolitho(auto)troph (Thiobacillus neapolitanus)
0 o
2
Energy source: S20 3
5
«
?
u
(or S ) =-> SO,
SPECIALIST I I
Chemo(organo)heterotroph (Spirillum G7)
°
2
0 2 4 6 8 10 12 % 16
time (h)
0 2 4 6 8 10 12 14 16 18 20
acetate (mM)
40 36 32 28 24 20 16 12 8 4 0
thiosulfate (mM)
Figure 3. Maximum substrate oxidation potentials and carbon dioxide fixation
potential of whole cells of T . A2 as a function of different acetate and thiosulfate
concentrations in the reservoir medium of the chemostat cultures. Key: · , Qo™*-
thiosulfate; A , QoJ™ *-acetate;
1
M, C0 -fixation potential. Reproduced, with per-
2
Table I I
Maximum s p e c i f i c growth r a t e P m a x (h ^)
t a t +a
T. neapolitanus 0.3
3 A 5 6 7 8
Volume changes
Figure 4. Competition in continuous culture between T. neapolitanus (the special
ist) and T. A2 (the generalist) for thiosulfate as the only growth-limiting substrate.
Key: Φ, relative cell number of Τ. A2; Ο , T. neapolitanus. Reproduced, with per
mission, from Ref. 4. Copyright 1979, Springer-Verlag.
1
The chemostat was run at a dilution rate of 0.05 h' with a 40 mM thiosulfate concentration in
the reservoir medium. Organisms had been pregrown separately in continuous culture at
1
Ό = 0.05 h' and at zero time mixed in a 1:1 rate.
(• glycollate)
J. neapolitanus
(•glycollate)
Oh -L -L JL
3 4 5 6 7 8 9 10
GLYCOLLATE OR ACETATE (mM)
mM THIOSULFATE
Figure 6. Results of competition between the generalist T . A2, and two specialists,
T. neapolitanus and Spirillum G7 for thiosulfate and acetate as growth-limiting
1
substrates in the chemostat at a dilution rate of 0.07 h' . Concentrations in the
inflowing medium ranged from 0-20 mM for acetate and from 0-40 mM for thio-
sulfate. After a steady state had been established relative cell numbers were
determined. Solid line, experimental data; dashed line, outcome of the competition
as predicted from mathematical modeling.
max
y S
U tA (8 ) = t A · t
0 2 4 6 8 10
acetate (mM)
Figure 7A. Cell density of the generalist (mixotroph T. A2, O) and the autotroph
(specialist T . neapolitanus, Φ) in mixed chemostat culture at steady state, growing
1
at a dilution rate of 0.075 h' . Growth was simultaneously limited by thiosulfate
(reservoir medium concentration, S° —40 mM) and by increasing concentrations
of acetate in the reservoir medium (S ° = 0-10 mM).
a
2 4 6 8
thiosulfate (mM)
Figure 7B. Cell density of the mixotroph (O) and the heterotroph (specialist
Spirillum G7, %) in mixed chemostat-culture at steady state, growing at a dilution
1
rate of 0.075 h . Growth was simultaneously limited by acetate (S ° = 20 mM)
a
-433
150
ι
125| protein
ο 100
ε 50
ο 25|
Ô ^ C02-f'xotion
CM
ο
4-+
* -1000k
<u -c I
•o J 80θ|
QQ£ - thiosulfate
S Ϊ X
Φ £ 600|
i -S 4001
QQ -9 acetate
1 | 200h — 0
(A)
1 _L
1
X I I
-2 k 6 8 10
t i m e (h)
a c e t a t e — ι — t h i o s u l f a t e —|!— a c e t a t e —!—-thiosulfate
American Chemical
Society Library
In Foundations 1155 16th St.,
of Biochemical N.W. Blanch, H., et al.;
Engineering;
Washington,
ACS Symposium Series; U.C. 20036
American Chemical Society: Washington, DC, 1983.
248 BIOCHEMICAL ENGINEERING
60,
°h . ι . • . • • • • ι . ι • Π
0 4 8 12 16 20 24 28
volume changes
Acknowledgements
Many thanks are due to my two former co-workers Dr. J . C . Gott-
schal and Dr. R.F. Beudeker who did most of the experimental work
described i n this paper. I thank Miss L . A . Robertson for correc-
ting the English and Mrs. C.M. Paanakker-Kuipers and Mrs. I . Hag-
man-v.d. Bulk for typing the manuscript.
Literature Cited
1 1
M. J. BAZIN, C. CURDS , A. DAUPPE , Β. Α. OWEN, and
P. T. SAUNDERS
Queen Elizabeth College, Department of Microbiology, London, England W87AH
H = k x Η - k PH
2 (1)
Ρ = -k Ρ 3 + k PH
4 (2)
1
Current address: British Museum (Natural History), Cromwell Road,
London SW7, England
0097-6156/83 /0207-0253$06.00/0
© 1983 American Chemical Society
λ - k 4 Η (3)
λ = λ H/(L + Η) (4)
m
where λ i s the maximum s p e c i f i c growth r a t e and L i s the
c o n c e n t r a t i o n of prey s u f f i c i e n t f o growth t s p e c i f i rat
2
V ·
Both the L o t k a - V o l t e r r a and the Monod functions f o r predator
s p e c i f i c growth r a t e are dependent upon a s i n g l e v a r i a b l e , the
c o n c e n t r a t i o n of prey organisms. A t h i r d f u n c t i o n , proposed by
Contois (3) f o r b a c t e r i a l growth as an a l t e r n a t i v e to that of
Monod, when a p p l i e d to predator growth takes the form:
λ = λ Η/(LP + Η) (5)
m
In t h i s case the s p e c i f i c growth rate i s a f u n c t i o n of both prey
and predator d e n s i t y .
Using the c i l i a t e protozoan Tetrahymena p y r i f o r m i s as a
predator and K l e b s i e l l a aerogenes as prey, Curds and Cockburn
(4) found that of the three f u n c t i o n a l forms suggested f o r
predator s p e c i f i c growth r a t e represented by equations (3) - ( 5 ) ,
the Contois equation gave the best f i t to t h e i r data. D i v i d i n g
the numerator and denomenator of equation (5) by Ρ gives
Ρ - ( λ - D) Ρ (6)
Η = ( u - D ) H (7)
W
where D = d i l u t i o n r a t e (the r a t e of flow through the c u l t u r e
v e s s e l d i v i d e d by i t s volume), u i s the s p e c i f i c growth r a t e of
the prey and W i s the y i e l d of predator per u n i t of prey consumed,
assumed to be constant.
When slime mould amoebae and b a c t e r i a l prey are grown
together i n a chemostat, the p o p u l a t i o n d e n s i t i e s of both
organisms f l u c t u a t e s i n u s o i d a l l y f o r s e v e r a l days (7, 8 ) .
We estimated the s p e c i f i c growth r a t e of the predator p o p u l a t i o n
i n such c u l t u r e s from the slope of the curve generated by
p l o t t i n g the logarithm of the predator d e n s i t y a g a i n s t time.
T h i s slope i s
Ρ/Ρ = λ - D (8)
Ρ = (λ - D ) Ρ (9)
so that at steady s t a t e
λ = D (10)
Methods
E s c h e r i c h i a c o l i B/r and D i c t y o s t e l i u m discoideum NC4 were
maintained r o u t i n e l y and grown i n s i n g l e - s t a t e chemostat c u l t u r e
as described p r e v i o u s l y ( 8 ) . The outflow of chemostat c u l t u r e s
was passed along the same l i n e as e x p e l l e d a i r and by t h i s means
was forced i n t o tubes r e s t i n g i n a r e f r i g e r a t e d f r a c t i o n
c o l l e c t o r . F r a c t i o n s were c o l l e c t e d at hourly i n t e r v a l s .
For two-stage continuous c u l t u r e , the f i r s t stage v e s s e l had
a maximum c a p a c i t y of 1 L while the v e s s e l used f o r the second
stage had a maximum c a p a c i t 1 500
upon the volume appropriat
v e s s e l s were f i t t e d wit , flange g
access p o r t s . Both v e s s e l s were mixed by means of magnetic
s t i r r e r s and immersed i n water maintained at 25°. F i l t e r -
s t e r i l i s e d a i r was supplied to both stages and the flow of n u t r i e n t
to the f i r s t v e s s e l and the flow from the f i r s t to the second
v e s s e l was regulated by p e r i s t a l t i c pumps. Two-stage chemostat
experiments were performed by i n o c u l a t i n g the f i r s t v e s s e l with
I>» c o l i and i n c u b a t i n g with the flow on u n t i l the system came to
steady s t a t e . This was considered to have occurred when no
s i g n i f i c a n t change i n the t u r b i d i t y of the e f f l u e n t c e l l suspension
could be detected. At t h i s time the second v e s s e l was f i l l e d with
c u l t u r e from the f i r s t and i n o c u l a t e d with a suspension of
D. discoideum spores. The c u l t u r e i n the second v e s s e l was
incubated under batch c o n d i t i o n s f o r about 48 h during which time
the spores germinated to form amoebae. Flow from the f i r s t
v e s s e l to the second was then i n i t i a t e d and the amoebae c u l t u r e d
on a continuous b a s i s . Samples were taken d i r e c t l y from the
culture vessels for analysis.
The c e l l number d e n s i t i e s of the b a c t e r i a and the amoebae i n
both systems was measured on a C o u l t e r Counter (Coulter
E l e c t r o n i c s L t d . , Harpenden, England). Mean c e l l volumes were
estimated using a C o u l t e r C1000 Channelyzer. A 30 um diameter
aperture was used f o r the b a c t e r i a and a 50 um aperture f o r the
amoebae. Samples were suspended e i t h e r i n c u l t u r e medium or
"Isoton" (Coulter E l e c t r o n i c s Ltd) immediately p r i o r to c o u n t i n g .
Biomass was estimated e i t h e r i n terms of biovolume, the product of
the mean c e l l volume and the number of c e l l s present, or, f o r
b a c t e r i a l biomass where appropriate, as t u r b i d i t y at 560 nm.
Within the range of readings made there i s a l i n e a r r e l a t i o n s h i p
between c e l l volume d e n s i t y and t u r b i d i t y at 560 nm f o r E. c o l i .
Sing-estage continuous c u l t u r e data was smoothed by the method of
cubic s p l i n e s using Nottingham Algorithms Routine E02AAF. A l l
computations were performed on a CDC 6600 d i g i t a l computer.
Results
Φ = D (Η χ - H )/P
2 (11)
7
io E
Ε ο
ο
w 10- Mo» ë
Φ
I
Φ °% .·/·· β
ο°;°ο·-
β % ν <- 1
1C
10-ï 8
; E
.2
*c
S
& 10 7 E
<
M00
co
E ÎaooE
a.
— ο·4Η
> >
o o
100.2
φ
O
E
<
100 200 300
TIME (h)
1
Smoothed prey number density ml"
Figure 2. Phase plane plot of predator and prey number densities. Data from a
single stage chemostat culture of D . discoideum and E . coli were smoothed by the
method of cubic splines, and the smoothed data were used to construct this figure.
The arrows indicate the direction of increasing time.
1
Smoothed prey biovolume density (μηι ml')
Figure 3. Phase plane plot of predator and prey densities constructed as for Figure
2, using biovolume density as the coordinates. The arrows indicate the direction
of increasing time.
008
i\ A
ooH
s Λ Λ
o • · · · / \ m
• · · · · \ S
o ooH f :
φ
\ - \/ v
w
υ
Φ
α
y
^ -004H
0
1
l
I
50 100 150 200 250
Time ( h )
Figure 4. Change in the specific rate of change of D. discoideum in a single-stage
chemostat as a function of time. Values were obtained from smoothed data. The
specific growth rate of the amoebae is obtained by adding the dilution rate of the
1
culture (0.065 h' ) to the values on the ordinate.
0.08 h
ο 0.04
-0.04
σ
-D
Discussion
6h
-6 4
Q.
10 20 30
Prey/predator ratio
Figure 7. Specific feeding rate plotted against the ratio of prey to predator in the
second vessel, estimated in terms of absorbance at 560 nm divided by the predator
ζ 1
biovolume density (μ/η mL ). Other conditions as in Figure 6.
0.08 h
• · · ·*··.
#
.·.·*. »
0.04 h
• ··
0 h
£ -0.04
ν..·.
LÎ!_
0.8 0.9 1.0 l.l 1.2
Ratio of prey t o predator population densities
Acknowledgments
Literature Cited
DOUGLAS A. LAUFFENBURGER
University of Pennsylvania, Department of Chemical Engineering,
Philadelphia, PA 19104
0097-6156/83/0207-0265 $08.00/0
© 1983 American Chemical Society
S t u d i e s o f m i c r o b i a l p o p u l a t i o n dynamics have
focused p r i m a r i l y on well-mixed, macroscopically
homogeneous s y s t e m s . T h i s emphasis i s e a s i l y
d i s c e r n i b l e from t h e c o n t e n t s o f t h i s symposium
volume. However, t h e s i t u a t i o n i n most n a t u r a l l y
o c c u r i n g m i c r o b i a l systems i s f a r from i d e a l
l a b o r a t o r y c o n d i t i o n s , so that understanding gained
a b o u t w e l l - m i x e d s y s t e m s may n o t p r o v i d e t h e
appropriate insight into ecological situations.
Environments which a r e not well-mixed can allow
formation o f s p a t i a l gradients o f chemical concentra-
t i o n s and c e l l d e n s i t i e s . Also, chemical d i f f u s i o n
and c e l l m o t i l i t y ( i . e . , s e l f - p r o p e l l e d movement
r e q u i r i n g energy expenditure) can replace convection
a s t h e d o m i n a n t mod
common c a t e g o r i z a t i o n
c o n t a i n a t l e a s t o n e m o t i l e s p e c i e s (1), including
many o f t h e commonly o c c u r i n g s p e c i e s . F u r t h e r , most
m o t i l e b a c t e r i a e x h i b i t c h e m o t a x i s , w h i c h i s most
s i m p l y d e f i n e d a s c e l l movement t o w a r d o r away f r o m
chemicals ( 2 ) , o r a s p r e f e r e n t i a l c e l l movement t o w a r d
h i g h e r o r lower c o n c e n t r a t i o n s o f a c h e m i c a l stimulus
{3). A c t u a l l y , t h e r e a r e a number o f d i f f e r e n t types
o f movement r e s p o n s e s w h i c h l e a d t o b e h a v i o r o f t h i s
g e n e r a l d e s c r i p t i o n ( 4 ) . For peritrichously flagellated bacteria,
w h i c h a p p e a r t o be t h e m o s t c o m m o n l y e n c o u n t e r e d group
(and o n w h i c h t h i s p a p e r w i l l a c c o r d i n g l y f o c u s ) ,
k l i n o k i n e s i s ( i n which the t u r n i n g frequency o f
swimming b a c t e r i a i s m o d u l a t e d b y s t i m u l u s c o n c e n t r a -
t i o n ) a p p e a r s t o be c l o s e s t t o o b s e r v e d b e h a v i o r ( 5 ) .
T h i s i s i l l u s t r a t e d i n F i g u r e 1. In the absence o f a
chemical stimulus gradient, these b a c t e r i a swim i n
roughly s t r a i g h t l i n e steps c a l l e d "runs" f o r a short
time (about 1 second) and t h e n s t o p and change
d i r e c t i o n , o r " t u m b l e " , f o r a b o u t 1/10 s e c o n d ( 6 ) .
The d i r e c t i o n c h a n g e i s p u r e l y r a n d o m , b u t t h e
p r o b a b i l i t y o f tumbling i s constant d u r i n g a r u n ,so
t h a t t h e r u n time d i s t r i b u t i o n i s P o i s s o n i a n ( 6 ) .
T h i s movement b e h a v i o r i s t e r m e d r a n d o m m o t i l i t y . In
the presence o f a g r a d i e n t , t h e d i r e c t i o n change
r e m a i n s random b u t t h e t u m b l i n g p r o b a b i l i t y decreases
f o r a c e l l swimming t o w a r d h i g h e r a t t r a c t a n t c o n c e n t r a -
t i o n s o r lower r e p e l l e n t c o n c e n t r a t i o n s (6), l e a d i n g
to n e t m i g r a t i o n i n t h e d i r e c t i o n o f t h e g r a d i e n t .
T h i s mechanism p r o v i d e s v e r y e f f i c i e n t response (]_) ;
i n an o p t i m a l g r a d i e n t t h e n e t m i g r a t i o n v e l o c i t y i s
r o u g h l y h a l f t h e l i n e a r c e l l swimming s p e e d ( 8 , 9 ) .
S t i m u l u s c o n c e n t r a t i o n s a r e m e a s u r e d by o c c u p a n c y
o f c e l l membrane r e c e p t o r s w h i c h c a n r e v e r s i b l y b i n d
stimulus molecules according to Michaelis-Menten
enzyme k i n e t i c s w i t h d i s s o c i a t i o n c o n s t a n t .
G r a d i e n t s a r e d e t e c t e d by a t e m p o r a l s e n s i n g m e c h a n i s m
by w h i c h r e c e p t o r o c c u p a n c y o v e r t h e e n t i r e c e l l i s
m o n i t o r e d d u r i n g a r u n (1Λ) . A spatial sensing
m e c h a n i s m by w h i c h d i f f e r e n c e s i n r e c e p t o r o c c u p a n c y
a c r o s s the c e l l l e n g t h i s measured i s i m p r a c t i c a l f o r
s u c h r a p i d l y swimming c e l l s , b e c a u s e o f f a l s e
a p p a r e n t g r a d i e n t s due t o c e l l m o t i o n i t s e l f (12).
A l a r g e v a r i e t y o f c h e m i c a l s c a n s e r v e as
chemotactic s t i m u l i
r e c e p t o r s a n d 10 r e p e l l e n
i d e n t i f i e d f o r E s c h e r i c h i a c o l i and Salmonella
t y p h i m u r i u m , t h e two m o s t w i d e l y - s t u d i e d s p e c i e s ( Γ 3 ) .
T h e s e r e c e p t o r s a r e s p e c i f i c f o r one o r two chemicals
at h i g h a f f i n i t i e s (low K ^ ) , but w i l l a l s o b i n d a
range o f r e l a t e d m o l e c u l e s w i t h lower a f f i n i t y (2).
Table 1 l i s t s a number o f t h e w e l l - k n o w n s t i m u l i f o r
a v a r i e t y of species, although t h i s i s c e r t a i n l y
i n c o m p l e t e a s new r e s p o n s e s a r e b e i n g d i s c o v e r e d
almost d a i l y . I n g e n e r a l , s u b s t a n c e s b e n e f i c i a l t o an
o r g a n i s m s u c h as n u t r i e n t s and o x y g e n ( i f a e r o b i c )
s e r v e as a t t r a c t a n t s w h i l e t o x i c compounds o r compounds
c a u s i n g pH e x t r e m e s a c t a s r e p e l l e n t s ( 2 ) . Some a m i n o
a c i d s a r e a t t r a c t a n t s and o t h e r s r e p e l l e n t s , d e p e n d i n g
upon t h e s p e c i e s . A l t h o u g h t h e r e i s n o t an e x a c t
correspondence between m e t a b o l i z a b l e compounds and
a t t r a c t a n t s n o r b e t w e e n u n f a v o r a b l e c o m p o u n d s and
r e p e l l e n t s (L4), the observed responses can g e n e r a l l y
be r a t i o n a l i z e d i n t e r m s o f p a r t i c u l a r s p e c i e s
b i o c h e m i c a l p a t h w a y s and by r e c o g n i t i o n o f some
p u z z l i n g s t i m u l i as a n a l o g u e s o f o t h e r s t i m u l i (2).
Speculation regarding a possible survival
advantage o f chemotaxis i s thus not s u r p r i s i n g .
Approximately 40 g e n e s a r e d e v o t e d s p e c i f i c a l l y t o t h e
c h e m o t a c t i c r e s p o n s e i n E ^ c o l i and S^ t y p h i m u r i u m (15) y
s t i m u l i , p r o b a b l y b o t h a t t r a c t a n t s and r e p e l l e n t s , a r e
present. M u l t i p l e s i g n a l s a r e a d d i t i v e i n some s e n s e
( 1 6 ) , and p r e s u m a b l y t h e b a c t e r i a a t t e m p t t o o p t i m i z e
t h e i r growth through p r e f e r e n t i a l m i g r a t i o n . F r o m an
e n g i n e e r i n g p e r s p e c t i v e , t h e r e i s no q u a n t i t a t i v e
b a s i s f o r p r e d i c t i o n o f the e f f e c t s o f c e l l m o t i l i t y
and c h e m o t a x i s o n m i c r o b i a l p o p u l a t i o n d y n a m i c s i n a n y
given s i t u a t i o n at present. T h i s paper i s devoted to
r e v i e w o f the s m a l l body o f i n f o r m a t i o n a v a i l a b l e i n
t h i s area at t h i s time.
Experimental Observations
T h e r e e x i s t s o n l y a s m a l l number o f p u b l i s h e d
experiments p e r t a i n i n t th effect f cell motilit
on p o p u l a t i o n g r o w t h
g i v e s one example w i t h o u
c o m p e t i t i o n b e t w e e n an a e r o t a c t i c ( i . e . , c h e m o t a c -
t i c a l l y a t t r a c t e d by o x y g e n ) P s e u d o m o n a s s p e c i e s a n d
an i m m o t i l e A c i n e t o b a c t e r s p e c i e s , f o r o x y g e n ( 1 7 ) .
When t h e g r o w t h medium i s w e l l - a e r a t e d t h e
A c i n e t o b a c t e r predominate, thus showing s u p e r i o r
g r o w t h k i n e t i c s on t h e r a t e - l i m i t i n g s u b s t r a t e
(presumably oxygen) s i n c e c e l l m o t i l i t y i s a p p a r e n t l y
irrelevant. But i n a non-mixed c u l t u r e the
Pseudomonas p r e d o m i n a t e . The c h e m o t a c t i c a b i l i t y of
the Pseudomonas s p e c i e s p r o v i d e s , i n t h i s s i t u a t i o n ,
enough o f a b e n e f i t t o overcome i t s growth k i n e t i c
inferiority.
The f i r s t l i t e r a t u r e r e p o r t i n t h i s a r e a was by
S m i t h a n d D o e t s c h (]J*) , who s t u d i e d c o m p e t i t i o n
b e t w e e n a e r o t a c t i c P s e u d o m o n a s f l u o r e s c e n s and an
i m m o t i l e m u t a n t s t r a i n o f t h e same s p e c i e s , f o r o x y g e n
(see Figure 2 ). In a e r a t e d mixed c u l t u r e b o t h s t r a i n s
g r e w t o a r o u g h l y 1:1 r a t i o o v e r a 2 4 - h o u r p e r i o d
i n d i c a t i n g t h a t t h e i r g r o w t h k i n e t i c p r o p e r t i e s were
i d e n t i c a l as e x p e c t e d . In n o n - a e r a t e d media, the
a e r o t a c t i c s t r a i n outgrew the mutant t o a f i n a l ratio
o f o v e r 10:1 a f t e r 24 h o u r s . U n f o r t u n a t e l y , the
a u t h o r s c r e d i t e d m o t i l i t y per se f o r t h i s a d v a n t a g e ,
e v e n t h o u g h i t i s not c l e a r whether random m o t i l i t y
without chemotaxis i s n e c e s s a r i l y always b e n e f i c i a l .
In the c o u r s e o f s t u d y i n g t h e r o l e o f f i m b r i a e i n
b a c t e r i a l g r o w t h , O l d and D u g u i d l o o k e d a t c o m p e t i t i o n
f o r o x y g e n b e t w e e n two n o n f i m b r i a t e s t r a i n s o f
Salmonella typhimurium: one a e r o t a c t i c and one
immotile (_19) (see Table 2 ) . In a e r o b i c shaken b r o t h ,
t h e a e r o t a c t i c s t r a i n m u l t i p l i e d by a f a c t o r o f 46
w i t h i n 48 h o u r s , w h i l e t h e i m m o t i l e s t r a i n m u l t i p l i e d
by a f a c t o r o f 52. Again the growth k i n e t i c
Theoretical Analyses
E a r l y attempts at t h e o r e t i c a l a n a l y s i s o f the
e f f e c t s o f c e l l m o t i l i t y on p o p u l a t i o n growth c e n t e r e d
on uptake o f n u t r i e n t by a s i n g l e c e l l i n a medium o f
i n f i n i t e e x t e n t (12, 24, 25, 2 6 ) . These a n a l y s e s have
J- J
6 8 10 12 H
Hours
6 8 10 12 H 2L
Hours
I 1 ι ι ι ι ι IK ι
0 2 4 6 8 10 12 Η 24
Hours
c 100 η
I 90
5 80
Ο
^ 70
° ~ 60
5 2 50 -
6 30-
ζ ζ
!: 20-1 < LU
< <
ο 10 CL
LU
° 0
<
DAY AFTER INFECTION
0 (inoculum) 0.0
-
1 44 (+ 27) 6
2 50 (+ 35) 11
3 39 <+ 36) 3
5 96 1
10 o r more 93 (+ 7.9) 11
R e p r o d u c e d w i t h p e r m i s s i o n f r o m R e f e r e n c e 21. C o p y r i g h t 1979,
American S o c i e t y f o r C l i n i c a l N u t r i t i o n .
3 J
3b _ b
+ G
3t 3x "b L
3s !^s
at ax s
over 0<x<L, w h e r e t h e n o t a t i o n i s a s f o l l o w s :
b (x,t) = v i a b l e c e l l d e n s i t y
s (x,t) = n u t r i e n t c o n c e n t r a t i o n
Jfc = viable cell flux
Js = nutrient flux
Gfc = net c e l l growth r a t e
Gs = n u t r i e n t consumption rate
The b o u n d a r y c o n d i t i o n s a r e
Jb = 0 s = s at χ = L
0
Jfc = 0 J s = 0 at χ = 0
i f the n u t r i e n t c o n c e n t r a t i o n at the source boundary
remains constant.
F o r t h e n e t c e l l g r o w t h r a t e , we u s e Monod's
m o d e l f o r g r o w t h and a f i r s t o r d e r r a t e o f l o s s o f
viability:
ks .
w h e r e k i s t h e g r o w t h r a t e c o n s t a n t , Κ i s t h e Monod
c o n s t a n t , and k i s the n o n - v i a b i l i t y r a t e constant.
e
The n u t r i e n t c o n s u m p t i o n r a t e i s
1
r _ ks t_
vas
*S -~ Ϋ77 K+S
where Y i s the y i e l d c o e f f i c i e n t .
In order to permit a n a l y t i c a l s o l u t i o n o f the
m o d e l e q u a t i o n s , we r e p l a c e t h e s e e x p r e s s i o n s by s t e p -
function approximations:
i(k - k ) e b
G
b= - k P b s <
γ kb s > s c
S c = (
kH^) Κ
T h i s a p p r o x i m a t i o n a l l o w s d i r e c t a n a l y s i s o f the model
p r e d i c t i o n s , and n u m e r i c a l c o m p u t a t i o n s show g o o d
q u a n t i t a t i v e agreement w i t h the o r i g i n a l e x p r e s s i o n s
(31) .
s
3x
where D i s t h e d i f f u s i v i t y . F o r t h e c e l l f l u x we use
an e x p r e s s i o n o r i g i n a l l y f o r m u l a t e d by K e l l e r and
Segel (32):
b y X
"3x âx
w h e r e μ i s t h e r a n d o m m o t i l i t y c o e f f i c i e n t and χ
i s the chemotaxis c o e f f i c i e n t . T h e r e a r e some
e s t i m a t e s o f μ and χ a v a i l a b l e ( 3 3 , 3±, 35)t although
a s a t i s f a c t o r y a n a l y s i s of a convenient assay for
t h e s e p a r a m e t e r s n e e d s t o be developed.
. Ys D
u = s_ v = b_ ξ = χ τ . Dt b = - ^
s 0
b
o L
L kL
k L* 1 u>u
= ML.
S
X 0
λ = K θ = -4- β = -^p F(u) =
D D
0 u<u c
we o b t a i n d i m e n s i o n l e s s model equations
If • λ
w ~ δ λ
i l (
2
3u _ 3 u
F ( U ) V
3T " ~
f f - 6 v f ! = 0 u « 1 at ζ - 1
|f - 0 - 0 at ς - 0
Using these equations, the steady-state
population
Β = Ifo£ ν
with 2
kL
V = / vdÇ 0
h a s b e e n a n a l y z e d f o r t h r e e c a s e s s o f a r : a) a s i n g l e
r a n d o m l y m o t i l e p o p u l a t i o n {3, 28), b) a s i n g l e
c h e m o t a c t i c p o p u l a t i o n (3, 2j)) , a n d c ) two r a n d o m l y
m o t i l e p o p u l a t i o n s g r o w i n g i n c o m p e t i t i o n {30, 31). A
s t e a d y s t a t e i s s e t up b y t h e b a l a n c e b e t w e e n c e l l
g r o w t h i n a n u t r i e n t - r i c h zone and l o s s o f v i a b i l i t y
i n a n u t r i e n t - p o o r zone, m e d i a t e d by c e l l f l u x from
t h e n u t r i e n t - r i c h zone t o t h e n u t r i e n t - p o o r zone ( s e e
F i g u r e 7 ) . The d i m e n s i o n l e s s p o s i t i o n , ω , o f t h e
d i v i s i o n b e t w e e n t h e s e two z o n e s i s t h e p o i n t a t w h i c h
u = u = s /s .
c ω i s g i v e n by t h e l a r g e s t r o o t , l e s s
c 0
α tanh αω = β t a n β(1-ω)
1 / 2 1 / 2
where α = (θ/λ) and β = ([κ - θ])/λ) .
Ambient
Substrate
Concentration
So
-Bacteria Density b (x)
Substrate
diffusion
Substrate Concentration s(x)
x= 0
and bacterial growth can be supported. In the depleted zone, 0 < £ < ω, u = u , c
T h u s , ω i n c r e a s e s a s κ i n c r e a s e s and λ d e c r e a s e s .
Although i n n a t u r a l systems such a steady s t a t e may
n o t u s u a l l y be a t t a i n e d , i t i s a g o o d i n d i c a t i o n o f
t h e s i t u a t i o n w h i c h w i l l be a p p r o a c h e d i f e n v i r o n
mental c o n d i t i o n s are not changed.
In the f i r s t c a s e , s i n g l e p o p u l a t i o n growth o f a
n o n c h e m o t a c t i c s p e c i e s (6 = 0 ) , t h e s t e a d y s t a t e
p o p u l a t i o n s i z e Β d e p e n d s u p o n two q u a n t i t i e s : λ/κ
and κ/θ (see Figure 8 ) . The l a t t e r q u a n t i t y i s t h e
r a t i o o f growth to n o n v i a b i l i t y r a t e c o n s t a n t s ,
r e p r e s e n t i n g t h e g r o w t h p o t e n t i a l a t any g i v e n n u t r i e n t
concentration. F i g u r e 11 shows t h a t f o r any v a l u e o f
λ/κ Β i n c r e a s e s a s κ/θ i n c r e a s e s . The f o r m e r q u a n t i t y
2
is equal to μ / k L , and t h u s i s t h e r a t i o o f t h e r a t e
o f c e l l movement a c r o s th regio t th growth r a t
constant. Figure 1
κ / θ , Β i r c r e a s e s a s λ/κ d e c r e a s e s . T h i s i s because
r a n d o m m o t i l i t y l e a d s t o d i s p e r s a l o f c e l l s away f r o m
the n u t r i e n t - r i c h zone. F r o m t h i s r e s u l t we e x p e c t
t h a t an i m m o t i l e s t r a i n c a n o u t g r o w a r a n d o m l y m o t i l e
s t r a i n , a l l o t h e r f a c t o r s b e i n g e q u a l , a s was observed
by F r é t e r e t a l . ( Z I ) . One c a n a l s o s e e f r o m F i g u r e
11 t h a t a s p e c i e s w i t h s m a l l e r g r o w t h r a t e c o n s t a n t
can outgrow a s p e c i e s with l a r g e r growth r a t e
c o n s t a n t , i f t h e random m o t i l i t y c o e f f i c i e n t o f t h e
former s p e c i e s i s s m a l l enough r e l a t i v e t o t h a t o f the
latter species. F i g u r e 11 a l s o s h o w s t h a t f o r λ/κ
much g r e a t e r t h a n 1, Β i s i n d e p e n d e n t o f t h e r a n d o m
m o t i l i t y c o e f f i c i e n t , because the system behaves
e s s e n t i a l l y l i k e a well-mixed system.
In the second c a s e , s i n g l e p o p u l a t i o n growth o f a
chemotactic s p e c i e s , Β a d d i t i o n a l l y d e p e n d s u p o n 6,
t h e r a t i o o f t h e c h e m o t a x i s c o e f f i c i e n t t o t h e random
m o t i l i t y c o e f f i c i e n t (see Figure 9 ) . Β i n c r e a s e s as 6
i n c r e a s e s , b e c a u s e c h e m o t a x i s c o u n t e r a c t s t h e random
d i s p e r s a l o f c e l l s away f r o m t h e n u t r i e n t - r i c h z o n e
(see F i g u r e 10 )· The e f f e c t o f c h e m o t a x i s b e c o m e s
s i g n i f i c a n t o n l y when δ b e c o m e s g r e a t e r t h a n a b o u t
1
10" . T h e s e c o m p u t a t i o n s were done u s i n g a p e r t u r b a
t i o n m e t h o d , s o t h a t r e s u l t s f o r 6> 1 a r e m e r e l y
extrapolations. N u m e r i c a l c o m p u t a t i o n s h a v e shown t h e
p e r t u r b a t i o n r e s u l t s t o be a c c u r a t e up t o a t l e a s t 6 =
1.1, however ( 3 1 ) .
An i n t e r e s t i n g i n f e r e n c e w h i c h c a n be d r a w n f r o m
F i g u r e 12 i s t h a t t h e r e i s a minimum v a l u e o f 6 that
m u s t be e x c e e d e d i n o r d e r f o r m o t i l i t y t o c o n f e r an
advantage i n t h i s c o n f i n e d growth s i t u a t i o n . I f λ χ
r e p r e s e n t s t h e B r o w n i a n m o t i o n c o e f f i c i e n t f o r an
Figure 9. Plot of dimensionless total steady state bacterial density vs. 8 for single
chemotactic populations. Extrapolations of perturbation computations beyond 8
= 1 shown ( ). Asymptotic values for δ = 0 shown (- · - · -).
i m m o t i l e s p e c i e s (_33) , t h e n a c h e m o t a c t i c s p e c i e s m u s t
have a l a r g e r v a l u e , say λ > λι.
2 By i t s e l f t h i s
w o u l d y i e l d a s m a l l e r v a l u e o f B. Increasing 6 from
0 i n c r e a s e s B, and t h e r e w i l l be a c r i t i c a l v a l u e , 6*,
a t w h i c h Β f o r λ2 and 6 b e c o m e s e q u a l t o Β f o r λ 1 #
O n l y f o r 6>6* w i l l t h e c h e m o t a c t i c s t r a i n o u t g r o w t h e
immotile s t r a i n . Thus, s p e c u l a t i o n t h a t a m o t i l e
c h e m o t a c t i c s t r a i n s h o u l d a l w a y s be s u p e r i o r t o an
immotile s t r a i n i s not n e c e s s a r i l y t r u e .
T h e s e s i n g l e p o p u l a t i o n r e s u l t s s u g g e s t some
i m p o r t a n t i m p l i c a t i o n s f o r c o m p e t i t i o n b e t w e e n two o r
more p o p u l a t i o n s g r o w i n g t o g e t h e r o n a s i n g l e r a t e -
limiting nutrient. I f one s p e c i e s h a s s u p e r i o r g r o w t h
k i n e t i c p r o p e r t i e s but t h e o t h e r has s u p e r i o r m o t i l i t y
p r o p e r t i e s , we m i g h
A n a l y s i s o f the t h i r
r a n d o m l y m o t i l e p o p u l a t i o n s , shows t h a t t h i s i s i n d e e d
possible. T h e r e a r e now a c t u a l l y t h r e e p e r m i s s i b l e
steady s t a t e s : 1) c o e x i s t e n c e , 2) s p e c i e s 1 o n l y , a n d
3) s p e c i e s 2 o n l y . For sake o f c l a r i t y , l e t the two
s p e c i e s have i d e n t i c a l p r o p e r t i e s e x c e p t t h a t
ki > k2« Then s p e c i e s 1 w i l l have a g r e a t e r growth
rate at a l l n u t r i e n t concentrations. In a d d i t i o n , the
t h r e s h o l d c o n c e n t r a t i o n f o r net growth o f s p e c i e s 2
u > u
must be g r e a t e r t h a n t h a t o f s p e c i e s 1; i . e . , c 2c i *
We c a n i m m e d i a t e l y see t h a t a n e c e s s a r y c o n d i t i o n f o r
> ω
coexistence i s that ω 2 * · The v a l u e s o f ω f o r
e a c h s p e c i e s a r e d e t e r m i n e d by t h e same e q u a t i o n a s i n
t h e s i n g l e p o p u l a t i o n c a s e , u n a f f e c t e d by t h e p r e s e n c e
o f the other s p e c i e s . We c a n , t h e r e f o r e , move
d i r e c t l y to a g r a p h i c a l d e s c r i p t i o n o f the steady-
s t a t e behavior f o r our c o m p e t i t i o n model. F i g u r e 11
shows i s o c l i n e s o f ω i n t h e p l a n e o f (κ,λ) v a l u e s . If
we s p e c i f y v a l u e s λ χ and κι f o r p o p u l a t i o n 1, t h i s
y i e l d s a value for ωι. We c a n t h e n immediately
d i s c o v e r t h e p e r m i s s i b l e s t e a d y - s t a t e s f o r any s p e c i e s
2 with parameter values λ 2 and κ 2 (see Figure 1 2 ) . If
κ 2 < K l r o n l y s p e c i e s 1 can s u r v i v e , u n l e s s λ2 i s such
t h a t o)2 > ω χ — which would a l l o w c o e x i s t e n c e . If
κ 2 > Κ χ , o n l y s p e c i e s 2 can s u r v i v e , u n l e s s λ 2 is
s u c h t h a t α)2 < ω ι , w h i c h a g a i n a l l o w s c o e x i s t e n c e .
S o , t h e c o m p e t i t i o n o u t c o m e c a n be p r e d i c t e d f r o m
t h e s i n g l e p o p u l a t i o n r e s u l t s , w i t h one m i n o r
modification. Remember t h a t t h e ω c r i t e r i o n i s o n l y a
necessary condition for coexistence. I t turns out
t h a t a s e c o n d , s l i g h t l y more r e s t r i c t i v e c o n d i t i o n i s
a l s o r e q u i r e d , t o e n s u r e t h a t t h e c e l l d e n s i t i e s and
n u t r i e n t c o n c e n t r a t i o n r e m a i n p o s i t i v e e v e r y w h e r e (3JL) .
The d i f f e r e n c e b e t w e e n t h e two c o n d i t i o n s i s s m a l l
Ω
1
10 e ! !
10*
I COEXISTENCE j
ΙΟ 2
SPECIES 1 !
ONLY ! /
/
ι ι ι •
8 ! 32
λ, =10"'
•*• II
Χ Ι
• I
ΙΟ" - 2
/
/ ι SPECIES 2
/ s ONLY
/ ι
-/ I
4
/ ι
,ο -ι Ι
! COEXISTENCE j
ίο j 6
J
I 1
K 1 = 12
Figure 12. Illustration of predicted results for competition between two randomly
motile populations with identical properties except for Κ and λ.
Conclusions
R e v i e w o f t h e l i t e r a t u r e r e v e a l s a s m a l l number
o f e x p e r i m e n t s t h a t show t h a t c e l l m o t i l i t y p r o p e r t i e s
c a n h a v e d r a m a t i c e f f e c t s o n p o p u l a t i o n g r o w t h and
c o m p e t i t i o n i n non-mixed systems. Simple mathematical
models have been d e v e l o p e d w h i c h p r o v i d e q u a l i t a t i v e
e x p l a n a t i o n f o r a l l t h e o b s e r v e d p h e n o m e n a , and yield
q u a n t i t a t i v e p r e d i c t i o n of the magnitude of e f f e c t s
w h i c h m i g h t be e x p e c t e d i n a v a r i e t y o f s i t u a t i o n s .
Ac k nowledgme n t s
T h i s work h a s b e e n p a r t i a l l y s u p p o r t e d by
NSF C h e m i c a l and B i o c h e m i c a l P r o c e s s e s Program
G r a n t CPE80-06701. D.A.L. w o u l d a l s o l i k e t o t h a n k
P a t Thompson f o r t y p i n g t h i s m a n u s c r i p t and R e n a t e
S c h u l t z f o r many o f t h e f i g u r e s .
L i t e r a t u r e Cited
J. A. ROELS
Delft University of Technology, Netherlands Central Organization for Applied
Scientific Research, T.N.O., P.O. Box 108, 3700 A C ZEIST, The Netherlands
The b i o s p h e r e o n e a r t h i s , t h e r m o d y n a m i c a l l y s p e a k i n g , i n a
c e r t a i n s e n s e an o p e n s y s t e m . I t r e c e i v e s e n e r g y f r o m t h e s u n
i n t h e f o r m o f r a d i a t i o n . The e n e r g y o f t h e p h o t o n s r e a c h i n g e a r t h
i s , i n p a r t , converted t o chemical energy i n a process c a l l e d
0097-6156/83/0207Ό295$08.00/0
© 1983 American Chemical Society
h e a t a t a l o w t e m p e r a t u r e l e v e l . As a r e s u l t t h e o r g a n i s m s c a n
m a i n t a i n a s t a t e o u t s i d e t h e r m o d y n a m i c e q u i l i b r i u m and c a n
continue performing the processes c h a r a c t e r i s t i c f o r t h e i r " l i f e " .
As o r g a n i s m s a r e s y s t e m s w h i c h e x i s t o u t s i d e t h e r m o d y n a m i c
e q u i l i b r i u m and i r r e v e r s i b l e p r o c e s s e s a r e t a k i n g p l a c e , t h e
f o r m a l i s m o f thermodynamics of i r r e v e r s i b l e p r o c e s s e s c o n s t i t u t e s
the l o g i c a l v e h i c l e t o t r e a t t h e i r b e h a v i o u r . I n the p r e s e n t
a r t i c l e t h e f o r m a l i s m w i l l be b r i e f l y s u m m a r i z e d f o r t h e p u r p o s e
o f i t s a p p l i c a t i o n t o m i c r o o r g a n i s m s engaged i n g r o w t h and
p r o d u c t f o r m a t i o n . F o r a more t h o r o u g h t r e a t m e n t o f t h e b a s i c
f o r m a l i s m t h e r e a d e r i s r e f e r r e d t o t h e s t a n d a r d t e x t s ( 2 - 4 ) and
e a r l i e r w o r k o f t h e p r e s e n t a u t h o r ( 5 , 6^).
\
HEAT
(1)
V V s
I n t h e f o r m u l a t i o n o f eqn. (1) i t i s assumed t h a t t h e volume and
t h e s u r f a c e a r e a o f t h e s y s t e m do n o t change. I n t h e p r e s e n t
a r t i c l e t h e d i s c u s s i o n w i l l be r e s t r i c t e d t o s y s t e m s i n a
s t a t i o n a r y s t a t e , i . e . systems f o r which the time d e r i v a t i v e
a p p e a r i n g a t t h e l e f t hand s i d e o f eqn. (1) has become z e r o . I n
s u c h a c a s e eqn. (1) c a n be s i m p l i f i e d t o :
(2)
V
W i t h respect to th
n o n - e q u i l i b r i u m system the e x t e n s i v e q u a n t i t i e s c h a r a c t e r i z i n g
t h e s y s t e m can be d i s t i n g u i s h e d i n t o two g r o u p s : c o n s e r v e d and
non-conserved q u a n t i t i e s . S o - c a l l e d conserved q u a n t i t i e s cannot
be p r o d u c e d o r consumed i n t h e t r a n s f o r m a t i o n p r o c e s s e s o p e n t o
a g i v e n s y s t e m . T h e r e f o r e , t h e f i r s t t e r m a t t h e r i g h t hand s i d e
of eqn. (1) i s n e c e s s a r i l y z e r o and eqn. (2) c a n be s i m p l y
w r i t t e n as :
(3)
E q u a t i o n (2) e x p r e s s e s t h e f a c t t h a t f o r e a c h c o n s e r v e d
q u a n t i t y t h e t r a n s p o r t t o a s y s t e m i n s t a t i o n a r y s t a t e must
e x a c t l y match t r a n s p o r t from t h a t system. For a non-conserved
q u a n t i t y such s i m p l i f i c a t i o n i s not p o s s i b l e .
The a p p l i c a t i o n o f t h e f o r m a l m a c r o s c o p i c t h e o r y t o t r a n s
f o r m a t i o n p r o c e s s e s i n o p e n s y s t e m s i s b a s e d on t h e f o r m u l a t i o n
o f b a l a n c e e q u a t i o n s f o r a number o f c o n s e r v e d q u a n t i t i e s and
an a d d i t i o n a l t h e r m o d y n a m i c c o n s t r a i n t a l l o w i n g t h e f o r m u l a t i o n
of a u s e f u l e f f i c i e n c y measure.
The e l e m e n t a l b a l a n c e e q u a t i o n s . In m i c r o b i a l conversion
p r o c e s s e s t h e amounts o f t h e v a r i o u s a t o m i c s p e c i e s a r e c o n s e r v e d .
T h i s o b s e r v a t i o n r e s u l t s i n the f o r m u l a t i o n of e l e m e n t a l balance
e q u a t i o n s . U s i n g eqn. (3) t h e e l e m e n t a l b a l a n c e e q u a t i o n f o r
a t o m i c s p e c i e s j c a n , a g a i n a s s u m i n g a s t a t i o n a r y s t a t e , be
e x p r e s s e d as ( 5 , 6 ) :
η
Σ Φ.«*. .= 0 (4)
1 1 J
i-i
I n e q n . ( 4 ) Φ. s t a n d s f o r t h e n e t m o l a r f l o w o f compound
i t o t h e s y s t e m , e. . s t a n d s f o r t h e number o f m o l e s o f a t o m i c
s p e c i e s j i n one m J l e o f compound i . E q u a t i o n s o f t h e t y p e o f
eqn. ( 4 ) p r o v i d e one c o n s t r a i n t t o t h e n e t exchange f l o w s f o r e a c h
atomic species considered.
Φ Ε = 0 (5
i n w h i c h Φ^ i s t h e n e t f l o w o f e n e r g y t o w a r d s t h e s y s t e m .
Thermodynamics show t h a t , f o r a n open s y s t e m o n w h i c h no work
i s p e r f o r m e d by e x t e r n a l f o r c e f i e l d s , t h e e n e r g y f l o w t o w a r d s
t h a t s y s t e m c a n be e x p r e s s e d as f o l l o w s :
Φ
Ε - Η Φ +
Σ Φ
Α ( 6 )
I n t h i s e q u a t i o n Φ^ i s t h e s o - c a l l e d h e a t f l o w o f P r i g o g i n e ( 2 )
and t h e h. a r e t h e p a r t i a l m o l a r e n t h a l p i e s o f t h e compounds
exchanged w i t h t h e environment.
I f eqns. ( 5 ) and ( 6 ) a r e combined t h e f a m i l i a r b a l a n c e
equation f o r enthalpy i s obtained. I t allows the c a l c u l a t i o n of
the heat exchanged w i t h t h e environment from t h e f o l l o w i n g
equation:
Φ Η = - Σ Φ h. (7)
1
i n w h i c h Tig i s t h e t o t a l e n t r o p y p r o d u c t i o n i n t h e s y s t e m , Φ<,
i s the f l o w o f entropy t o the system.
The s e c o n d l a w o f t h e r m o d y n a m i c s p o s e s an i m p o r t a n t
r e s t r i c t i o n t o Tig » w h i c h must n e c e s s a r i l y e x c e e d z e r o f o r any
possible process:
Π δ > 0 (9)
Furthermore, t h e r m o d y n a m i c s show t h a t t h e e n t r o p y f l o w c a n
be w r i t t e n as :
Φ / Τ + ΣΦ.β. < 0
υ (11)
η . 1 1
1
Σ Φ 8· > 0 ί (12)
i
The p a r t i a l m o l a r f r e e e n t h a l p y i s d e f i n e d by:
g = h T s ( 1 3 )
i i " i
E q u a t i o n (12) w i l l be shown t o a l l o w t h e f o r m u l a t i o n o f an
e f f i c i e n c y m e a s u r e , w h i c h c a n be used t o a n a l y s e g r o w t h and
p r o d u c t f o r m a t i o n i n m i c r o o r g a n i s m s , i t s d e v e l o p m e n t w i l l be
undertaken i n the next s e c t i o n .
The t h e r m o d y n a m i c efficiency.
The e n e r g y and e n t r o p y c o n t e n t o f c h e m i c a l s u b s t a n c e s .
The t h e r m o d y n a m i c t h e o r y o u t l i n e d above c a n , i n p r i n c i p l e , be
s t r a i g h t f o r w a r d l y a p p l i e d t o t h e d e s c r i p t i o n o f m i c r o b i a l growth
and p r o d u c t f o r m a t i o n . I n o r d e r t o p e r f o r m s u c h an a n a l y s i s ,
t h e r m o d y n a m i c d a t a a r e needed r e g a r d i n g t h e compounds w h i c h a r e
exchanged w i t h t h e environment, i . e . t h e p a r t i a l molar e n t h a l p i e s
and e n t r o p i e s o f t h e compounds i n v o l v e d i n t h e p r o c e s s e s i n t h e
s y s t e m need t o be known. I n p r i n c i p l e t h e p a r t i a l m o l a r
q u a n t i t i e s , e n t h a l p y a s w e l l as e n t r o p y , a r e f u n c t i o n s o f t h e
c o n c e n t r a t i o n s o f e a c h and e v e r y c h e m i c a l compound p r e s e n t , i . e .
d e f i n i t e v a l u e s c a n n o t be a t t r i b u t e d t o a s i n g l e compound. I n
the approximation t o the e n e r g e t i c s o f m i c r o b i a l growth presented
h e r e , i d e a l i t y o f t h e m i x t u r e o f compounds i n v o l v e d w i l l be
assumed. T h i s i m p l i e s t h e p a r t i a l m o l a r t h e r m o d y n a m i c q u a n t i t i e s
to be e q u a l t o t h e s p e c i f i c m o l a r q u a n t i t i e s , t h e s e l a t t e r
q u a n t i t i e s depend o n i n t e n s i v e v a r i a b l e s l i k e t e m p e r a t u r e and
p r e s s u r e and t h e c o n c e n t r a t i o n o f t h e compound c o n s i d e r e d o n l y .
To a good d e g r e e o f a p p r o x i m a t i o n , e n t h a l p y c a n , a t a g i v e n
t e m p e r a t u r e and p r e s s u r e , be assumed i n d e p e n d e n t o f t h e c o n c e n t r a
t i o n o f t h e compound u n d e r c o n s i d e r a t i o n . The f r e e e n t h a l p y i s ,
h o w e v e r , by v i r t u e o f t h e n t r o p c o n t r i b u t i o t that quantit
(eqn. 1 3 ) , d e f i n i t e l y c o n c e n t r a t i o
relationship holds:
g L = g? + R T l n C i (14)
i n w h i c h g? i s t h e f r e e e n t h a l p y a t a g i v e n t e m p e r a t u r e and
p r e s s u r e and u n i t c o n c e n t r a t i o n o f t h e compound, w h i c h i s c a l l e d
standard free enthalpy. C. i s t h e c o n c e n t r a t i o n o f compound i .
As a f i r s t a p p r o a c h s t a n d a r d q u a n t i t i e s , i . e . a s s u m i n g u n i t
9
c o n c e n t r a t i o n s , w i l l be u s e d i n t h e p r e s e n t e v a l u a t i o n o f g r o w t h
and product formation.
One f u r t h e r c o n v e n i e n t c o n v e n t i o n needs t o be d i s c u s s e d .
E n e r g y , and hence a l s o d e r i v e d q u a n t i t i e s l i k e e n t h a l p y and f r e e
e n t h a l p y , c a n n o t be a t t r i b u t e d a d e f i n i t e v a l u e ; i t s m a g n i t u d e
c a n o n l y be d e f i n e d w i t h r e s p e c t t o a g i v e n r e f e r e n c e s t a t e w h i c h
i s a t t r i b u t e d a zero energy l e v e l . A convenient r e f e r e n c e s t a t e
f o r t h e e v a l u a t i o n o f g r o w t h and p r o d u c t f o r m a t i o n i n m i c r o
o r g a n i s m s i s o b t a i n e d i f C 0 , H 0 , 0 and N a r e a s s i g n e d a z e r o
2 2 2 2
m o t i v a t e d by t h e f a c t t h a t m i c r o o r g a n i s m s c a n u n d e r no c i r c u m
stances d e r i v e u s e f u l energy from processes i n which o n l y C 0 , 2
H 0 , 0 and N a r e i n v o l v e d .
2 2 2
The m o l a r f r e e e n t h a l p i e s and e n t h a l p i e s _ o f c o m b u s t i o n a t
0
s t a n d a r d c o n d i t i o n s w i l l be termed Ag°. and A h , r e s p e c t i v e l y . ^
C 1
From e q n . (7) i t i s c l e a r t h a t t h e e n f n a l p y o f c o m b u s t i o n , A h ° ^ ,
equals t h e heat o f combustion.
0
The f r e e e n t h a l p y o f c o m b u s t i o n , A g . , i s m a r k e d l y d e p e n d e n t
on t h e c o n c e n t r a t i o n s o f t h e r e a c t a n t s i n v o l v e d ( e q n . ( 1 4 ) ) and
most b i o l o g i c a l p r o c e s s e s t a k e p l a c e i n aquous s o l u t i o n s a t a
h y d r o g e n i o n c o n c e n t r a t i o n c o r r e s p o n d i n g t o a pH o f 7 r a t h e r t h a n
a t u n i t c o n c e n t r a t i o n o f t h e H - i o n , c o r r e s p o n d i n g t o a pH o f
z e r o . The f r e e e n t h a l p i e s o f c o m b u s t i o n t o l i q u i d w a t e r , t h e HC0 3
i o n (the p r e d o m i n a n t f o r m i n w h i c h C 0 e x i s t s a t a pH o f 7) and
2
N a t a pH o f 7 c a n t h u s be c o n s i d e r e d more r e l e v a n t t o b i o l o g i c a l
2
t r a n s f o r m a t i o n s . The f r e e e n t h a l p y o f c o m b u s t i o n u n d e r t h e s e
0
c o n d i t i o n s w i l l be i n d i c a t e d A g ! . The e n t h a l p y o f c o m b u s t i o n
i s h a r d l y a f f e c t e d by t h e pH. T^e f r e e e n t h a l p i e s and e n t h a l p i e s
o f c o m b u s t i o n a r e known t o obey r e g u l a r i t i e s ( 6 , 8 ) . T h e s e c a n
be t r e a t e d u s i n g t h e c o n c e p t o f t h e d e g r e e o f r e d u c t i o n a s i n t r o
d u c e d by M i n k e v i c h and E r o s h i n (_7) and e x t e n d e d and g e n e r a l i z e d
by t h e p r e s e n t a u t h o r ( 5 , 6 ) . The g e n e r a l i z e d d e g r e e o f r e d u c t i o n ,
γ., o f a compound w i t h r e s p e c t t o m o l e c u l a r n i t r o g e n i s d e f i n e d
by:
Ύ = 4 + a 2 b ( 1 5 )
ί i" i
I n w h i c h a. and b. a r e t h e number o f m o l e s o f Η and 0 p r e s e n t
i n one C-mole ( b e i n g t h e amount c o n t a i n i n g 12 grams o f c a r b o n )
o f coumpound i . F o r a component i t h e n t h a l p i e d fre
e n t h a l p i e s o f combustio
Ag°. r e s p e c t i v e l y , a r e approximation uniqu
f u n c t i o n o f t h e d e g r e e o f r e d u c t i o n , γ., as i n t r o d u c e d i n e q n .
( 1 5 ) . A s t a t i s t i c a l a n a l y s i s o f d a t a f o r some 60 o r g a n i c
compounds o f b i o l o g i c a l s i g n i f i c a n c e revealed the existence of
the f o l l o w i n g r e g u l a r i t i e s :
0
Ah . = 115γ. (16)
CL ' 1
Ag°. = 94.4γ. + 86.6 (17)
The r e s i d u a l e r r o r o f t h e e s t i m a t e i s 18 k J f o r b o t h e q n s . ( 1 6 )
and ( 1 7 ) . E q u a t i o n (16) s t a t e s t h a t t h e h e a t o f c o m b u s t i o n p e r
C-mole i s more o r l e s s d i r e c t l y p r o p o r t i o n a l t o t h e d e g r e e o f
r e d u c t i o n . As i s a p p a r e n t f r o m eqn. (17) s u c h a s i m p l e p r o p o r
t i o n a l i t y r e l a t i o n does n o t a p p l y t o f r e e e n t h a l p i e s o f
combustion.
E q u a t i o n s (16) and (17) show t h a t s y s t e m a t i c d e v i a t i o n s
b e t w e e n f r e e e n t h a l p i e s and h e a t s o f c o m b u s t i o n must e x i s t . F o r
s u b s t r a t e s o f a low degree o f r e d u c t i o n the f r e e e n t h a l p i e s o f
combustion exceed t h e heats o f combustion, f o r s u b s t r a t e s of a
h i g h d e g r e e o f r e d u c t i o n t h e r e v e r s e a p p l i e s . T h i s phenomenon
c a n be i l l u s t r a t e d i f t h e e n t r o p y c o n t r i b u t i o n t o t h e f r e e
0
enthalpy o f combustion, T A s . , i s c a l c u l a t e d . I t i s obtained from
the equation:
0 0
Ag°. = Ah . - TAs . (18)
6
C1 CI CI
I n f i g . 2 t h e r e l a t i o n i s shown b e t w e e n t h e s a i d e n t r o p y c o n t r i
b u t i o n and t h e d e g r e e o f r e d u c t i o n u s i n g d a t a f o r a v a r i e t y o f
o r g a n i c compounds. A d e f i n i t e t r e n d c a n i n d e e d be shown t o e x i s t
( a p a r t f r o m an i n c i d e n t a l o u t l y e r ) : The e n t r o p y c o n t r i b u t i o n
i n c r e a s e s w i t h i n c r e a s i n g degree o f r e d u c t i o n . I n v i e w o f the
o b s e r v a t i o n t h a t f r e e e n t h a l p y changes a t a pH o f 7 may w e l l be
more r e l e v a n t i n m i c r o b i o l o g i c a l p r o c e s s e s , t h e e n t r o p y c o n t r i b u -
501 ι ι ί
ο 2.5 5 7.5
Γ
0 f
t i o n t o t h e f r e e e n t h a l p y o f c o m b u s t i o n a t a pH o f 7, T A s , was
a l s o c a l c u l a t e d . T h e s e v a l u e s a r e p l o t t e d as a f u n c t i o n o?
t h e d e g r e e o f r e d u c t i o n i n f i g . 3. The f e a t u r e s o f f i g s . 2 and
3 a r e s e e n t o be v e r y much a l i k e , e x c e p t f o r t h e a c i d s , w h i c h
0 f
have a m a r k e d l y h i g h e r T A s a t a pH o f 7, i . e . t h e i r f r e e
e n t h a l p y o f c o m b u s t i o n i s îower a t t h a t pH. T h i s d i f f e r e n c e
b e t w e e n s t a n d a r d c o n d i t i o n s and a s i t u a t i o n i n w h i c h t h e c o n c e n
t r a t i o n s d i f f e r f r o m u n i t y , e.g. a t a pH o f 7, i s c h a r a c t e r i s t i c
f o r a l i m i t a t i o n o f t h e use o f s t a n d a r d f r e e e n t h a l p y changes
i n t h e a n a l y s i s o f p r o c e s s e s , where t h e c o n c e n t r a t i o n s a t t h e
l o c a l e o f t h e p r o c e s s may d i f f e r f r o m u n i t y : t h e Ag° v a l u e s c a n
o n l y be a p p l i e d t o an a p p r o x i m a t e a n a l y s i s . F o r d e t a i l e d
c o n s i d e r a t i o n s the A g v a l u e s at the c o n c e n t r a t i o n s at the l o c a l e
Q
o f e n e r g y g e n e r a t i o n a r e n e e d e d . S t i l l , as w i l l be shown, t h e
approximate analyses g r e a t l c o n t r i b u t t th understandin
of m i c r o b i a l energetics
T h e r e a l s o e x i s t compounds o f w h i c h t h e e n e r g y r e l e a s e d on
t h e i r c o m b u s t i o n does d e f i n i t e l y n o t f o l l o w t h e t r e n d s i n d i c a t e d
by e q n s . (16) and ( 1 7 ) . E x a m p l e s a r e o x y g e n and n i t r i c a c i d w h i c h
have a " h e a t o f c o m b u s t i o n " w h i c h i s c l o s e t o z e r o and a d e g r e e o f
r e d u c t i o n o f -4 and -5 r e s p e c t i v e l y . By v i r t u e o f t h i s f e a t u r e
t h e s e compounds can s e r v e as v e r y e f f i c i e n t " e l e c t r o n a c c e p t o r s " .
The t h e r m o d y n a m i c e f f i c i e n c y . The t h e r m o d y n a m i c t h e o r y
d e v e l o p e d e a r l i e r was shown t o r e s u l t i n eqn. ( 1 2 ) , a r e s t r i c t i v e
e q u a t i o n r e g a r d i n g the f l o w s o f m a t t e r exchanged w i t h the e n v i r o n
ment by an open s y s t e m . On eqn. (12) a d e f i n i t i o n o f t h e t h e r m o
d y n a m i c e f f i c i e n c y c a n be b a s e d i f t h e d i s s i p a t i o n , ΤΠ , i s
compared t o t h e t o t a l o f t h e f l o w s o f f r e e e n t h a l p y e n t e r i n g t h e
s y s t e m . However, a p r o b l e m w h i c h was a l r e a d y i n d i c a t e d above
has t o be t a c k l e d . The amount o f e n e r g y c a n n o t be s p e c i f i e d i n
a u n i q u e way and i t can o n l y be d e f i n e d w i t h r e f e r e n c e t o a b a s e
l e v e l , w h i c h i s a r b i t r a r i l y a t t r i b u t e d z e r o e n e r g y c o n t e n t . As
s o o n as s u c h a r e f e r e n c e s t a t e has been c h o s e n , t h e t h e r m o d y n a m i c
e f f i c i e n c y i s e a s i l y c a l c u l a t e d . The p r o c e d u r e i s i l l u s t r a t e d
i n f i g . 4. The t h e r m o d y n a m i c e f f i c i e n c y , fL,» i s d e f i n e d e q u a l
t o t h e r a t i o o f t h e f r e e e n t h a l p y g a i n e d i f t h e compounds l e a v i n g
t h e s y s t e m were t r a n s f o r m e d t o t h e r e f e r e n c e s t a t e , t o t h a t ,
w h i c h w o u l d be o b t a i n e d i f t h i s p r o c e d u r e were a p p l i e d t o t h e
compounds e n t e r i n g t h e s y s t e m . I t i s e a s i l y u n d e r s t o o d t h a t
η , i s c o n s t r a i n e d between z e r o , i f a l l t h e f r e e e n t h a l p y e n t e r i n g
trie s y s t e m i s d i s s i p a t e d and u n i t y i f Ag!j e q u a l s Ag^ , i . e . i f t h e
d i s s i p a t i o n equals zero. I t i s important to r e a l i z e t h a t the
f o r m e r c o n s t r a i n t s t r o n g l y depends on t h e c o r r e c t c h o i c e o f t h e
reference state.
A p p l i c a t i o n s of the theory
A e r o b i c g r o w t h w i t h o u t p r o d u c t f o r m a t i o n . The a p p l i c a t i o n
of the theory to a e r o b i c growth without f o r m a t i o n of products
k J/C-mole
100
50
Π
Ο
Δ*
* *
*
**
* *
-50
2.5 7.5
Υ
Φ Ag°
n'th
. u - — ^ — ^ (19
cN
i n which Φ , and Φ^ a r e t h e f l o w s o f b i o m a s s ( C - m o l e / h r ) ,
s u b s t r a t e ^ C - m o l e / h r ) and n i t r o g e n s o u r c e ( m o l e / h r ) t o o r f r o m
t h e s y s t e m ( a s i n d i c a t e d i n f i g u r e 5 ) . Ag° and A g ° a r e t h e
g
f r e e e n t h a l p i e s _ o f c o m b u s t i o n o f a C-mole o i b i o m a s s and s u b s t r a t e
r e s p e c t i v e l y . A g ^ i s t h e f r e e e n t h a l p y o f combustion o f a mole
of t h e n i t r o g e n source.
E q u a t i o n (19) c a n a l s o be w r i t t e n a s :
Ag°
η,. — (20)
A o / r -
g s + ( /c )Ai»
C ) 4 N
γ " = φ /φ (21)
SX X s
A n o t h e r u s e f u l e f f i c i e n c y measure i s t h e s o - c a l l e d e n t h a l p y
e f f i c i e n c y o f g r o w t h , η^, i t i s by a n a l o g y o b t a i n e d i f t h e Ag°.
0
i n eqn. (20) a r e r e p l a c e d by t h e r e s p e c t i v e A h . . As was alreaày
i n d i c a t e d t h e e n t h a l p y e f f i c i e n c y does n o t have t h e f u n d a m e n t a l
p r o p e r t i e s o f t h e thermodynamic e f f i c i e n c y a s t h e r e s t r i c t i o n
f o l l o w i n g from t h e a p p l i c a t i o n o f t h e second law o f thermodynamics
( s e e e q n . ( 1 1 ) ) d o e s n o t pose an u p p e r l i m i t t o Φ^. H e n c e , p r o c e s s e s
f o r w h i c h η„ e x c e e d s u n i t y c a n by no means be e x c l u d e d . I t c a n
e a s i l y b e shown t h a t t h e e f f i c i e n c y m e a s u r e s d e v e l o p e d a b o v e ,
i . e . η^, and η^, a l l o w t h e f o r m u l a t i o n o f e x p r e s s i o n s f o r t h e
d i s s i p a t i o n ΤΠ and t h e h e a t p r o d u c t i o n (-Φ„). The f o l l o w i n g
S n
FREE ENTHALPY
ENTERING SYSTEM
FREE ENTHALPY
LEAVING SYSTEM
_\h°-0
c
co 2
_H 0
2
cs
W W CHa b
i° i i Nc
Figure 5. System and flows for thermodynamic analysis of aerobic growth without
formation of products.
equations hold:
(22)
(23)
I n eqn. (22) D e q u a l s t h e d i s s i p a t i o n ΤΠ .
For a e r o b i c growth w i t h o u t product f o r m a t i o n the a b s o l u t e
m a g n i t u d e second t e r m a p p e a r i n g a t t h e l e f t hand s i d e o f eqn.
(11) c a n be shown t o be s m a l l as compared t o Φ^/Τ, i . e . t h e
exchange o f h e a t l a r g e l y e x c e e d s t h e exchange o f " c h e m i c a l
e n t r o p y " ( 5 , 6^. T h i s i m p l i e s t h a t D and a r e , t o a good a p p r o x
a n a
i m a t i o n e q u a l and t h i s , o f c o u r s e , a l s o a p p l i e s t o η * Π^·
I n t h i s c a s e an a n a l y s i s b a s e d on an e n t h a l p y e f f i c i e n c y o f
g r o w t h i s more o r l e s s v a l i
of t h e s e c o n d law as t
As has been shown e a r l i e r (6) t h e v e r y f a c t t h a t eqn. (16)
i s more o r l e s s v a l i d i m p l i e s t h a t h e a t p r o d u c t i o n and oxygen
consumption, Φ , are p r o p o r t i o n a l a c c o r d i n g to the f o l l o w i n g
equation :
Φ„ = 460 Φ (24)
ο
The a p p r o x i m a t e v a l i d i t y o f t h i s e q u a t i o n i s e a s i l y u n d e r s t o o d
as f o l l o w s . The d e g r e e o f r e d u c t i o n γ. i n d i c a t e s t h e number o f
moles o f e l e c t r o n s a v a i l a b l e f o r t r a n s f e r t o o x y g e n on c o m p l e t e
c o m b u s t i o n o f a C-mole o f a compound t o C0 , H 0 and N . On
2 2 2
t r a n s f e r t o oxygen t h e e n e r g y o f t h e e l e c t r o n s i s d i s s i p a t e d ,
r e s u l t i n g i n a h e a t p r o d u c t i o n o f 115 k J p e r mole o f e l e c t r o n s .
On a e r o b i c g r o w t h p a r t o f t h e e n e r g y c o n t e n t o f t h e s u b s t r a t e
and the n i t r o g e n source i s conserved i n the form o f newly
s y n t h e s i z e d biomass, the e l e c t r o n s c o r r e s p o n d i n g to the remainder
a r e t r a n s f e r r e d t o o x y g e n and p r o v i d e d i s s i p a t i o n . As f o u r moles
o f e l e c t r o n s a r e a c c e p t e d by one m o l e o f o x y g e n eqn. (16) shows
460 k J t o be g e n e r a t e d f o r e a c h m o l e o f oxygen consumed.
The v a l i d i t y o f eqn. (24) shows t h a t a t r e a t m e n t can a l s o
be b a s e d on o x y g e n e f f i c i e n c y o f g r o w t h C5"\7) ·
A s u b s t a n t i a l body o f d a t a on a e r o b i c g r o w t h w i t h o u t p r o d u c t
f o r m a t i o n s u p p o r t e d by ammonia as a n i t r o g e n s o u r c e has r e c e n t l y
been r e v i e w e d ( 9 ) . From t h e s e d a t a t h e v a l u e s o f η (- η ) and
t h e d i s s i p a t i o n p e r u n i t b i o m a s s p r o d u c e d (D/Φ , k J / C - m o l e ) were
c a l c u l a t e d . The r e s u l t s were a v e r a g e d f o r e a c h o f t h e c a r b o n
s o u r c e s c o n s i d e r e d and a r e shown i n f i g s . 6 and 7.
A l t h o u g h , not u n e x p e c t e d l y , i t i s c l e a r t h a t c o n s i d e r a b l e
s c a t t e r i s e x i s t e n t i n b o t h f i g s . 6 and 7,some g l o b a l r e g u l a r i t i e s
seem t o be p r e s e n t , w h i c h a r e i n d i c a t e d i n t h e f i g u r e s . F o r s u b
s t r a t e s w i t h a d e g r e e o f r e d u c t i o n l o w e r t h a n about 5, t h e thermo
dynamic e f f i c i e n c y a v e r a g e s 0.58 ( t h e o n l y s i g n i f i c a n t o u t l y e r
b e i n g t h e v e r y low e f f i c i e n c y o b s e r v e d f o r g r o w t h s u p p o r t e d by
0.25
A Î 1270 1240 I·
o x a l i c a c i d ) , t h i s corresponds to a d i s s i p a t i o n of roughly
400 k J / m o l e o f b i o m a s s p r o d u c e d . F o r s u b s t r a t e s w i t h a d e g r e e
o f r e d u c t i o n e x c e e d i n g 5 t h e thermodynamic e f f i c i e n c y
p r o g r e s s i v e l y d e c r e a s e s and t h e d i s s i p a t i o n i n c r e a s e s up t o 1400
kJ/C-mole f o r t h e c a s e o f g r o w t h s u p p o r t e d by methane.
I n f i g s . 6 and 7 t h e r e s t r i c t i o n s o f u n i t e f f i c i e n c y and
z e r o d i s s i p a t i o n r e s p e c t i v e l y , a s imposed by t h e s e c o n d law a r e
i n d i c a t e d as w e l l as a l i m i t imposed by a l i m i t a t i o n o f a
d i f f e r e n t n a t u r e , i . e . c a r b o n l i m i t a t i o n . The l a t t e r l i m i t m e r i t s
a more t h o r o u g h d i s c u s s i o n . The d e g r e e o f r e d u c t i o n , γ , o f b i o
mass f r o m a v a r i e t y o f s o u r c e s a v e r a g e s 4.8 (5,6,10) and hence
by v i r t u e o f eqn. (16) i t s e n e r g y c o n t e n t i s a b o u t 550 k J / C - m o l e .
I f g r o w t h i s s u p p o r t e d by a s u b s t r a t e o f a d e g r e e o f r e d u c t i o n
e x c e e d i n g 4.8 i t s e n e r g y c o n t e n t w i l l e x c e e d t h e s a i d 550
k J / C - m o l e and h e n c e , eve i f a l lsubstrat carbo fixed
i n b i o m a s s , a thermodynami
be o b t a i n e d . A c o m p l e t e x p l o i t a t i o energy p r e s e n
t h e s u b s t r a t e w o u l d r e q u i r e f i x a t i o n o f a d d i t i o n a l low e n e r g y
c a r b o n e.g. f r o m C0 . T h i s i s , however, e x c l u d e d , as o n l y one
2
c a r b o n s o u r c e i s assumed t o be s u p p l i e d .
On o b s e r v a t i o n o f f i g . 6 i t becomes c l e a r t h a t t h e t r e n d s
o b s e r v e d i n t h e e x p e r i m e n t a l v a l u e s o f t h e thermodynamic
e f f i c i e n c y mimic t h e shape o f t h e t h e o r e t i c a l r e s t r i c t i o n s a t
a l e v e l o f a b o u t 60%. The d e v i a t i o n between t h e t h e o r e t i c a l l i m i t
and t h e v a l u e s a c t u a l l y o b s e r v e d i s q u i t e e a s i l y u n d e r s t o o d i n
g e n e r a l terms i n t h e r e g i o n o f low d e g r e e s o f r e d u c t i o n , where
the energy a v a i l a b l e i n the s u b s t r a t e r a t h e r than i t s carbon
M
c o n t e n t l i m i t s the v a l u e s o f Y . The t h e o r e t i c a l l i m i t o f u n i t y
sχ ·
c a n n e v e r be r e a c h e d as any p r o c e s s needs a n o n - z e r o d i s s i p a t i o n
t o proceed at a non-zero r a t e (2-4). In f a c t the r a t e at which
a p r o c e s s p r o c e e d s , e.g. t h e r a t e o f g r o w t h o f t h e amount o f
biomass, i s to a c e r t a i n extent i n c r e a s i n g w i t h i n c r e a s i n g
d i s s i p a t i o n . V a r i o u s o p t i m a l i t y p r i n c i p l e s ( 1 1 ) , may d i c t a t e
an o p t i m a l thermodynamic e f f i c i e n c y o f r o u g h l y t h e m a g n i t u d e
o b s e r v e d h e r e ( 1 1 , 1 2 ) . The b e h a v i o u r a t h i g h d e g r e e s o f r e d u c t i o n
i s l e s s e a s i l y u n d e r s t o o d on f u n d a m e n t a l g r o u n d s . The t h e r m o
dynamic e f f i c i e n c y c o u l d w e l l a p p r o a c h t h e t h e o r e t i c a l l i m i t c l o s e r
with a s u f f i c i e n t l y l a r g e d i s s i p a t i o n . A p p a r e n t l y o t h e r phenomena
t o w h i c h t h e f o r m a l m a c r o s c o p i c t r e a t m e n t p r o v i d e s no c l u e s ,
l i m i t t h e maximum c o n s e r v a t i o n o f s u b s t r a t e c a r b o n i n b i o m a s s
t o a b o u t 2/3 o f t h e maximum. O b v i o u s l y one has t o r e s o r t t o b i o
c h e m i c a l t h e o r y t o o b t a i n c l u e s t o the n a t u r e o f t h e mechanisms
u n d e r l y i n g t h e phenomenon.
T h e r e e x i s t s s t i l l a n o t h e r u s e f u l q u a n t i t y , w h i c h has been
used t o s y s t e m a t i z e t h e e x p e r i m e n t a l d a t a on t h e e f f i c i e n c y
1
o f g r o w t h s u p p o r t e d by v a r i o u s c a r b o n s o u r c e s ; i t i s P a y n e s
y i e l d on a v a i l a b l e e l e c t r o n s , Υ , ( 1 3 ) . I t i s d e f i n e d as t h e
amount o f b i o m a s s p r o d u c e d p e r mole o f e l e c t r o n s a v a i l a b l e f o r
t r a n s f e r t o o x y g e n on c o m p l e t e c o m b u s t i o n . N u m e r i c a l l y t h e number
o f m o l e s o f e l e c t r o n s a v a i l a b l e f o r t r a n s f e r t o o x y g e n on
c o m b u s t i o n o f a C-mole o f a g i v e n s u b s t r a t e i s e q u a l t o t h e
d e g r e e o f r e d u c t i o n , γ., as d e f i n e d by eqn. ( 1 5 ) . As a f i r s t
a p p r o x i m a t i o n , t a k i n g i n t o account the f a c t t h a t biomass from
a«-·• vVW
a.
rAi.^
eWtWy^ νo fΛ. I
mUX
i.V
c.rLVUo b iJLaj.
a l sIo7uWUr1c.eU^t7
s can
COU beVJ t.w eW lI— lJ. J- r e p r e s e n t e d by t h e
composition formula C H O . 5 N 0 . 2 » l e 8 0
Y
/ C c a n be c o n s i d e r e d
p r o p o r t i o n a l t o X]^ ( o r r a t h e r n ) (§, 6 ) H
e
F i g u r e 8 shows t h e d a t a d e p i c t e d i n f i g s . 6 and 7 i n a p l o t o f
Y . v e r s u s t h e d e g r e e o f r e d u c t i o n γ. The t r e n d s and t h e o r e t i c a l
l i m i t s a r e a l s o shown.
Growth w i t h e l e c t r o
preceeding s e c t i o n a cas
e l e c t r o n a c c e p t i n g m o i e t y . Oxygen i s , h o w e v e r , by no means t h e
o n l y compound, w h i c h c a n p e r f o r m s u c h f u n c t i o n . O t h e r examples
a r e n i t r a t e and s u l p h a t e . The c h a r a c t e r i s t i c f e a t u r e o f an
e l e c t r o n a c c e p t o r i s t h a t f r e e e n t h a l p y i s gained i n the o v e r a l l
p r o c e s s o f t r a n s f e r o f an e l e c t r o n f r o m a g i v e n s o u r c e (a
s u b s t r a t e ) to the acceptor. T h i s f r e e enthalpy i s p a r t l y
d i s s i p a t e d t o p r o v i d e t h e n e c e s s a r y i r r e v e r s i b i l i t y and i t c a n
be u s e d t o t r a n s f e r c a r b o n ( - d i o x i d e ) f r o m a low e n e r g y l e v e l
t o a h i g h e n e r g y one. I n t a b l e I a summary i s p r o v i d e d o f t h e
e n e r g y b e c o m i n g a v a i l a b l e on t r a n s f e r o f one m o l e o f e l e c t r o n s
f r o m g l u c o s e t o a g i v e n e l e c t r o n a c c e p t o r engaged i n a g i v e n
reduction process.
Table I. F r e e e n t h a l p y g a i n e d a t pH = 7 and s t a n d a r d
c o n d i t i o n s on t r a n s f e r o f one m o l e o f e l e c t r o n s
f r o m g l u c o s e t o a g i v e n e l e c t r o n a c c e p t o r (14)
0 1
e l e c t r o n acceptor moles of e l e c t r o n s Ag ^ ^ ^Sai/^
+ * +
accepted kJ kjfmole
N 2 (NH ) 4 6 79.1 13.2
S (HS~) 2 27.7 13.9
2-
SO, (HS ) 8 150.7 18.8
4 9
S 0
(HS~) 6 171.9 28.7
3
N0 ~ 2 (NH ) 4
+
6 435.5 72.6
N0 " 3 (NH ) 4
+
8 598.4 74.8
N0 " 3 (N )
2
5 559.5 111.9
(H 0)
2
4 473.9 118.5
°2
* I n b r a c k e t s reduced form of e l e c t r o n a c c e p t o r
'av/e
g DM/mole
6h SECOND
4H
2h
1h
the second law and C-limitation. Average of experimental data for various sub-
strates, symbols as in Figure 6.
The v a l u e s o f t h e f r e e e n t h a l p y g a i n e d p e r m o l e o f e l e c t r o n s
t r a n s f e r r e d a r e a r r a n g e d i n o r d e r o f i n c r e a s i n g m a g n i t u d e . The
" e f f i c i e n c y " o f an e l e c t r o n a c c e p t o r t h u s i n c r e a s e s f r o m t h e t o p
to t h e b o t t o m o f t h e t a b l e and f a i t h i n l i f e ' s u n i v e r s a l s t r i v i n g
f o r more o p t i m a l g r o w t h w o u l d assume p r e f e r e n c e f o r t h e e l e c t r o n
a c c e p t o r s a t t h e b o t t o m o f t h e t a b l e i f two o r more e l e c t r o n
acceptors are simultaneously a v a i l a b l e .
Growth w i t h o u t e x t e r n a l l y s u p p l i e d e l e c t r o n a c c e p t o r s . I n
a number o f c a s e s g r o w t h i s o b s e r v e d t o t a k e p l a c e w i t h o u t an
i d e n t i f i a b l e seperate e l e c t r o n a c c e p t o r b e i n g p r e s e n t . I n such
case t h e s u b s t r a t e o r a s u b s t r a t e d e r i v e d moiety i s both donor
and a c c e p t o r o f e l e c t r o n s . F o r s i m p l i c i t y ' s s a k e o n l y t h e c a s e
o f a n a e r o b i c g r o w t h on a s i n g l e c a r b o n s o u r c e w i t h f o r m a t i o n o f
a s i n g l e p r o d u c t and NH^ as t h e n i t r o g e n s o u r c e w i l l be t r e a t e d
I t i s e a s i l y understoo
be b a s e d o n a b a l a n c e o
eqn. ( 1 6 ) . T a b l e I I shows t h e f l o w s o f t h e v a r i o u s compounds and
t h e i r c o n t e n t o f e l e c t r o n s a v a i l a b l e f o r t r a n s f e r t o oxygen on
f o r m a t i o n o f Φ C-moles of b i o m a s s .
χ
Table I I . A degree o f r e d u c t i o n balance f o r anaerobic
growth w i t h o u t e x t e r n a l e l e c t r o n a c c e p t o r s
By v i r t u e o f t h e f a c t t h a t no e x t e r n a l e l e c t r o n a c c e p t o r s
are present the c o n t r i b u t i o n s t o the degree o f r e d u c t i o n balance
as shown i n t h e l a s t c o l u m n o f t a b l e 2 must add up t o z e r o , i t
follows :
Φ (26)
<w v SX ( Y
+ 3 C
I
Φ = Φ {(1 γ /γ ) / Υ " - 1 3c,/γ • γ /γ } (27)
c χ ρ χ ρ
s 'ρ SX
I t becomes c l e a r t h a t on t h e a n a e r o b i c g r o w t h p r o c e s s an
amount o f p r o d u c t Φ , g i v e n by eqn. ( 2 6 ) , i s formed. I t
c o r r e s p o n d s t o t h e E r a n s f e r o f (γ /Y" + 3c, - γ )Φ moles o f
s sχ . 1 χ χ
e l e c t r o n s from the s u b s t r a t e to r e s u l t i n g i n the f o r m a t i o n
of the p r o d u c t . C l e a r l y , the d r i v i n g f o r c e of the r e a c t i o n , w h i c h
m u s t , amongst o t h e r s , p r o v i d e t h e n e c e s s a r y d i s s i p a t i o n , must
r e s t i n th e f a c t t h a t t h e e l e c t r o n s i n t h e s u b s t r a t e a r e a t a
h i g h e r f r e e e n t h a l p y l e v e l t h a n t h o s e i n t h e p r o d u c t . The
mechanism o f t h i s p r o c e s s becomes c l e a r e r i f eqns. (16) and (17)
a r e o b s e r v e d c l o s e r . The e n t h a l p y o f c o m b u s t i o n i s , f o r compounds
o f d i f f e r e n t d e g r e e o f r e d u c t i o n ^ a p p a r e n t l y t o a v e r y good d e g r e e
o f a p p r o x i m a t i o n e q u a l p e r mole o f e l e c t r o n s a v a i l a b l e f o r
t r a n s f e r t o o x y g e n . H e n c e , i f e n t h a l p y w e r e , as i n t h e c a s e o f
a e r o b i c g r o w t h , t o be t h e s o u r c e p r o v i d i n g dissipât i o n , a n a e r o b i c
growth would to a degre approximatio involv negligibl
d i s s i p a t i o n and be i m p o s s i b l
view. E q u a t i o n ( 1 7 ) , however, enthalpy
o f a s u b s t r a t e p e r mole o f a v a i l a b l e e l e c t r o n s d e c r e a s e s , as a
g e n e r a l t e n d e n c y , w i t h i n c r e a s i n g d e g r e e o f r e d u c t i o n . Hence,
t h e d i s s i p a t i o n n e c e s s a r y f o r e f f i c i e n t g r o w t h c a n be g a i n e d i f
a product of a h i g h e r degree of r e d u c t i o n i s , w i t h f o r m a t i o n of
CO^, formed f r o m a s u b s t r a t e w i t h a low d e g r e e o f r e d u c t i o n 0 6 ) .
T h i s c a n be shown t o be t h e g e n e r a l t e n d e n c y u n d e r l y i n g a n a e r o b i c
g r o w t h o f t h e t y p e t r e a t e d h e r e . The g e n e r a l t e n d e n c y u n d e r l y i n g
a n a e r o b i c growth i s t h a t , c o n t r a r y t o the s i t u a t i o n i n a e r o b i c
g r o w t h , t h a t i s d r i v e n by t h e e n t h a l p y r a t h e r t h a n the " c h e m i c a l
entropy" c o n t r i b u t i o n to the d i s s i p a t i o n , i t i n v o l v e s a l a r g e
c o n t r i b u t i o n of "chemical entropy" d i s s i p a t i o n at a g e n e r a l l y
m i n o r c o n t r i b u t i o n due t o h e a t p r o d u c t i o n . F o r p r a c t i c a l p u r p o s e s ,
t h i s means t h a t on t h e s e a n a e r o b i c p r o c e s s e s c l o s e t o 100% o f
the energy of combustion o f the s u b s t r a t e s u p p l i e d i s conserved
i n t h e p r o d u c t and b i o m a s s f o r m e d , t h e i r r e v e r s i b i l i t y b e i n g
0
p r o v i d e d by t h e c o n t r i b u t i o n , T A s .
The t e n d e n c y s k e t c h e d above o n l y a c c o u n t s f o r t h e m a i n
f e a t u r e s , another important aspect b e i n g t h a t anaerobic growth
p r o c e e d s t o compounds, w h i c h have a low e n t h a l p y a t a g i v e n d e g r e e
o f r e d u c t i o n , i . e . a n a e r o b i c g r o w t h i s a l s o d r i v e n by t h e
d e v i a t i o n s from e q n s . (16) and (17) r a t h e r t h a n by t h e g e n e r a l
tendencies these equations d e s c r i b e .
The r e a s o n t h a t t h e l i m i t e d a c c u r a c y o f eqns. (16) and (17)
becomes i m p o r t a n t h e r e , w h i l s t b e i n g o f v i r t u a l l y no i m p o r t a n c e
i n a e r o b i c growth, r e s t s i n the f a c t t h a t l a r g e f l o w s of s u b s t r a t e
and p r o d u c t a r e i n v o l v e d i n a n a e r o b i c g r o w t h . T h u s , d i s s i p a t i o n
becomes t h e d i f f e r e n c e o f two l a r g e numbers and m i n o r e r r o r s i n
t h e s e c o n t r i b u t i o n s become q u i t e s i g n i f i c a n t .
As a t y p i c a l example o f t h e f e a t u r e s e x h i b i t e d by a n a e r o b i c
g r o w t h the f o l l o w i n g q u a n t i t i e s a r e w o r t h w h i l e . On a n a e r o b i c
g r o w t h o f a y e a s t on g l u c o s e w i t h f o r m a t i o n o f e t h a n o l t y p i c a l l y
a b o u t 90 k J o f h e a t a r e g e n e r a t e d p e r C-mole o f b i o m a s s p r o d u c e d ;
t h i s v a l u e i s s u b s t a n t i a l l y l o w e r t h a n t h e h e a t p r o d u c t i o n on
a e r o b i c g r o w t h o f t h a t same y e a s t , w h i c h t y p i c a l l y amounts t o
300 - 350 k J / C - m o l e . The f r e e e n t h a l p y d i s s i p a t i o n on a n a e r o b i c
g r o w t h i s , h o w e v e r , c l o s e t o 300 k J / C - m o l e p r o d u c e d , i . e . s l i g h t l y
l o w e r t h a n o r e q u a l t o t h a t on a e r o b i c g r o w t h .
An e a r l i e r s t u d y p e r f o r m e d by t h e p r e s e n t a u t h o r (5_, 6^)
showed t h a t , i f a r e a l i s t i c r e f e r e n c e s t a t e f o r e n e r g y c o n t e n t
i s c h o s e n , a n a e r o b i c g r o w t h p r o c e e d s as a g e n e r a l t e n d e n c y w i t h
a t h e r m o d y n a m i c e f f i c i e n c y o f t h e same o r d e r o f m a g n i t u d e as t h a t
o b s e r v e d on a e r o b i c g r o w t h . T h i s p r o v i d e s t h e n u c l e u s t o a
p o s t u l a t e t h a t w i l l be somewhat more c l o s e l y i n v e s t i g a t e d i n t h e
l a s t s e c t i o n o f t h i s p a p e r : Growth y i e l d s can as a r u l e o f thumb
be e s t i m a t e d f r o m t h e a s s u m p t i o n o f a c o n s t a n t t h e r m o d y n a m i c
e f f i c i e n c y o r , e q u i v a l e n t l y , a constant d i s s i p a t i o n per u n i t
biomass produced.
The constant e f f i c i e n c
F o r g r o w t h s u p p o r t e d by d i f f e r e n t s o u r c e s and s i n k s o f
e l e c t r o n s , e.g. a e r o b i c g r o w t h , g r o w t h s u p p o r t e d by e l e c t r o n
a c c e p t o r s o t h e r t h a n o x y g e n o r g r o w t h p r o c e s s e s i n w h i c h no
e x t e r n a l e l e c t r o n a c c e p t o r s are p r e s e n t , w i d e l y v a r y i n g s u b s t r a t e
y i e l d s , e.g. e x p r e s s e d as Υ" , a r e o b s e r v e d . As was a l r e a d y
i n d i c a t e d w i t h r e f e r e n c e t o a e r o b i c g r o w t h , m a t t e r s can be
g e n e r a l i z e d to a c e r t a i n extent i f y i e l d s are expressed i n
terms o f Y / > t h e y i e l d e x p r e s s e d p e r mole o f s u b s t r a t e
a v e
e l e c t r o n s a v a i l a b l e f o r t r a n s f e r t o o x y g e n . However, as was
p o i n t e d o u t i n d i s c u s s i n g t a b l e I , l a r g e l y d i f f e r e n t amounts
o f e n e r g y may r e s u l t from t h e t r a n s f e r o f one mole o f e l e c t r o n s
i n dependence on t h e d o n o r / a c c e p t o r c o m b i n a t i o n . T a b l e I I I
p r o v i d e s a summary o f l i t e r a t u r e d a t a . F o r a number o f g r o w t h
p r o c e s s e s t h e y i e l d on a v a i l a b l e e l e c t r o n s , Y , , as w e l l
ο
1
av / e
as t h e Ag , , t h e f r e e e n t h a l p y o b t a i n e d on t r a n s f e r o f one
m o l e o f eîecfrons f r o m t h e s u b s t r a t e t o t h e e l e c t r o n a c c e p t o r
( s t a n d a r d c o n d i t i o n s and a pH = 7 ) , a r e g i v e n .
Even a s u p e r f i c i a l s t u d y o f t a b l e I I I c l e a r l y shows t h e
e x i s t e n c e o f a r e l a t i o n s h i p between Y . and A g ° ,. T h i s
1
r e l a t i o n s h i p can be s y s t e m a t i z e d as f o l l o w s . I f t K e t h e r m o d y n a m i c
e f f i c i e n c y , η , , i s known eqn. (22) a l l o w s t h e c a l c u l a t i o n
of the d i s s i p a t i o n per u n i t biomass produced. Furthermore i t
has t o be r e c o g n i z e d t h a t t h e d i s s i p a t i o n p e r mole o f b i o m a s s
p r o d u c e d can be c a l c u l a t e d f r o m :
0
D/Φ = A g ' , (25.8/Y - 4.8) + 65.8 (28)
χ °av/e av/e
I n w h i c h t h e c o n v e r s i o n f a c t o r 25.8 a c c o u n t s f o r t h e
c o n v e r s i o n o f g.DM. t o C-moles, 4.8 i s a c o r r e c t i o n a c c o u n t i n g
f o r t h e number o f m o l e s o f a v a i l a b l e e l e c t r o n s c o n s e r v e d i n
one C-mole o f b i o m a s s f o r m e d , and 65.3 a c c o u n t s f o r t h e f r e e
e n t h a l p y o f c o m b u s t i o n o f t h e ammonia u s e d i n the p r o c e s s o f
Table I I I . Y i e l d s on a v a i l a b l e e l e c t r o n s and e n e r g y
a v a i l a b l e p e r mole o f e l e c t r o n s t r a n s f e r r e d
for various processes
0 t
25.8Ag , &
av/e
Y
a v / e ( 2 9 )
536(1 - n t h )/n t h + 4.8Ag- / e - 65.8
4h
av/e
g D.MV
/note av/e
r/ th = 0 65
10 50 100
9 [kJ molej
^ av e
and the energy gained per mole electrons transferred to the electron acceptor,
A g a , / r ' (kJ/mole).
0
Expected relation if the thermodynamic efficiency were
constant at 0.65 (—). Experimental data are shown for aerobic growth on glucose
( ), succinate (®), and gluconate (®). Data are shown for growth with Ν Of
as an electron acceptor on succinate ( @ ), and on gluconate ( @ ). A naerobic growth
on glucose is shown with formation of ethanol (*), lactate (®), propionic/acetic
acids ( Δ J, and methane/acetic acid (O). Anaerobic growth is shown with formation
of methane from acetic acid (\7), formic acid (V), propionic acid (A), and
methanol (•,).
As°. p a r t i a l e n t r o p y o f c o m b u s t i o n o f a C-mole o f ( k J / C - m o l e K)
compound i a t s t a n d a r d c o n d i t i o n s
As°. p a r t i a l e n t r o p y o f c o m b u s t i o n o f a C-mole o f ( k J / C - m o l e K)
compound i a t a pH = 7
Τ absolute temperature Κ
3
ι
V vo 1 urne m
Y
sx yi^d f° r
b i o m a s s on s u b s t r a t e (Omole/C-mole)
1
Y , J
y i e l d on t h e s u b s t r a t e s available electrons (g.D.M./mole)
&
av/e
d e g r e e o f r e d u c t i o n o f compound i (-)
f
η M i n k e v i c h and E r o s h i n s e f f i c i e n c y (-)
η enthalpy e f f i c i e n c
Η
η ^ thermodynamic e f f i c i e n c y (-)
TTg entropy production r a t e (kJ/Ks)
r a t e o f f l o w o f compound i t o t h e s y s t e m (mole/s)
Φ_ r a t e o f f l o w o f energy to the system (kJ/s)
heat f l o w t o the system (kJ/s)
Subscripts :
c carbon d i o x i d e
Ν nitrogen source
ο oxygen
ρ product
s substrate
χ biomass
Literature Cited
ERICH HEINZ
Cornell University Medical College, New York, NY 10021
0097-6156/83/0207-0323$06.00/0
© 1983 American Chemical Society
T h e o v e r a l l effectiveness of a n y c o u p l i n g m e c h a n i s m m a y b e
a s s e s s e d i n t e r m s o f i t s ( e n e r g e t i c ) e f f i c i e n c y a s w e l l a s i n t e r m s of i t s
(kinetic) e x p e d i t i o u s n e s s . T h e e n e r g e t i c e f f i c i e n c y i s l i m i t e d b y the
e x t e n t of u n a v o i d a b l e l e a k a g e s , w h i c h a r e p a r t l y due to p a s s i v e permea-
b i l i t y of the m e m b r a n e b a r r i e r to the t r a n s p o r t e d s o l u t e (outer l e a k a g e )
a n d p a r t l y r e s i d e i n the t r a n s p o r t m e c h a n i s m i t s e l f ( i n n e r l e a k a g e ) i n
that " s l i p p i n g " a n d " b a c k c y c l i n g " m a y p e r m i t the e s c a p e of u n u t i l i z e d
i n p u t e n e r g y (1 ). T o o l i t t l e i s k n o w n about the e x t e n t of l e a k a g e s w i t h -
i n the c h e m i o s m o t i c m e c h a n i s m o f p h o s p h o r y l a t i o n to c o m p a r e i t s
e n e r g e t i c e f f i c i e n c y w i t h that o f i t s c h e m i c a l c o u n t e r p a r t .
N o t m u c h m o r e i s known about the e x p e d i t i o u s n e s s w h i c h , i n
t h i s c o n t e x t , m a y be d e f i n e d a s the r a t e at w h i c h the p r o t o n m o t i v e
f o r c e r i s e s a f t e r the o n s e t of the p u m p , but s o m e g e n e r a l p r e d i c t i o n s
c a n be m a d e . The expeditiousnes
r a t e of p r o t o n s i s r e l a t i v
tend to shunt the e l e c t r o g e n i c p u m p effect, i n o t h e r w o r d s , to m a i n t a i n
electroneutrality. W e s e e that the two c o m p o n e n t s of the p r o t o n m o t i v e
force, the e l e c t r i c a l a n d the c h e m i c a l P D , a r e c o n t r o l l e d b y d i f f e r e n t
f a c t o r s a n d m a y t h e r e f o r e d e v e l o p at d i f f e r e n t r a t e s , a s i s i l l u s t r a t e d
b y the f o l l o w i n g two e x t r e m e c o n d i t i o n s :
If the p a s s i v e c o n d u c t a n c e i s e x t r e m e l y l o w , the e l e c t r i c a l
p o t e n t i a l w i l l b e g e n e r a t e d p u r e l y e l e c t r o g e n i c a l l y , i . e. without
a p p r e c i a b l e net m o v e m e n t of p a s s i v e i o n s . A s the e l e c t r i c c a p a c i t y of
the m e m b r a n e i s r a t h e r l o w , ^F/cm , 2
e l e c t r o g e n i c e x p u l s i o n of 3 0 0 -
400 p r o t o n s p e r c e l l , p a s s i v e s h u n t i n g effect b e i n g n e g l i g i b l e at t h i s
stage, s h o u l d s u f f i c e to r a i s e the e l e c t r i c a l P D b y 1 m v o l t .
O n the o t h e r h a n d , i f the p a s s i v e c o n d u c t a n c e i s v e r y h i g h , i t
w i l l k e e p e l e c t r i c a l e f f e c t s s m a l l but a l l o w i n s t e a d a n e q u i v a l e n t r i s e
o f the c h e m i c a l P D of p r o t o n s . T h i s r i s e w i l l d e p e n d o n the b u f f e r
c a p a c i t y o f the m e d i u m , w h i c h i s n o r m a l l y m u c h h i g h e r than the
e l e c t r i c a l capacity. F r o m i t s v a l u e i n the m i t o c h o n d r i a we w o u l d e s t i -
m a t e that about a m i l l i o n p r o t o n s h a v e to be e x p e l l e d p e r 1 m v o l t r i s e
of the c h e m i c a l P D , a n d h e n c e o f the p r o t o n m o t i v e f o r c e . Obviously,
the r i s e i n c h e m i c a l P D i n t h i s c a s e i s t h o u s a n d o r m o r e t i m e s s l o w e r
than the e l e c t r o g e n i c one i n the f o r m e r c a s e . A t the r a t e a s s u m e d for
the m i t o c h o n d r i a l p r o t o n p u m p , we would p r e d i c t that the c r i t i c a l
protonmotive force, i . e . about 200 m v o l t s , c o u l d be r e a c h e d w i t h i n a
few h u n d r e d m i l l i s e c o n d s i n the f i r s t c a s e , but would take a few
h u n d r e d s e c o n d s i n the l a s t c a s e . It f o l l o w s that the e x p e d i t i o u s n e s s of
the c o u p l i n g , i . e. the s p e e d at w h i c h t h i s c r i t i c a l p r o t o n m o t i v e f o r c e
i s r e a c h e d a f t e r the i n i t i a t i o n of the p u m p d e p e n d s on the r e l a t i v e c o n -
t r i b u t i o n of the i n i t i a l p u m p i n g r a t e r e l a t i v e to the s h u n t i n g p a t h w a y s
for passive i o n s .
T o a p p r o a c h t h i s p r o b l e m q u a n t i t a t i v e l y , we m a y f o r s i m p l i c i t y
r e a s o n s f o l l o w the c o n v e n t i o n a l p r o c e d u r e u s e d b y e l e c t r o p h y s i o l o g i s t s
i n d e a l i n g with e l e c t r o g e n i c p u m p s in t h e i r b i o l o g i c a l s y s t e m s , e t c . In
the e q u i v a l e n t c i r c u i t s u s e d b y t h e m to v i s u a l i z e the e l e c t r i c p h e n o m e n a
of b i o l o g i c a l s y s t e m s , an electrogenic ion p u m p i s u s u a l l y represented
e i t h e r as a constant voltage s o u r c e o r as a constant c u r r e n t s o u r c e ,
b a s e d o n the s i m p l i f y i n g a s s u m p t i o n that d u r i n g the e x p e r i m e n t a l
o b s e r v a t i o n e i t h e r the d r i v i n g f o r c e o f the p u m p o r the p u m p i n g r a t e
remains fairly constant. T h e d i f f e r e n c e b e t w e e n t h e s e two
a l t e r n a t i v e s c o r r e s p o n d s w i t h s o m e c r u d e a p p r o x i m a t i o n to the a b o v e
d i s c u s s e d d i f f e r e n c e b e t w e e n two k i n d s o f c o u p l i n g m o d e l s ; the c o n s t a n t
v o l t a g e s o u r c e n e t w o r k m o s t c l o s e l y w o u l d a c c o u n t f o r the e x p e d i t i o u s -
n e s s , but l e s s s t a b l e c o u p l i n g m o d e l , w h e r e a s the c o n s t a n t c u r r e n t
s o u r c e w o u l d d o s o f o r th
most cases, it i s difficul
f o r e i t h e r one s o m e p l a u s i b l e e x p l a n a t i o n c a n b e g i v e n . As a conse-
q u e n c e , the t r e a t m e n t b y e l e c t r o p h y s i o l o g i s t s i n t U s r e s p e c t i s n o t
u n i f o r m , b u t a s l o n g a s the s t e a d y s t a t e i s c o n c e r n e d , e i t h e r one m a y
give reasonable a n s w e r s . T h e d i f f e r e n c e b e t w e e n the two m o d e l s
becomes, however, c r u c i a l i n transient states, for instance, i m m e d i -
a t e l y a f t e r the p u m p h a s b e e n t u r n e d o n o r off.
M i t c h e l l ( 8 ) was p r o b a b l y the f i r s t t o i n v e s t i g a t e t h e s e questions»
a n d b y r i g o r o u s c a l c u l a t i o n s , b a s e d on the a s s u m p t i o n o f c o n s t a n t
p u m p i n g r a t e , h e p r e d i c t e d that the e l e c t r i c a l P D r i s e s v e r y f a s t but
i n s i g n i f i c a n t l y h i g h w h i l e the f u l l d e v e l o p m e n t o f the p r o t o n m o t i v e f o r c e
h a s to a w a i t the g e n e r a t i o n o f a s u f f i c i e n t l y h i g h c o n c e n t r a t i o n g r a d i e n t
w h i c h w o u l d t a k e m o r e t h a n 30 s e c o n d s . The same approach, except
f o r the a s s u m p t i o n that the d r i v i n g f o r c e , r a t h e r t h a n the r a t e o f the
p u m p r e m a i n s c o n s t a n t , g a v e a g r e a t l y d i f f e r e n t r e s u l t (10, 11 ).
D e p e n d i n g o n the p u m p i n g r a t e r e l a t i v e to the p a s s i v e i o n m o b i l i t i e s ,
t h e i n i t i a l s u r g e o f the e l e c t r i c a l p o t e n t i a l m a y i n d e e d b e h i g h e n o u g h
to a n t i c i p a t e m o s t o f the final p r o t o n m o t i v e f o r c e a t s t a t i c h e a d
( F i g . 2). O b v i o u s l y , a c o n s t a n t p u m p i n g r a t e i m p l i e s that the d r i v i n g
f o r c e i s i n i t i a l l y l o w but r i s e s a c c o r d i n g l y a s the o p p o s i n g p r o t o n m o t i v e
force grows. B y contrast, at constant d r i v i n g force, the p u m p i n g r a t e
s h o u l d i n i t i a l l y b e v e r y h i g h a n d l a t e r d e c l i n e a c c o r d i n g l y a s the
opposing protonmotive force r i s e s . A p r i o r i , both models a r e i n s o m e
w a y p l a u s i b l e a n d o n l y the e x p e r i m e n t c a n d e c i d e w h i c h one i s the v a l i d
one.
So f a r , e x p e r i m e n t a l t e s t s a r e s c a r c e , p a r t l y due t o the
d i f f i c u l t i e s to m e a s u r e r a p i d l y e n o u g h c h a n g e s i n e l e c t r i c a l m e m b r a n e
p o t e n t i a l (or p r o t o n m o t i v e f o r c e ) w i t h s m a l l c o m p a r t m e n t s s u c h a s
mitochondria and b a c t e r i a . T h e usually applied chemical probes for
p o t e n t i a l c h a n g e s a r e e i t h e r too s l o w o r too n o i s y . Recently, however,
,,ιι
H + -pump M II
on
,,1
Η -pump o f f
II
Figure 2. The calculated response of the protonmotive force, and its electrical and
chemical components, to the sudden initiation or stopping of an electrogenic proton
pump.
Ordinate: protonmotive force (X )
H in relative units. Abscissa: time. Solid line, electric PD;
dashed line, chemical PD of H*; dotted line, X . H Top: constant rate of pumping (constant
current source). Bottom: constant driving force of the pump (constant voltage source). It is
seen that only under the condition of constant driving force is the initial rise of the electrical
PD, and hence of the total protonmotive force, high enough to make pumping power available
almost instantaneously after the start of the pump. It also is seen that the pump is more stable
under constant pumping rate, because after the pump is turned off, the PMF drops almost
instantaneously in the lower curve whereas it declines gradually in the upper curve. Reproduced,
with permission, from Ref. 11. Copyright 1982, Academic Press, Inc.
a m e t h o d that a l l o w s r a p i d m o n i t o r i n g o f s u c h c h a n g e s b a s e d o n a c h a n g e
i n l i g h t a b s o r p t i o n o f the n a t u r a l l y p r e s e n t s u b s t a n c e , bacteriorhodopsm,
h a s b e e n d e v i s e d f o r the l i g h t - d r i v e n p r o t o n p u m p s i n h a l o b i u m (12 ).
B y this m e t h o d , i t c o u l d be shown with envelope v e s i c l e s of this b a c
t e r i u m , that a f t e r i n i t i a t i n g the p u m p b y i l l u m i n a t i o n , the e l e c t r i c a l
p o t e n t i a l (or the p r o t o n m o t i v e f o r c e ) r i s e s w i t h a h a l f t i m e of a b o u t
2 0 m s e c to a v a l u e c l o s e to 2 0 0 m v e v e n t h o u g h the b u f f e r c a p a c i t i e s o f
a l l p e r m e a n t i o n s i n s i d e a n d o u t s i d e was k e p t h i g h e n o u g h to p r e c l u d e
a p p r e c i a b l e c h a n g e s i n i o n d i s t r i b u t i o n d u r i n g that t i m e (13 ). This
r e s u l t c l o s e l y a g r e e s w i t h what h a s b e e n p r e d i c t e d o n the b a s i s of the
c o n s t a n t d r i v i n g f o r c e m o d e l , a n d t h u s c o n f i r m s the h y p o t h e s i s o f the
c r u c i a l r o l e o f the e l e c t r i c a l p o t e n t i a l f o r the e x p e d i t i o u s n e s s o f the
coupling system.
This result also confirm
d i n g to the f o l l o w i n g e q u a t i o n d e r i v e d i n t e r m s o f n o n - e q u i l i b r i u m
thermodynamics (14):
W h e t h e r the'higfr e x p e d i t i o u s n e s s r e s u l t i n g f r o m the e l e c t r o g e n i c
P D r i s e w o u l d b e a n a d v a n t a g e f o r a n y s y s t e m i s h a r d to p r e d i c t . Owing
to the l o w e l e c t r i c m e m b r a n e c a p a c i t y the i o n m o t i v e f o r c e at t h i s s t a g e ,
being predominantly electrogenic i s v e r y sensitive towards changing the
driving force. B y contrast a m o r e slowly developing ionmotive force
g e n e r a t e d b y i o n t r a n s l o c a t i o n i s c e r t a i n l y m o r e s t a b l e a s the energy
s t o r e d i n i t w o u l d s t i l l b e a v a i l a b l e f o r s o m e t i m e a f t e r the p u m p h a s
stopped. If the s y s t e m w e r e t o s u p p l y e n e r g y - r i c h c o m p o u n d s a t a
s t e a d y r a t e i n s p i t e of h i g h l y f l u c t u a t i n g e n e r g y i n p u t , o b v i o u s l y the
s l o w e r but m o r e stable a r r a n g e m e n t would be m o r e suitable. If, o n the
o t h e r h a n d , the s y s t e m w e r e to a d j u s t i t s a c t i v i t y to a r a p i d l y c h a n g i n g
r e q u i r e m e n t at a r a t h e r steady input of e n e r g y , the m o r e expeditious
but l e s s stable a r r a n g e m e n t would be m o r e suitable.
Acknowledgment
T h e s e studies were supported by a U S P H S - N I H grant No. R O I G M
26554-01.
Literature Cited
M . MOO-YOUNG
University of Waterloo, Department of Chemical Engineering,
Waterloo, Ont. N2L 3G1 Canada
H . W. B L A N C H
University of California, Department f Chemical Engineering Berkeley CA 94720
0097-6156/83/0207-0335$06.00/0
© 1983 American Chemical Society
7. V e r y low c o n c e n t r a t i o n s o f r e a c t a n t s a n d / o r p r o d u c t s i n
a c q u e o u s m e d i a a r e n o r m a l l y i n v o l v e d i n b i o r e a c t o r s so
c o n c e n t r a t i o n d r i v i n g f o r c e s f o r mass t r a n s f e r a r e o f t e n
severely limited.
8. M i c r o b i a l growth r a t e s a r e s u b s t a n t i a l l y lower t h a n
c h e m i c a l r e a c t i o n r a t e s so t h a t r e l a t i v e l y l a r g e r e a c t o r
v o l u m e s and r e s i d e n c
A s a n i l l u s t r a t i o n o f some o f t h e p r o b l e m s i m p o s e d by t h e
a b o v e c o n s t r a i n t s * we n o t e t h a t a n a d e q u a t e o x y g e n s u p p l y r a t e t o
growing c e l l s i s o f t e n c r i t i c a l i n a e r o b i c f e r m e n t a t i o n s . Because
o f i t s low s o l u b i l i t y i n w a t e r * g a s e o u s o x y g e n * u s u a l l y i n t h e
f o r m o f a i r * m u s t be s u p p l i e d c o n t i n u o u s l y t o t h e f e r m e n t a t i o n
medium i n s u c h a way t h a t t h e o x y g e n a b s o r p t i o n r a t e a t least
equals the oxygen consumption r a t e of the c e l l s . Even temporary
d e p l e t i o n o f d i s s o l v e d o x y g e n c o u l d mean i r r e v e r s i b l e c e l l damage.
I n t h i s r e s p e c t * i t i s w o r t h n o t i n g t h a t t h e same m i c r o b i a l
s p e c i e s may show l a r g e v a r i a t i o n s i n i t s o x y g e n r e q u i r e m e n t s *
depending on t h e o x y g e n c o n c e n t r a t i o n t o w h i c h t h e y have b e e n
adapted.
P r e v i o u s s t u d i e s i n w h i c h t h e o x y g e n s u p p l y t o a submerged
g r o w i n g m i c r o b i a l c u l t u r e was s t o p p e d * h a v e s h o w n a l i n e a r
decrease i n oxygen c o n c e n t r a t i o n with time over a large
concentration range (Finn* 1954). Below a c e r t a i n oxygen
c o n c e n t r a t i o n * c a l l e d the " c r i t i c a l oxygen tension*** the decrease
f o l l o w e d a h y p e r b o l i c p a t t e r n compatible with Michaelis-Menton
kinetics. O f t e n d e v i a t i o n s f r o m t h e l i n e a r and h y p e r b o l i c o x y g e n
concentration decrease patterns are found. The r a t e c o n t r o l l i n g
s t e p i n a f e r m e n t a t i o n p r o c e s s may s h i f t t h e o x y g e n s u p p l y r a t e
i n t o the b u l k l i q u i d t o t h e demand r a t e i n s i d e the c e l l i f c e l l
a g g r e g a t e s a r e f o r m e d w h i c h a r e l a r g e r t h a n a few h u n d r e d t h s o f
millimeters. U s u a l l y * t h i s i s s e e n as an i n c r e a s e d v a l u e o f t h e
c r i t i c a l oxygen t e n s i o n o r t o t a l a b s e n c e o f a l i n e a r p a r t of the
oxygen c o n c e n t r a t i o n d e c r e a s e c u r v e * s h o w i n g d e p e n d e n c e o n t h e
c o n c e n t r a t i o n d r i v i n g force at the c e l l surface*
RATE-CONTROLLING STEPS
F i g u r e 1 g i v e s a g e n e r a l i z e d p h y s i c a l r e p r e s e n t a t i o n of a
b i o r e a c t o r s u b s y s t e m i n v o l v i n g two o r m o r e p h a s e s * An important
example o f t h i s r e p r e s e n t a t i o n i s an a e r o b i c f e r m e n t a t i o n i n w h i c h
Gas ( 0 , C 0 , C H , e t c . )
2 2 4 Cells
1
Route 1 Reactant Supply and U t i l i z a t i o n
ι
Route 2 P r o d u c t Removal . and Formation
Figure 1. Generalization of biochemical reactor conditions illustrating the importance of aqueous
phase transfer steps for mass and heat. Reproduced, with permission, from Ref. 38.
Copyright 1981, Springer-Verlag.
a m i c r o b e u t i l i z e s o x y g e n ( s u p p l i e d by a i r b u b b l e s w h i c h a l s o
d e s o r b t o x i c c a r b o n d i o x i d e ) and o t h e r d i s s o l v e d n u t r i e n t s
(sugars* e t c . ) t o grow and p r o d u c e s o l u b l e e x t r a c e l l u l a r
metabolites. E i g h t r e s i s t a n c e s i n t h e mass t r a n s f e r p a t h w a y s f o r
t h e n u t r i e n t s s u p p l y and u t i l i z a t i o n and f o r m e t a b o l i t e excretion
and r e m o v a l * a r e p o s s i b l e as i n d i c a t e d i n t h e i l l u s t r a t i o n a t t h e
f o l l o w i n g l o c a t i o n s : (1) i n a g a s - f i l m (2) at the g a s - l i q u i d
i n t e r f a c e (3) i n a l i q u i d - f i l m a t t h e g a s - l i q u i d i n t e r f a c e ( 4 ) i n
b u l k l i q u i d (5) i n a l i q u i d - f i l m s u r r o u n d i n g the s o l i d (6) a t the
l i q u i d - s o l i d i n t e r f a c e ( 7 ) i n t h e s o l i d phase c o n t a i n i n g t h e c e l l s
(8) a t t h e s i t e s o f t h e b i o c h e m i c a l r e a c t i o n s . I t s h o u l d be n o t e d
t h a t a l l t h e pathways e x c e p t t h e l a s t one are purely p h y s i c a l .
I f we c o n s i d e r a f l u i d p a r t i c l e ( g a s o r l i q u i d ) m o v i n g
r e l a t i v e l y t o a c o n t i n u o u s l i q u i d p h a s e , t h e r e a r e two p o s s i b l e
e x t r e m e s o f i n t e r f a c i a l m o v e m e n t as c l a s s i f i e d b e l o w . (For
c o n v e n i e n c e * we w i l l c o n s i d e r a s p h e r e ) .
(a) T h e r e i s no i n t e r n a l c i r c u l a t i o n w i t h i n t h e p a r t i c l e .
These p a r t i c l e s behave e s s e n t i a l l y as i f t h e y a r e s o l i d
with r i g i d surfaces. We w i l l r e f e r t o t h e s e as p a r t i c l e s
with "rigid" surfaces.
I l l u s t r a t i o n s of v e l o c i t y v e c t o r s f o r both k i n d s of these
p a r t i c l e s a r e i l l u s t r a t e d i n F i g u r e 2. T h i s c o n c e p t h a s been
u s e f u l i n e x p l a i n i n g many d r o p and b u b b l e phenomena. F o r e x a m p l e ,
i t has been f o u n d t h a t t r a c e amounts o f s u r f a c e - a c t i v e materials
c a n h i n d e r t h e d e v e l o p m e n t of i n t e r n a l c i r c u l a t i o n by means of a
d i f f e r e n t i a l surface pressure. S m a l l b u b b l e s r i s i n g slowly are
apt t o behave l i k e p a r t i c l e s w i t h r i g i d s u r f a c e s . T h i s phenomenon
can l e a d t o a d e c r e a s e i n k a s t h e age o f a b u b b l e increases
( L e v i c h , 1956). L a r g e r b u f e b l e s , r i s i n g more q u i c k l y * may sweep
t h e i r f r o n t s u r f a c e f r e e of t r a c e i m p u r i t i e s and t h e r e f o r e e s c a p e
the c o n t a m i n a t i o n e f f e c t o f s u r f a c t a n t s as i l l u s t r a t e d i n F i g u r e
3· These e f f e c t s lead to s i g n i f i c a n t v a r i a t i o n s of k with L
In fermentation p r a c t i c e , c l e a n b u b b l e systems a r e
p r o b a b l y r a r e l y a c h i e v e d and i t i s c o n s e r v a t i v e l y s a f e t o b a s e a
d e s i g n on c o n t a m i n a t e d r i g i d i n t e r f a c e b e h a v i o r .
F o r non-moving s u b m e r g e d p a r t i c l e s ( w i t h r i g i d o r m o b i l e
s u r f a c e ) i n a s t a g n a n t médium» mass t r a n s f e r o c c u r s o n l y by r a d i a l
diffusion. Re = Gr = 0
Sh = Nu = 2 (1)
As t h e l o w e r l i m i t f o r S h . we w i l l s e e t h a t t h i s v a l u e u s u a l l y
v a n i s h e s f o r b u b b l e mass t r a n s f e r , b u t i t may become s i g n i f i c a n t
when a p p l i e d t o s m a l l l i g h t p a r t i c l e s , e . g . , m i c r o b i a l c e l l s .
Pseudostagnant l i q u i d environments can exist i n viscous
fermentations and/or w i t h w e l l d i s p e r s e d s i n g l e c e l l s as
i l l u s t r a t e d i n c a s e ( a ) o f F i g u r e 3.
Sh = 0 . 9 9 R e 1 / 3
Sc 1 / 3
= 0.99 P e 1 / 3
(2)
Sh = 0 . 9 5 R e 1 / 2
Sc 1 / 3
(3)
At high a g i t a t i o n i n t e n s i t i e s , t u r b u l e n c e i s expected t o
a f f e c t t h e mass t r a n s f e r r a t e s a t s o l i d p a r t i c l e surfaces.
H o w e v e r , i n t h e s e c a s e s t h e a c t u a l p a r t i c l e v e l o c i t y i s unknown
and c o n v e n t i o n a l R e y n o l d s numbers c a n n o t be d e d u c e d .
An a p r o p r i a t e Re-number e x p r e s s i o n c h a r a c t e r i s t i c o f l o c a l
i s o t r o p i c t u r b u l e n c e c a n be d e r i v e d u s i n g t h e r o o t - m e a n - s q u a r e
f l u c t u a t i n g v e l o c i t y p o s t u l a t e d by B a t c h e l o r ( 1 9 5 1 ) .
α b e
C a l d e r b a n k and M o o - Y o u n g ( 1 9 6 1 ) d e v e l o p e d a c o r r e l a t i o n for
r i g i d - s u r f a c e p a r t i c l e m a s s t r a n s f e r i n b i o r e a c t o r s i n terms of
the e n e r g y i n p u t t o t h e s y s t e m as f o l l o w s :
3 / 4 1 / 3
Sh = 0.13 Re Sc (5)
e
l y
where k i s s e e n t o be
L d e p e n d e n t on ( P / V ) ^ » a dependence w h i c h
may be m a s k e d by t h e e f f e c t o f p o w e r on i n t e r f a c i a l a r e a , as
discussed l a t e r .
Mobile surface f l u i
l e s s s p h e r e - l i k e t h a n t h a t of r i g i d - s u r f a c e p a r t i c l e s .
By v i s c o u s i n t e r a c t i o n w i t h t h e c o n t i n u o u s phase» o s c i l l a t i n g
shape v a r i a t i o n s o f l i q u i d d r o p s and gas b u b b l e s o c c u r * and f o r Re
1» m o b i l e s u r f a c e f l u i d p a r t i c l e s i n f r e e - r i s i n g o r falling
c o n d i t i o n s move i n a w o b b l i n g o r s p i r i a l - l i k e manner, w h i c h has a
marked i n f l u e n c e on mass t r a n s f e r r a t e s . As b e f o r e , we can a r r i v e
at d i f f e r e n t c o r r e l a t i o n s f o r d i f f e r e n t bulk flow regions. These
a r e summarized b e l o w :
1 / 2 1 / 2
Sh = 0.65( £ ) Pe (6)
^d
1 / 2
Sh = 1.13 Pe (7)
I n t h i c k v i c o u s l i q u i d s (μ > 70 cp) l a r g e s p h e r i c a l - c a p
b u b b l e s are f r e q u e n t l y e n c o u n t e r e d and t h e r e l e v a n t correlation
is :
1 / 2
Sh = 1.31 Pe (8)
Non-Newtonian. F l o y Effects
When t h e l i q u i d phase e x h i b i t s n o n - N e w t o n i a n b e h a v i o r , t h e
mass t r a n s f e r c o e f f i c i e n t w i l l c h a n g e due t o a l t e r a t i o n s i n t h e
f l u i d v e l o c i t y p r o f i l e a r o u n d t h e submerged p a r t i c l e s . The t r e n d s
f o r b o t h m a s s t r a n s f e r and d r a g c o e f f i c i e n t a r e a n a l o g o u s . As
b e f o r e f o r N e w t o n i a n f l u i d s , two t y p e s o f i n t e r f a c i a l b e h a v i o r
need t o be c o n s i d e r e d .
F o r r i g i d s u r f a c e b e h a v i o r , t h e mass t r a n s f e r c o e f f i c i e n t s
c a n be o b t a i n e d f r o m t h e r e s u l t s o f W e l l e k a n d H u a n g ( 1 9 7 0 ) .
M o o - Y o u n g a n d H i r o s e ( 1 9 7 2 ) showed t h a t an a d d i t i o n a l e f f e c t o f
" i n t e r f a c i a l s l i p " by a d d i t i v e s can o c c u r i n p r a c t i c e .
In a d d i t i o n t
c o e f f i c i e n t , k » and t h
L
In low v i s c o s i t y l i q u i d s i t i s r e a s o n a b l y w e l l e s t a b l i s h e d
t h a t i n s m a l l s t i r r e d - t a n k s t h e l i q u i d phase can be c o n s i d e r e d t o
be " p e r f e c t l y m i x e d " ( W e s t e r t e r p , e t a l . . 1 9 6 3 ) . Under t h e s e
c o n d i t i o n s , t h e g a s p h a s e h a s a l s o g e n e r a l l y b e e n assumed t o be
w e l l m i x e d i n t a n k s o p e r a t i n g above c r i t i c a l i m p e l l e r s p e e d . In
l a r g e t a n k s , h o w e v e r , t h e s i t u a t i o n i s l e e s c l e a r , and c a r e must
be t a k e n t o e s t a b l i s h t h e b e h a v i o r o f b o t h p h a s e s . I n c a s e s where
the d e g r e e o f gas a b s o r p t i o n i s h i g h , t h e a s s u m p t i o n s o f w e l l
m i x e d o r p l u g - f l o w o f t h e g a s p h a s e may p r e d i c t s i g n i f i c a n t l y
d i f f e r e n t gas a b s o r p t i o n r a t e s . S h a f t l e i n and R u s s e l l ( 1 9 6 8 )
p r e s e n t d e s i g n e q u a t i o n s f o r v a r i o u s m o d e l s o f g a s and liquid
flows.
General Concepts
In some b i o c h e m i c a l s y s t e m s t h e l i m i t i n g m a s s t r a n s f e r
s t e p s h i f t s from a g a s - l i q u i d or s o l i d - l i q u i d i n t e r f a c e (as
discussed e a r l i e r ) to the i n t e r i o r of s o l i d p a r t i c l e s . The most
i m p o r t a n t c a s e s a r e s o l i d s u b s t r a t e s and c e l l a g g r e g a t e s ( s u c h as
m i c r o b i a l f l o e s , c e l l u l a r t i s s u e s , e t c . ) and i m m o b i l i z e d enzymes
(gel-entrapped or s u p p o r t e d i n s o l i d m a t r i c e s ) . In the former,
d i f f u s i o n of o x y g e n ( o r o t h e r n u t r i e n t s ) t h r o u g h t h e p a r t i c l e
l i m i t s m e t a b o l i c r a t e s , w h i l e i n the l a t t e r , s u b s t r a t e r e a c t a n t or
p r o d u c t d i f f u s i o n i n t o o r out o f t h e enzyme c a r r i e r o f t e n l i m i t s
the o v e r a l l g l o b a l b i o r e a c t i o n r a t e s .
M a r s h a l l and A l e x a n d e r (1960) d i s c o v e r e d t h a t f o r s e v e r a l
p e l l e t - f o r m i n g f u n g i a "cube r o o t " g r o w t h curve f i t t h e i r data
s i g n i f i c a n t l y b e t t e r t h a n t h e s t a n d a r d e x p o n e n t i a l growth model.
I t was s u g g e s t e d t h a t t h i s was p r o b a b l y due t o t h e e f f e c t s o f
i n t r a p a r t i c l e d i f f u s i o n ; a n u t r i e n t was n o t d i f f u s i n g i n t o t h e
p a r t i c l e f a s t enough t o m a i n t a i n u n r e s t r i c t e d g r o w t h . I t was s o o n
r e a l i z e d t h a t o x y g e n was t h e l i m i t i n g n u t r i e n t .
P h i l l i p s (1966) m e a s u r e d o x y g e n d i f f u s i o n i n p e l l e t s of
ΡΡΠ i ci 11 i iim chrysogenum by f i r s t a s s u m i n g t h a t d i f f u s i o n i s t h e
mechanism that s u p p l i e
p e l l e t and t h a t t h e mas
comparatively small. Y a n o e t . a l . ( 1 9 6 1 ) and Koyayashi» e t a l .
(1973 b) d i d t h e same w i t h A s p e r g i l l u s n i g e r p e l l e t s . By t a k i n g
i n t o a c c o u n t t h e e f f e c t o f i n t r a p a r t i c l e diffusion» K o b a y a s h i and
S u z u k i ( 1 9 7 6 ) w e r e a b l e t o c h a r a c t e r i z e t h e k i n e t i c b e h a v i o r of
t h e e n z y m e g a l a c t o s i d a s e w i t h i n m o l d p e l l e t s o f HûJLLiexfillâ.
vinacea.
K o b a y a s h i , e t a l . ( 1 9 7 3 b) s t u d i e d t h r e e c a s e s : (1)
u n i f o r m r e s p i r a t i o n a c t i v i t y t h r o u g h o u t m y c e l i a l p e l l e t s * (2)
r e s p i r a t i o n a c t i v i t y as a f u n c t i o n of age d i s t r i b u t i o n w i t h i n t h e
p e l l e t * (3) r e s p i r a t i v e a c t i v i t y a d a p t a t i o n to the l o c a l oxygen
c o n c e n t r a t i o n w i t h i n the p e l l e t . I n c a s e 1» i t was f o u n d t h a t t h e
e f f e c t i v e n e s s f a c t o r ( E ) i s e q u a l t o t h e r a t i o of the s p e c i f i c
f
r e s p i r a t i o n r a t e of a p e l l e t to the r e s p i r a t i o n r a t e of w e l l
dispersed filamentous mycelia. The t h e o r e t i c a l and e x p e r i m e n t a l
r e s u l t s f o r e a c h c a s e a r e g i v e n i n F i g u r e 4. I t i s seen t h a t the
t h r e e c a s e s c o n s i d e r e d gave s i m i l a r r e s u l t s and i t i s d i f f i c u l t t o
d i s c r i m i n a t e b e t w e e n them w i t h the l i m i t e d e x p e r i m e n t a l d a t a
available.
I n t r a p a r t i c l e d i f f u s i o n c a n h a v e a s i g n i f i c a n t e f f e c t on
t h e k i n e t i c b e h a v i o r of enzymes i m m o b i l i z e d o n s o l i d c a r r i e r s o r
entrapped i n gels. In t h e i r b a s i c a n a l y s i s of t h i s p r o b l e m .
M o o - Y o u n g and K o b a y a s h i ( 1 9 7 2 ) d e r i v e d a g e n e r a l m o d u l u s and
effectiveness factor. The r e s u l t s a l s o p r e d i c t e d possible
m u l t i p l e s t e a d y - s t a t e s as w e l l as u n s t a b l e s i t u a t i o n s f o r c e r t a i n
s y s t e m s . W h i l e t h e s e r e s u l t s a r e v e r y i n t e r e s t i n g i t s h o u l d be
r e m e m b e r e d t h a t t h e y a r e p r i m a r i l y m a t h e m a t i c a l and a w a i t
extensive experimental support data.
When t h e s u b s t r a t e i n a b i o r e a c t o r i s a w a t e r - s o l u b l e
material (e.g., c e l l u l o s e , o i l s ) , the e f f e c t s of i n t r a p a r t i c l e
m a s s t r a n s f e r may be i m p o r t a n t . In such systems, e x t r a c e l l u l a r
enzymes c a n b r e a k down s u b s t r a t e e v e n t u a l l y i n t o w a t e r s o l u b l e
c o m p o n e n t s , as p r o d u c t s , o r as i n t e r m e d i a t e s f o r c o n s u m p t i o n by
m i c r o - o r g a n i s m s ( O k a z a k i and Moo-Young, 1 9 7 8 ) .
I f a s o l i d s u b s t r a t e i s s u f f i c i e n t l y p o r o u s , t h e enzyme
can d i f f u s e i n t o i t and d e g r a d a t i o n can proceed i n s i d e the
m a t e r i a l . The w a t e r - s o l u b l e s u b s t r a t e f r a g m e n t s must a l s o d i f f u s e
out o f t h e s o l i d m a t r i x t h r o u g h t h e s e same p o r e s i n t o t h e b u l k
s o l u t i o n w h e r e t h e y may be s u b j e c t t o f u r t h e r e n z y m a t i c a t t a c k .
The r e a c t i o n may c o n c u r r e n t l y p r o c e e d a t t h e e x t e r i o r o f t h e
m a t r i x s u r f a c e and f o r
much o f t h e d e g r a d a t i o n
t h e e f f e c t i v e n e s s f a c t o r and g e n e r a l m o d u l u s i s c o n v e n i e n t i n
s o l v i n g the r e l e v a n t d i f f e r e n t i a l e q u a t i o n s .
Bubble-Columns
P n e u m a t i c a l l y a g i t a t e d g a s - l i q u i d r e a c t o r s may show w i d e
v a r i a t i o n s i n height to d i a m e t e r r a t i o s . In the p r o d u c t i o n of
baker's y e a s t , a tank-type c o n f i g u r a t i o n w i t h a r a t i o of 3 to 1 i s
common i n d u s t r i a l l y . Tower t y p e s y s t e m s have h e i g h t t o d i a m e t e r
r a t i o s of 6 to 1 or more. As w o u l d be e x p e c t e d , t h e b e h a v i o r o f
b o t h gas and l i q u i d p h a s e s may be q u i t e d i f f e r e n t i n t h e s e c a s e s .
I n g e n e r a l t h e gas phase r i s e s through the l i q u i d phase i n
p l u g - f l o w , under the a c t i o n of g r a v i t y , i n both types of system.
A v a r i e t y o f c o r r e l a t i o n s f o r k.a a r e a v a i l a b l e i n t h e
l i t e r a t u r e f o r b u b b l e - c o l u m n s . G e n e r a l l y ^ h e y a r e of t h e f o r m
n
k a
L = const. V (9)
where η i s u s u a l l y i n t h e r a n g e o f 0 . 9 t o u n i t y . Y o s h i d a and
A k i t a ( 1 9 6 5 ) c o r r e l a t e t h e o v e r a l l mass t r a n s f e r c o e f f i c i e n t s i n
b u b b l e columns i n t h e f o r m :
k
i °
a 2
„ 1/2 rri 0.62 _3 2 0.31
- V = o
- 6
^ 1
Φ - 1 ( 1 0 )
A r e c e n t r e v i e w o f b u b b l e - c o l u m n s by S c h u g e r l . e t a l .
( 1 9 7 7 . 1978) examines s i n g l e and m u l t i s t a g e columns and a v a r i e t y
of l i q u i d phases. A c o r r e l a t i o n f o r k a i s proposed f o r porous
T
and p e r f o r a t e d p l a t e s p a r g e r s (cgs u n i t s ) :
1 , 5 8
k.a = 0.0023 (V / d ) R (11)
L S D
Many o f t h e a v a i l a b l e c o r r e l a t i o n s f o r k a h a v e b e e n
L
o b t a i n e d on s m a l l s c a l e e q u i p m e n t , and have n o t t a k e n c o g n i z a n c e
o f t h e u n d e r l y i n g l i q u i d hydrodynamics. B h a v a r a j u , e t a l . (1978)
propose a design procedure b a s e d on t h e d i f f e r e n c e i n bubble s i z e
c l o s e t o t h e o r i f i c e and i n t h e l i q u i d b u l k . P r o v i d e d t h e l i q u i d
i s t u r b u l e n t * t h e e q u i l i b r i u m b u b b l e s i z e i n t h e b u l k w i l l be
independent of t h e s i z e a t f o r m a t i o n . The h e i g h t o f t h e r e g i o n
around t h e o r i f i c e where t h e bubble f o r m a t i o n p r o c e s s o c c u r s i s a
f u n c t i o n o f s p a r g e r g e o m e t r y and gas f l o w r a t e and i n l a b o r a t o r y
s c a l e e q u i p m e n t t h i s h e i g h t may be s i g n i f i c a n t f r a c t i o n o f t h e
t o t a l l i q u i d h e i g h t (up t o 3 0 % ) . I n p l a n t s c a l e e q u i p m e n t *
h o w e v e r , t h i s g e n e r a l l y r e p r e s e n t s l e s s t h a n 5% o f t h e t o t a l
l i q u i d height. Thus c o r r e l a t i o n s d e v e l o p e d on s m a l l s c a l e
a p p a r a t u s need t o be r e v i e w e
a r e a w i t h column h e i g h t
Systems w i t h S t a t i o n a r y Intpmak
A c o n s i d e r a b l e l i t e r a t u r e e x i s t s on d r a f t - t u b e columns*
w h e r e l i q u i d i s c i r c u l a t e d due t o a b u l k d e n s i t y d i f f e r e n c e
b e t w e e n t h e i n n e r c o r e a n d t h e s u r r o u n d i n g a n n u l a r s p a c e . The
downcoming l i q u i d i n t h e a n n u l a r s p a c e e n t r a i n s a i r b u b b l e s * and
t h u s h o l d u p i n t h e c e n t r a l c o r e and a n n u l a r r e g i o n w i l l be
different. S e v e r a l r e p o r t s o n s m a l l s c a l e a i r l i f t columns as
b i o r e a c t o r s have a p p e a r e d . C h a k r a v a r t y . e t a l . (1973) examined
gas h o l d u p a t v a r i o u s p o s i t i o n s i n a 10 cm d i a m e t e r c o l u m n . A
r e c t a g u l a r a i r l i f t h a s b e e n r e p o t e d ( G a s n e r , 1974) and a i r l i f t s
w i t h e x t e r n a l r e c i r c u l a t i o n ( K a n a z a w a . 1 9 7 5 ) have been p r o p o s e d .
The o v e r a l l mass t r a n s f e r c o r r e l a t i o n s f o r a i r l i f t d e v i c e s a r e
u s u a l l y expressed a s :
k. a = const. V (12)
L· S
I n d u s t r i a l l y , p l a n t s c a l e a i r l i f t d e v i c e s h a v e b e e n u s e d by
J a p a n e s e , B r i t i s h , F r e n c h a n d USA c o m p a n i e s m a i n l y f o r SCP
p r o d u c t i o n and w a s t e t r e a t m e n t
S t a t i c m i x i n g e l e m e n t s have been i n c o r p o r a t e d i n t o a i r l i f t
devices with the o b j e c t i v e s o f p r o v i d i n g a d d i t i o n a l m i x i n g and
h e n c e g r e a t e r mass t r a n s f e r c a p a b i l i t i e s . S t a t i c mixers are
b e c o m i n g i n c r e a s i n g l y m o r e common i n o x i d a t i o n ponds f o r
b i o l o g i c a l wastewater t r e a t m e n t . H e r e , f i n e b u b b l e s may be
produced as t h e g a s - l i q u i d m i x t u r e passes through t h e m i x i n g
element. These a r e u s u a l l y 18-24 i n c h e s i n d i a m e t e r , and a r e
placed over sparger pipes. A f a i r l y intense l i q u i d c i r c u l a t i o n
c a n be d e v e l o p e d b y s u c h m i x e r s d u e t o e n t r a i n m e n t b y t h e
g a s - l i q u i d j e t r i s i n g from the m i x i n g element.
Non-Viscous Systems.
The p r e v i o u s s e c t i o n s d e a l t w i t h g a s - l i q u i d c o n t a c t i n g
w i t h o u t m e c h a n i c a l a g i t a t i o n i n such d e v i c e s a s b u b b l e - c o l u m n s a n d
a i r l i f t towers. To o b t a i n b e t t e r g a s - l i q u i d contacting,
mechanical a g i t a t i o n i s o f t e n r e q u i r e d . The d i s c u s s i o n i s
confined to baffled sparge
A e r a t i o n by s u r f a c t i o
wastewater treatment f a c i l i t i e s ( Z l o k a r n i k , 1978).
For p a r t i c u l a t e s , such as c e l l s , i n s o l u b l e s u b s t r a t e s , o r
i m m o b i l i z e d - e n z y m e s , t h e i n t e r f a c i a l a r e a c a n be d e t e r m i n e d f r o m
d i r e c t a n a l y s e s s u c h as by m i c r o s c o p i c e x a m i n a t i o n . F o r gas
b u b b l e s and l i q u i d d r o p s , a c a n be e v a l u a t e d f r o m s e m i - e m p i r i c a l
correlations. Two c a s e s a r e i d e n t i f i e d :
4
Ρ "°· 0 5
a = 0.55 φ V s- 0 5
and
-0.17 0 2 7 ,
U , Z /
d n = 0.27 C l ) V + 9 χ 10 *
Β γ s
0.7 0.3
a = 0.15 (I) V
and
B = °· Ψ 89
V
1 7
d Ρ "°· 0 17
3
I n t h e e q u a t i o n , (P/V) i s i n W a t t s / m , and V g i s i n m/s.
American Chemical
Society Library
1155 16th St., N.W.
In Foundations of Biochemical Engineering; Blanch, H., et al.;
Washington,
ACS Symposium Series; D.C. Society:
American Chemical 20036Washington, DC, 1983.
348 BIOCHEMICAL ENGINEERING
A v e r y h i g h gas f l o w r a t e » l i q u i d b l o w - o u t f r o m t h e v e s s e l
may o c c u r * In a d d i t i o n , t h e above equations are applicable
p r o v i d e d t h a t t h e i m p e l l e r i s n o t f l o o d e d by t o o h i g h a g a s flow
r a t e and t h e r e i s no g r o s s s u r f a c e - a e r a t i o n due t o g a s b a c k - m i x i n g
at the surface of the l i q u i d . Equations are available to
c a l c u l a t e these c r i t e r i a (Calderbank, 1959; W e s t e r t e r p » 1963)·
m
ρ
k a L = const. (^) V g
n
(17)
I n r e c e n t y e a r s , e x t e n s i v e s t u d i e s have b e e n c a r r i e d o u t
u s i n g p h y s i c a l r a t h e r than c h e m i c a l r e a c t i o n measurements f o r t h e
evaluation of k . a On t h i s b a s i s . S m i t h V a n t R i e t and M i d d l e t o n
f
(197 7) f o u n d t n a t i n g e n e r a l , t h e f o l l o w i n g c o r r e l a t i o n s a p p l y t o
a w i d e v a r i e t y o f a g i t a t o r t y p e s , s i z e s and D/T r a t i o s :
k a = 0.01 ^)
L ν β
0
· 4
(18)
Ρ ° · 4 7 5
0 4
l ^ a = 0 . 0 2 (|) ν β
ϋ
·* (19)
-1 3
Both e q u a t i o n s a r e i n SI u n i t s k . a i n s » (P/V) i n W/m · V in g
i n p u t s * t h e m a g n i t u d e o f k ^ a o b t a i n e d i s a b o u t t h e same w h e t h e r
m i x i n g i s done m e c h a n i c a l l y i n s t i r r e d - t a n k s o r p n e u m a t i c a l l y i n
bubble-columns or a i r l i f t d e v i c e s . However, only mechanically
a g i t a t e d systems are capable of a t t a i n i n g h i g h v a l u e s of k a L
r q u i r e d ( e . g . * i n some a n t i b i o t i c f e r m e n t a t i o n s and t h e a c t i v a t e d
s l u d g e method o f t r e a t i n g w a s t e w a t e r ) . Non-mechanically a g i t a t e d
s y s t e m s would r e s u l t i n l i q u i d blowout b e f o r e r e a c h i n g these h i g h
aeration rates.
Viscous Systems
(b) p o l y s a c c h a r i d e f e r m e n t a t i o n s w h e r e t h e v i s c o s i t y i s due t o
polymers i n the continuous aqueous p h a s e r e s u l t i n g i n an
e s s e n t i a l l y homogeneous* v i s c o u s l i q u i d .
The f i r s t t y p e o f f e r m e n t a t i o n b r o t h c a n be s i m u l a t e d b y
m a t e r i a l s u c h as p a p e r p u l p * w h i c h has a m a c r o s c o p i c s t r u c t u r e
analogous t o f u n g a l h y p h a e * s u s p e n d e d i n w a t e r . The second t y p e
may be s i m u l a t e d by aqueous p o l y m e r s o l u t i o n s o f known p r o p e r t i e s .
D L
A
<pD >
L
(
σ > (
M a > V
A d d i t i o n a l d i m i n s i o n l e s s g r o u p s be i n c o r p o r a t e d t o a c c o u n t f o r
geometric v a r i a b l e s * e . g . H / T » D/T. L T h e a b o v e e q u a t i o n c a n be
a l t e r e d t o r e l a t e k a t o t h e s p e c i f i c power i n p u t .
L
L o u c a i d e s and McManamey ( 1 9 7 3 ) e x a m i n e d r a t e s i n p a p e r
pulp supensione s i m u l a t i n g filamentous fermentation media. Tank
g e o m e t r i e s and i m p e l l e r g e o m e t r i e s were b o t h v a r i e d ; vessel
v a r i a t i o n s i n tank diameter*
k a = C j (T/H ) Ν D ζ-Τ .5
L L
(21)
NOMENCLATURE
a s p e c i f i c i n t e r f a c i a l area (based on d i s p e r s i o n
volume)
D D i l u t i o n r a t e ; i m p e l l e r diameter
L i q u i d phase d i f f u s i v i t y
Bubble diameter
Effectiveness factor
L i q u i d height i n r e a c t o r
L i q u i d phase mass t r a n s f e r c o e f f i c i e n t
C o n t i n u e d on n e x t page
m An e x p o n e n t
Ν Speed o f agitator
V Volume o f r e a c t o r c o n t e n t s
V Superficial gas v e l o c i t y
6
VVM Volume
&£££k L e t t e r s
Ρ Viscosity ( d y n a m i c ) o f c o n t i n u o u s phase
P
a Apparent v i s c o s i t y (dynamic)
ν K i n e m a t i c v i s c o s i t y o f c o n t i n u o u s phase
Ρ D e n s i t y o f c o n t i n u o u s phase
Φ Holdup o f d i s p e r s e d phase.
A b b r e v i a t i o n s f o r D i m e n s i o n l e s s Groups
Gr G r a s h o f number f o r mass t r a n s f e r ( b a s e d o n
p a r t i c l e environment d e n s i t y d i f f e r e n c e )
Nu N u s s e l t number
Pe P e c l e t number ( s i n g l e particles)
Pr P r a n d t l number
Re R e y n o l d s number f o r p a r t i c l e s
Re R e y n o l d s numbers f o r i s o t r o p i c turbulence
e
Sh Sherwood number
Se S c h m i d t number
Literature Cited
14. Hamer, G., Blakebrough, Ν., J . Appl. Chem., 1963, 13, 517
15. Higbie, R., Trans. Amer. Inst. Chem. Engrs., 1935, 31, 365.
16. Hirose, T., Moo-Young, M., Can. J . Chem. Eng., 1969, 47,
265.
17. Kanazawa, M., in "SCP II," Tannenbaum and Wang (Eds.), MIT
Press, 1975.
18. Kobayashi, H., Suzuki, M., Biotechnol. Bioeng., 1976, 18, 37.
22. Loucaides, R., McManamey, W.J., Chem. Eng. Sci., 1973, 28,
2165.
28. Schugerl, K., Oels, U., Lucke, J., Adv. Biochem. Eng.,
1977, 7, 1.
29. Schugerl, Κ., Lucke, J., Lehman, J., Wagner, F., Adv.
Biochem. Eng., 1978, 8, 63.
33. Wellek, R.M., Huang, C.C., Ind. Eng, Chem. Fund., 1970,
2 , 480.
35. Yano, T., Kodama, T., Yamada, K., Agr. Biol. Chem., 1961,
25, 589.
ALES PROKOP
Kuwait Institute for Scientific Research, Biotechnology Department,
P.O. Box 24885, Kuwait
On l e a v e f r o m : I n s t i t u t e o f M i c r o b i o l o g y , C z e c h o s l o v a k Academy
K I S R 620 o f S c i e n c e s , 142 20 P r a g u e 4, C z e c h o s l o v a k i a
0097-6156/83/0207-0355$06.50/0
© 1983 American Chemical Society
Table I S t r u c t u r a l and F u n c t i o n a l H i e r a r c h i e s
of a B i o t e c h n o l o g i c a l Process
I n t e r n a t . J . G e n e r a l Systems 1982, j $ ( l ) , T a b l e V I I ( 1 )
Molecular Level
1.1. C a t a b o l i c and a n a b o l i c m a n i f e s t a t i o n s o f l i v i n g systems
1.1.1. E n e r g y f o r m a t i o n
1.1.2. S y n t h e t i c p r o c e s s e s
1.1.3. M a c r o m o l e c u l a r d e c a y
1.2. C o n t r o l mechanisms
1.2.1. R e a c t i o n mechanisms
1.2.2. E p i g e n e t i
1.2.3. B i o c h e m i c a
1.2.4. R e p r o d u c t i o n mechanisms
S t r u c t u r a l H i e r a r c h y o f C e l l s (Compartments)
2.1. T r a n s l o c a t i o n o f s u b s t a n c e s
2.2. Complex b i o c h e m i c a l n e t w o r k
2.3. M o r p h o l o g i c a l d i f f e r e n t i a t i o n
2.4. C e l l c y c l e
Population
3.1. D i s p e r s e d growth
3.1.1. P u r e c u l t u r e
3.1.2. M i x e d c u l t u r e
3.2. A g g r e g a t e d g r o w t h
B i o r e a c t o r and O t h e r E q u i p m e n t
4.1. M a c r o - and m i c r o - m i x i n g o f e q u i p m e n t c o n t e n t s
4.2. Mass t r a n s f e r
4.3. Heat e x c h a n g e
4.4. B i o r e a c t o r c o n t r o l and o f o t h e r e q u i p m e n t
4.5. S c a l e - u p
Technological Line
5.1. B a t c h p r o c e s s e s
5.2. C o n t i n u o u s p r o c e s s e s
l e v e l ( m o l e c u l a r ) i n T a b l e I I i s c o n s i d e r e d quasihomogeneous
( r e a c t i o n s o c c u r r i n g i n a s o l u t i o n ) , the second i s represented by
a c e l l c o n s i d e r e d a s a two-phase s y s t e m , composed o f o r g a n e l l e s
h o m o g e n e o u s l y d i s p e r s e d i n t h e c y t o p l a s m . The p o p u l a t i o n l e v e l i s
m a n i f e s t e d b y d i f f e r e n t m o r p h o l o g i c a l s t a t e s ( d i s p e r s e d and a g g r e
g a t e d ) o f a p o p u l a t i o n . The l e v e l s o f t h e r e a c t o r and t e c h n o l o g i
c a l l i n e a r e d i s t i n g u i s h e d by fundamental f u n c t i o n s t y p i c a l f o r
chemico-technological processes.
The r e a c t o r l e v e l i s d i s t i n g u i s h e d b y t h e f o l l o w i n g f u n d a
m e n t a l e v e n t s and f u n c t i o n s : m a c r o - and m i c r o - m i x i n g o f r e a c t o r
( e q u i p m e n t ) c o n t e n t s and t h e c o r r e s p o n d i n g mass t r a n s f e r a n d h e a t
e x c h a n g e i n v o l v e t h e d e t e r m i n a t i o n o f e l e m e n t a r y zones o f r e a c t o r ,
mass t r a n s f e r and h e a t e x c h a n g e b e t w e e n them and a l s o b e t w e e n
i n d i v i d u a l phases ( l i q u i d , gaseous, s o l i d ) ; b i o r e a c t o r c o n t r o l
and s c a l e - u p ( 2 ) . I f n o n - h o m o g e n e i t i e s o f r e a c t o r p e r f o r m a n c e i n
s p a c e and t i m e a r e e x p e c t e d
and momentum t r a n s p o r t
As a s o l u t i o n t o momentum t r a n s p o r t ( N a v i e r - S t o k e s e q u a t i o n ) i s
u s u a l l y n o t a v a i l a b l e f o r b i o r e a c t o r v e l o c i t y f i e l d , we a r e l e f t
w i t h mass and h e a t b a l a n c e s . A l o c a l b a l a n c e d e s c r i b i n g m i x i n g ,
f l o w and i n t e r f a c e t r a n s f e r , i n c l u d i n g r e a c t i o n i s known a s t h e
d i s p e r s e d model ( 3 ) :
— — + Vc.. u + V D V C . + r..
at ij ij ij
-k-id«) \ \ ( c
iks - °ik > • ° ( i )
Hydrodynamics
E l e m e n t a r y r e g i o n s o f b i o r e a c t o r . A more s i m p l e d e s c r i p t i o n
of the flow c h a r a c t e r i s t i c s o f a r e a c t o r than that by a l o c a l
mass b a l a n c e i s done b y i n t r o d u c i n g t h e r e s i d e n c e t i m e d i s t r i b u
t i o n c o n c e p t (RTD), w h i c h p r o v i d e s m a c r o - m i x i n g c h a r a c t e r i s t i c s
(determination of the elementary regions o f the r e a c t o r ) . I n
f a c t , u s i n g t h e r e s i d e n c e t i m e c o n c e p t a p a r t i c u l a r s i t u a t i o n may
be d e s c r i b e d b y m i x e d - t y p e m o d e l s c o n s i d e r i n g " t h e r e a c t o r a s
c o n s i s t i n g o f i n t e r c o n n e c t e d f l o w s b e t w e e n and a r o u n d t h e s e r e
g i o n s " ( 6 ) . The f o l l o w i n g k i n d s o f e l e m e n t a r y r e g i o n s a r e u s e d i n
t h e c o n s t r u c t i o n o f m i x e d m o d e l s : two t y p e s o f i d e a l z o n e s ( p l u g -
f l o w and p e r f e c t l y s t i r r e d = b a c k m i x e d ) , d i s p e r s e d p l u g f l o w and
deadwater. I n a d d i t i o n t o these r e g i o n s , the f o l l o w i n g k i n d s o f
f l o w may be u s e d : b y - p a s s , r e c y c l e and c r o s j s - f l o w . The above n o n -
i d e a l i t i e s i n m i x i n g may be d e s c r i b e d e i t h e r b y means o f d i s p e r
sed o r d i s c r e t e ( s t a g e - w i s e ) models, t h e l a t t e r c o n s i d e r i n g a
s e r i e s o f i d e a l l y mixed v e s s e l s .
Mass T r a n s f e r
Mass t r a n s f e r i n v o l v e s e s t a b l i s h i n g a t r a n s f e r b e t w e e n t h e
e l e m e n t a r y r e g i o n s o f t h e r e a c t o r and b e t w e e n i n d i v i d u a l p h a s e s
( i n t e r f a c i a l mass t r a n s f e r c o e f f i c i e n t s : gas phase mass t r a n s f e r ,
l i q u i d p h a s e mass t r a n s f e r , mass t r a n s f e r w i t h r e a c t i o n , l i q u i d -
s o l i d mass t r a n s f e r ) , a s w e l l a s o t h e r e l e m e n t a r y phenomena and
p r o c e s s e s c o n n e c t e d w i t h mass t r a n s f e r : g a s p h a s e phenomena and
p r o c e s s e s ( g a s h o l d - u p , b u b b l e s i z e , i n t e r f a c i a l a r e a and b u b b l e
c o a l e s c e n c e / r e d i s p e r s i o n ) , v o l u m e t r i c mass t r a n s f e r and power
c o n s u m p t i o n d u r i n g mass t r a n s f e r ( 2 ) .
I n t e r f a c i a l mass t r a n s f e r c o e f f i c i e n t s . T h e o r e t i c a l and
e x p e r i m e n t a l i n v e s t i g a t i o n s o f many a u t h o r s h a v e shown t h a t d u r i n g
t h e a b s o r p t i o n o f s p a r i n g l y s o l u b l e g a s e s s u c h as o x y g e n , t h e gas
phase c o e f f i c i e n t ( k ) i s s e v e r a l o r d e r s i n m a g n i t u d e h i g h e r t h a n
ç
t h e l i q u i d phase mass t r a n s f e r c o e f f i c i e n t ( k _ ) . B e c a u s e o f t h e
above f a c t and a l s o due t o t h e h i g h v a l u e o f H e n r y ' s Law c o n s t a n t
f o r o x y g e n , t h e l i q u i d p h a s e mass t r a n s f e r c o e f f i c i e n t d e t e r m i n e s
t h e o v e r a l l r a t e o f mass t r a n s f e r . I n d e a l i n g w i t h i n t e r f a c i a l
phenomena, two e x t r e m e c a s e s a r e e n c o u n t e r e d : mass t r a n s f e r by
m o l e c u l a r d i f f u s i o n and c o n v e c t i o n f r o m r i g i d i m m o b i l e s p h e r e s ,
r e p r e s e n t e d by gas b u b b l e s w i t h c o n t a m i n a t e d s u r f a c e s and d e s c r i
bed by t h e F r B s s l i n g e q u a t i o n ( e . g . , i n ( 1 2 ) ) , and mass t r a n s f e r
by c o n v e c t i o n f r o m m o b i l e s p h e r e s , d e s c r i b e d by t h e B o u s s i n e s q
e q u a t i o n (e.g., i n ( 1 3 ) ) . In p r a c t i c e , the l a t t e r system i s r a r e
l y a c h i e v e d s i n c e f e r m e n t a t i o n media w i t h e l e c t r o l y t e s p r o t e i n s
p r o d u c t s o f m e t a b o l i s m an
m a t e r i a l may h i n d e r t h
gas b u b b l e s . V a r i o u s t h e o r e t i c a l and e x p e r i m e n t a l c o r r e l a t i o n s
f o r p r e d i c t i n g k^ f o r b u b b l e s w i t h i m m o b i l e i n t e r f a c e s h a v e b e e n
p r e s e n t e d i n r e v i e w s ( 1 4 , 15) i n v o l v i n g s i t u a t i o n s w i t h c r e e p i n g
f l o w ( t y p i c a l f o r a i r - l i f t r e a c t o r s ) and l o w - h i g h Re numbers, and
a l s o e f f e c t s o f n o n - N e w t o n i a n f l u i d on mass t r a n s f e r f r o m m o v i n g
b u b b l e s . These c o r r e l a t i o n s a r e o f i m p o r t a n c e i n p o l y s a c c h a r i d e
and a n t i b i o t i c p r o d u c t i o n s . The e f f e c t o f s u r f a c e - a c t i v e compounds
on k^ and a i s d i f f e r e n t i a l b e c a u s e o f t h e i r d i f f e r e n t i n f l u e n c e
on s u r f a c e t e n s i o n ( i n c r e a s e o r d e c r e a s e ) . T h e s e phenomena h a v e
not y e t been i n c o r p o r a t e d i n t o a s u f f i c i e n t l y g e n e r a l t h e o r y .
Mass t r a n s f e r w i t h r e a c t i o n . The p r e c e d i n g s i t u a t i o n i n v o l
v e d o n l y a p h y s i c a l mass t r a n s f e r b e t w e e n g a s - l i q u i d o r s o l i d - l i
q u i d i n t e r f a c e s . In the presence of microorganisms, oxygen uptake
by r e s p i r i n g c e l l s w i t h i n t h e r e l a t i v e l y s t a g n a n t l i q u i d r e g i o n
a d j a c e n t t o b u b b l e s submersed i n a c e l l s u s p e n s i o n may l e a d t o
e n h a n c e d o x y g e n t r a n s f e r r a t e s . U s i n g an enhancement c o n c e p t o f
A s t a r i t a , B l a n c h (16) p r e s e n t e d r u l e s f o r i t s p r e d i c t i o n : t h e
d i f f u s i o n t i m e f o r t h e o x y g e n o f gas b u b b l e s a s c e n d i n g t h r o u g h a
l i q u i d , e s t i m a t e d f r o m b u b b l e d i a m e t e r and a s c e n d i n g a i r v e l o c i t y ,
dg / u R , s h o u l d be g r e a t e r o r a t l e a s t o f t h e same o r d e r as t h e
r e a c t i o n t i m e , e v a l u a t e d from d i s s o l v e d oxygen c o n c e n t r a t i o n ,
o x y g e n u p t a k e r a t e and b i o m a s s d e n s i t y , C ^ / ( Q Q X ) . S o b o t k a e t a l .
(17) h a v e shown t h a t t h e c e l l f l o t a t i o n 2 mechanism c a n be
s u c c e s s f u l l y u t i l i z e d to obtain a b e t t e r f i t f o r experimental
growth d a t a , c o n s i d e r i n g g a s - s o l i d ( c e l l s ) a b s o r p t i o n of oxygen
( i t may amount up t o a h a l f o f t o t a l a b s o r p t i o n ) i n a d d i t i o n t o
the p r e v a i l i n g t y p i c a l oxygen path t o c e l l s v i a d i s s o l v e d oxygen
( F i g u r e 1 ) . C o n c l u s i o n of t h i s work, p r o v i d i n g enhanced a b s o r p t i
on ( r e d u c e d b u b b l e s l i p v e l o c i t y due t o e l e c t r o l y t e , a l c o h o l s and
o t h e r s u r f a c e - a c t i v e compounds; o x y g e n l i m i t a t i o n o f g r o w t h ; h i g h
r e s p i r a t i o n r a t e ; h i g h c e l l d e n s i t y a t the i n t e r f a c e ) are i n f u l l
a c c o r d w i t h B l a n c h ' s a r g u m e n t a t i o n . Enhanced a b s o r p t i o n i s , i n
f a c t , o n l y a p p a r e n t , b e c a u s e o f t h e p a r a l l e l pathway o f o x y g e n
utilization.
I n t r a p a r t i c l e mass t r a n s f e r . F o r some p r a c t i c a l s i t u a t i o n s
mass t r a n s f e r l i m i t i n g s t e p i s l o c a l i z e d i n t h e i n t e r i o r o f a s o
l i d phase. T h i s i s t h e case f o r c e r t a i n m y c e l i a l f e r m e n t a t i o n s
where t h e o x y g e n t r a n s f e r v i a p e l l e t o r m y c e l i a l clump i n t e r i o r
may l i m i t g r o w t h o r p r o d u c t i o n p r o c e s s e s . T h i s s i t u a t i o n , e m p l o y
i n g t h e e f f e c t i v e n e s s c o n c e p t , i s r e v i e w e d b y Moo-Young and
B l a n c h ( 1 4 ) . I n t h e f o l l o w i n g , some o t h e r e l e m e n t a r y phenomena
and p r o c e s s e s c o n n e c t e d w i t h mass t r a n s f e r a r e r e v i e w e d .
p r e s e n t s a p r o b l e m . The same a p p l i e s t o a n a s s e s s m e n t o f c o a l e s -
c e n c e / r e d i s p e r s i o n r a t e s and mechanisms a l t h o u g h some methods f o r
c o a l e s c e n c e f r e q u e n c y measurement h a v e b e e n s u g g e s t e d ( 1 9 , 2 0 ) .
Some f u r t h e r t h e o r e t i c a l c o n s i d e r a t i o n s and c o r r e l a t i o n s f o r p r e
d i c t i n g b u b b l e s i z e , gas h o l d - u p a n d i n t e r f a c i a l a r e a were
r e v i e w e d ( 1 6 , 1 5 ) . Gas h o l d - u p and p r e s s u r e d r o p a r e o f i m p o r t a n
ce i n a r a t i o n a l d e s i g n o f a i r - l i f t r e a c t o r s ( 2 1 ) .
1 5 0 6 2 3 1
Sh ( a D c ) = 0.6 e j ' , c°' . Bo '
S . Ga°' (2)
c o n t a i n i n g d i m e n s i o n l e s s Sherwood, S c h m i d t , B o d e n s t e i n and G r a s -
h o f numbers, i n t e r f a c i a l a r e a a , b u b b l e c o l u m n d i a m e t e r D and
gas h o l d - u p ε . A s i m i l a r e q u a t i o n may be a p p l i c a b l e t o b u b b l e
c o l u m n s p r o v i d e d w i t h t h e two-phase n o z z l e ( e j e c t o r ) , a s i n d i c a
t e d f r o m k ^ a v s . ε^ dependence ( F i g u r e 2 ) . Two-phase n o z z l e s and
m u l t i - o r i f i c e spargers a r e s u i t e d f o r i n d u s t r i a l r e a c t o r s because
of h i g h v o l u m e t r i c mass t r a n s f e r c o e f f i c i e n t s . The m a i n f e a t u r e
of two-phase n o z z l e i s i n p r e s e r v i n g t h e e n e r g y o f t h e j e t , u t i
l i z e d f o r the p r o d u c t i o n o f a h i g h i n t e r f a c i a l a r e a , by suppres
s e d c o a l e s c e n c e i n s u i t a b l e m e d i a and b y a s u i t a b l e g u i d e t u b e
(momentum t r a n s f e r t u b e , ( 2 6 ) ) . The i n d u s t r i a l a p p l i c a t i o n s i n
v o l v e t h o s e i n SCP p r o d u c t i o n ( 2 7 ) a n d i n w a s t e w a t e r t r e a t m e n t
( 2 8 ) . The c o r r e l a t i o n o f t h e t y p e o f e q . ( 2 ) c a n , p e r h a p s , be o f
use i n p r e d i c t i n g k ^ a f o r s t i r r e d - t a n k r e a c t o r s , b a s e d o n a
s i n g l e m e a s u r e d v a l u e (gas h o l d - u p ) . C o r r e l a t i o n s b a s e d o n power
i n p u t and s u p e r f i c i a l v e l o c i t y a r e t h e most f r e q u e n t ( 2 9 ) .
E f f e c t o f v i s c o s i t y o n k ^ a a n d power c o n s u m p t i o n d u r i n g mass
t r a n s f e r . The e f f e c t o f v i s c o s i t y o n k ^ a i s o f i m p o r t a n c e i n
s e v e r a l h i g h l y v i s c o u s N e w t o n i a n and n o n - N e w t o n i a n f e r m e n t a t i o n s .
Some c o r r e l a t i o n s d e s c r i b i n g l ^ a d e c r e a s e w i t h v i s c o s i t y have
+ - V L = 1.70 10 ms
S
3 1
A - V L 0.86. 1Ô ms
S
S 3 1
1 ' ' 1
4 6 8 10 20 30
b e e n s u m m a r i z e d i n ( 1 5 ) . From t h i s p a p e r , t h e H e n z l e r ' s c o r r e l a
t i o n f o r 1-2% CMC s o l u t i o n s i n b u b b l e c o l u m n s ( f l o w b e h a v i o u r r e p
resentative f o r p e n i c i l l i n fermentation) i s u s e f u l :
where v ^ i s t h e s u p e r f i c i a l a i r v e l o c i t y and υ k i n e m a t i c v i s c o s i
g
t y . R e u s s e t a l . ( 3 0 ) s u g g e s t e d a much s i m p l e r e q u a t i o n f o r s t i r -
red-tank reactor:
V T Ρ
a — = f ( § ) (4)
GV
V L S > ( U
0 0 6 4 0 1 5 6 0 3 8
Ρ /Ρ = 0.0312 R e ' Fr" ' Na" ' ( A _ )<>.8 ( 5 )
g o ai
i n v o l v i n g R e y n o l d s a n d F r o u d e numbers o f i m p e l l e r , a e r a t i o n num
b e r Na and r a t i o o f t a n k a n d i m p e l l e r d i a m e t e r . The a p p l i c a b i l i t y
of eq.(4) f o r p r e d i c t i n g k^a d u r i n g f e r m e n t a t i o n o f P.chrysogenum
and A . n i g e r i s shown i n F i g u r e 3. T h u s , t h e b a s i c p h y s i c a l p r o
p e r t i e s o f c u l t i v a t i o n broths ( i n t h i s case v i s c o s i t y ) a r e i n c o r
p o r a t e d i n t o c o r r e l a t i o n s f o r t r a n s p o r t phenomena t o g e t h e r w i t h
k e y d e s i g n p a r a m e t e r s ( k ^ a and power c o n s u m p t i o n ) .
N i s h i k a w a e t a l . (31; have r e c e n t l y proposed t h e t w o - r e g i o n
model ( b u b b l i n g - and a g i t a t i o n - c o n t r o l l e d ) f o r e s t i m a t i n g k a i n T
H e a t e x c h a n g e i n v o l v e s h e a t g e n e r a t i o n and t r a n s f e r i n a n d
between e l e m e n t a r y r e g i o n s o f a b i o r e a c t o r and t h a t o f i n d i v i d u a l
phases o f a r e a c t o r ( 2 ) . A r a t i o n a l s t a r t f o r these c o n s i d e r a t i
ons i s t h e g e n e r a l l o c a l h e a t b a l a n c e , s i m i l a r t o t h a t o f mass
transfer:
tations. Reprinted, with permission, from Ref. 30. Copyright 1981, Pergamon
Press Ltd.
nozzles (3-5 mm ID), electrolyte solution plus ethanol (0-0.1 % v/v) (25).
3 ρ . C . T.
+ V . c .
P T . . +Vk . V T . + ΣΔΗ r..
J PJ iJ ej j n j ij
a t
f
(6)
-k-l(k«) \ \ T
< j s - T
j > - 0
where Ρ i s s p e c i f i c d e n s i t y , C i s m o l a r h e a t , Τ i s a b s o l u t e tem
perature, k i s e f f e c t i v e thermal c o n d u c t i v i t y , Δ Η i s heat of
r e a c t i o n , h i s h e a t t r a n s f e r c o e f f i c i e n t and T. i s t h e t e m p e r a
t u r e a t t h e i n t e r f a c e a r e a . The f i r s t t e r m o f tftîs b a l a n c e i s a n
a c c u m u l a t i o n term a c c o u n t i n g f o r heat c a p a c i t y ; t h e second term
i s heat t r a n s p o r t due t o c o n v e c t i o n , w h i c h i s i m p o r t a n t i n t u b u
l a r r e a c t o r s . The t h i r d t e r r e p r e s e n t h e a t b a c k m i x i n d
be i m p o r t a n t i n b i o r e a c t i o n
nes o r i n a deep l a y e r clumps
f o u r t h t e r m c h a r a c t e r i s e s h e a t g e n e r a t i o n due t o a r e a c t i o n and
the f i f t h i s t h e i n t e r f a c e h e a t t r a n s f e r . I n b i o r e a c t o r s t h a t a r e
t h o r o u g h l y m i x e d o n l y t h e a c c u m u l a t i o n and h e a t g e n e r a t i o n terms
are s i g n i f i c a n t and t h e t e m p e r a t u r e f i e l d i s u n i f o r m . The b o u n d a
ry c o n d i t i o n s o f t h i s s i m p l i f i e d e q u a t i o n a r e r e p r e s e n t e d by the
o v e r a l l heat t r a n s f e r from the c u l t i v a t i o n b r o t h t o the c o o l i n g
o r h e a t i n g ( s t e r i l i z a t i o n ) medium.
The amount o f h e a t g e n e r a t e d i n a b i o r e a c t o r c a n be e a s i l y
e v a l u a t e d f r o m t h e above s i m p l i f i e d h e a t b a l a n c e ^ r e s u l t i n g i n a A
c e l l s a s t h e s o l e r e a c t i o n p r o d u c t , M i n k e v i c h and E r o s h i n ( 3 3 )
derived a t h e o r e t i c a l formula:
y
= 818 Na = 818 k a —
T (7)
f L H
w h e r e ^ N a ^ i s t h e maximum o x y g e n t r a n s f e r o f a b i o r e a c t o r ( i n
g.lt .h ) , y i s t h e mole f r a c t i o n o f oxygen i n t h e e x i t gas
2
vity ( X ) : y
k
Q
f = ( k
i " ~ T — > υ x ( 8 )
x/s
where k^ and k^ a r e c o n s t a n t s t h a t c a n be d e r i v e d f r o m t h e h e a t s
of c o m b u s t i o n o f s u b s t r a t e and c e l l s . T h u s , c a n be a s s e s s e d
The o v e r a l l d e s c r i p t i o n (model) o f a r e a c t o r i s o b t a i n e d
through p r o c e s s s y n t h e s i s by combining models o f r e a c t o r hydrody
n a m i c s , mass t r a n s f e r and h e a t e x c h a n g e w i t h an a p p r o p r i a t e c e l l
( s u b c e l l u l a r ) o r p o p u l a t i o n model ( 1 ) . D e s c r i p t i o n o f a p o p u l a t i o n
should take i n t o c o n s i d e r a t i o n p o s s i b l e d i s p e r s e d o r aggregated
(the d i s t i n c t morphological appearances o f a c u l t u r e : p e l l e t s ,
m y c e l i u m , f l o c k s , g r o w t h on r e a c t o r w a l l i n t h e f o r m o f m i c r o b i a l
f i l m ) forms o f p o p u l a t i o n . Biomass s u p p o r t p a r t i c l e s a r e g a i n i n g
a p p r e c i a b l e importance i n a e r o b i c (40) as w e l l as i n a n a e r o b i c
processes.
The d e s c r i p t i o n h i e r a r c h y a c q u i r e s d i f f e r e n t w a y s : p h y s i c a l
p r o c e s s e s i n mass and e n e r g y b a l a n c e s ; i n f o r m a t i o n p r o c e s s i n g and
c o n t r o l ; o p t i m i z a t i o n o f the system a t t h e r e a c t o r l e v e l o r as
a w h o l e ( 4 1 ) . The most e l a b o r a t e c o n c e p t o f d e s c r i p t i v e h i e r a r c h y ,
i n c l u d i n g a s e t o f p r o g r a m s , was p u b l i s h e d and r o u t i n e l y u s e d b y
K l i r ( 4 2 ) . As d e s c r i p t i v e h i e r a r c h y u s u a l l y f o l l o w s s t r u c t u r a l
and f u n c t i o n a l h i e r a r c h y , i t i s t h u s a s s u r e d t h a t t h e m o d e l l e d
o b j e c t p o s s e s s e s enough s t r u c t u r a l and f u n c t i o n a l f e a t u r e s , o t h e r
w i s e t h e r e a c t o r c o n t r o l b y means o f s u c h m o d e l s w o u l d n o t s a t i s
f a c t o r i l y respond t o changing c o n d i t i o n s .
Reactor Control
s e n s i t i v i t y o f t h e s e v a r i a b l e s , d e f i n e d as a r e l a t i v e o r a b s o l u t e
change i n an o p t i m i z a t i o n c r i t e r i o n on t h e i r change ( 4 4 ) , i s r e
q u i r e d . The d e c i s i v e p a r a m e t e r s s e r i o u s l y a f f e c t i n g t h e s y s t e m
a r e d e f i n e d i n t h i s way. The c r i t e r i o n o f c o n t r o l and o p t i m i z a
t i o n i s c o n v e n i e n t l y s e t up, e.g., as t h e maximum b i o l o g i c a l
t i t r e , maximum p r o d u c t i v i t y , l o w e s t r u n n i n g c o s t s , e t c .
C o n t r o l and d y n a m i c h i e r a r c h i e s may s e r v e as a good g u i d e l i
ne f o r s e t t i n g up p r o p e r c o n t r o l . C o n t r o l h i e r a r c h y o f b i o l o g i c a l
l e v e l s i s i n T a b l e I I I , c o n t r o l l o o p s and g o a l f u n c t i o n s ( c o n t r o l
and o p t i m i z a t i o n c r i t e r i a ) o f b i o l o g i c a l l e v e l s a r e i n T a b l e I V .
The c o m p l e x i t y o f b i o l o g i c a l phenomena i s r e f l e c t e d i n t h e p r e s e n
ce o f m u l t i p l e l o c a l g o a l f u n c t i o n s o f d i f f e r e n t s u b s y s t e m s , w h i c h
may a c t i n an a c c o r d o r o p p o s i t e l y . N o t e t h a t T a b l e IV has b e e n
d e v e l o p e d f o r e c o s y s t e m s . The i m p l e m e n t a t i o n o f c o n t r o l h i e r a r c h y
i n t h e m o d e l l i n g and c o n t r o l f b i o l o g i c a l s y s t e m ha t t
b e e n c a r r i e d o u t . The c o n t r o
namic h i e r a r c h y of tim
d i v i d u a l s u b s y s t e m s ) . The r e l a x a t i o n t i m e i s c o n s i d e r e d t o be t h e
t i m e f o r a change f r o m one s t a t e t o a n o t h e r (due t o r e a c t i o n ,
d i f f u s i o n , change i n t h e a d j o i n i n g l e v e l , e t c . ) . T a b l e V p r e s e n t s
a v e r a g e r e l a x a t i o n t i m e s o f some c e l l u l a r s u b s y s t e m s . As a p p e a r s
f r o m t h i s t a b l e , s l o w (and l i m i t i n g ) p r o c e s s e s g i v e r i s e t o s t r u c
t u r a l e l e m e n t s , s e r v i n g as b a s i s f o r f a s t p r o c e s s e s . U s u a l l y ,
responses of h i g h e r l e v e l s of the system t o d i s t u r b a n c e s coming
from the s u p e r i o r l e v e l are s l o w e r than responses a t the n e x t
lower l e v e l . B e s i d e s r e l a x a t i o n times of b i o l o g i c a l subsystems of
c e l l s , t h e s e l e c t i o n o f s y s t e m i c phenomena may p r o c e e d v i a an
u t i l i z a t i o n o f , e.g., c e l l g e n e r a t i o n t i m e , r a t e o f s u b s t r a t e
c o n s u m p t i o n , r e s p i r a t i o n r a t e , o x y g e n t r a n s f e r and o f t h e homoge-
n i z a t i o n t i m e o f a r e a c t o r , t o m e n t i o n some.
The above d i s c u s s i o n may i n v o l v e b o t h c o n t r o l and d y n a m i c s
o f s t e a d y s t a t e ( s i n g l e and m u l t i p l e ) , u n s t e a d y s t a t e ( t r a n s i t i o n
b e t w e e n two s t a t e s ) and u n s t a b l e o p e r a t i o n s o f t h e b i o r e a c t o r
r e s u l t i n g from t y p i c a l n o n - l i n e a r response of b i o l o g i c a l systems.
In s p i t e of the f a c t t h a t the s t a b i l i t y a n a l y s i s of n o n - l i n e a r
systems i s q u i t e advanced, e x p e r i m e n t a l c o n f i r m a t i o n of m u l t i p l e
s t e a d y s t a t e s and i n s t a b i l i t i e s l a g s b e h i n d ( f o r a r e v i e w o f
t h e o r y and e x p e r i m e n t s see ( 4 5 ) ) . An e x c e l l e n t e x a m p l e o f e x p e r i
mental demonstration of unstable operation of a continuous
reactor i s i n (46).
Reactor Scale-Up
On t h e b a s i s o f a r e a c t o r m o d e l , i n v o l v i n g enough s t r u c t u r a l
and f u n c t i o n a l f e a t u r e s o f b i o l o g i c a l and e n g i n e e r i n g (physical)
p r o p e r t i e s o f a b i o t e c h n o l o g i c a l p r o c e s s , new c r i t e r i a f o r a
s c a l e - u p may e v o l v e . The employment o f o n l y one c r i t e r i o n , t a k e n
f r o m one a r b i t r a r y l e v e l o f a h i e r a r c h y , i n the m o d e l l i n g and
s u b s e q u e n t s c a l e - u p f r e q u e n t l y f a i l s , as i s a p p a r e n t f r o m numerous
3 S t r u c t u r a l dynamics Organization
( c h a n g i n g number o f s t r u c t u r a l
elements )
S t r u c t u r a l H i e r a r c h y
Molecular Cellular Population Trophic
Table V Dynamic H i e r a r c h
. . 2 4
C e l l m u l t i p l i c a t i o n , DNA r e p l i c a t i o n 10 - 10
3 4
mRNA s y n t h e s i s on o p e r o n 10 - 10
* 1 3
T r a n s l o c a t i o n of substances i n t o c e l l s 10 - 10
1 2
P r o t e i n s y n t h e s i s i n polysomes 10 - 10
Allosteric control o f enzyme s y n t h e s i s 10°
Glycolysis i n cytoplasm 10 * - 10
-2
Oxidative phosphorylation i n mitochondria 10
—6 —3
E n z y m a t i c r e a c t i o n and t u r n o v e r 10 - 10
Bond b e t w e e n enzyme and s u b s t r a t e 10
I n t e r n a t . J . G e n e r a l Systems 1982, 8 ( 1 ) , T a b l e V I ( 1 )
examples of a p p l i c a t i o n s of e n g i n e e r i n g c r i t e r i a ( e . g . , i n ( 2 9 ) ) .
The n e g l e c t o f b i o l o g i c a l f e a t u r e s o f b i o t e c h n o l o g i c a l p r o c e s s e s
f r e q u e n t l y l e a d s t o an u n r e s o l v e d c a s e o f s i m u l t a n e o u s m a i n t e n a n
ce o f t h e n u m e r i c a l v a l u e s o f a l l d i m e n s i o n l e s s g r o u p s o f p h y s i
c a l p a r a m e t e r s (mass t r a n s f e r , power i n p u t p e r u n i t v o l u m e , m i x i n g
time, e t c . ) . For example, a s i n g l e c r i t e r i o n of constant m i x i n g
t i m e w o u l d v i o l a t e an i n v a r i a n c y o f gas p h a s e d y n a m i c s . M a c r o -
m i x i n g o f t h e f l u i d phase d e t e r m i n e s , t o a c o n s i d e r a b l e e x t e n t ,
t h e b e h a v i o u r o f t h e gas p h a s e , i . e . , t h e r e s i d e n c e t i m e d i s t r i
b u t i o n o f gas b u b b l e s . I n r e a c t o r s c a l e - u p , a c o n s i d e r a b l e d i f f e
r e n c e i n gas p h a s e r e s i d e n c e t i m e c a n be e x p e c t e d as t h e l i q u i d
f l o w p a t h s ( l o o p s ) g r o w w i t h i n c r e a s i n g r e a c t o r s c a l e . The m a i n t e
n a n c e o f m i x i n g t i m e w o u l d r e s u l t i n an i n c r e a s e i n o x y g e n u t i l i
z a t i o n e f f i c i e n c y . Some l i m i t e d e x p e r i m e n t a l d a t a f o r d i f f e r e n t
r e a c t o r designs are a v a i l a b l e i n (47)
Q u a l i t a t i v e l y new c r i t e r i a
scheme o f t h e h i e r a r c h i c a
c e s s , suggest a need f o r m a i n t a i n i n g s i m i l a r i t y between systems
o f d i f f e r e n t s i z e s o f l a b o r a t o r y and p l a n t l e v e l s , i . e . , t h e
i d e n t i t y o f v a l u e s o f p a r a m e t e r s and c o n s t a n t s (1,2) · Such a
scale-up procedure would r e q u i r e a r e p e t i t i o n of systemic f e a t u
res of d e c i s i v e l e v e l s of h i e r a r c h y i n v o l v i n g those of b i o t e c h n o
l o g i c a l and p h y s i c a l n a t u r e on t h e l a b o r a t o r y and p l a n t s c a l e
and w o u l d assume t h e i n v a r i a n c e o f t h e s y s t e m i c phenomena o f a
g i v e n l e v e l w i t h r e s p e c t t o t h e s c a l e . The m e t h o d o l o g y o f s c a l e -
up v i a m o d e l l i n g and s i m u l a t i o n i s y e t t o be d e v e l o p e d .
Conclusions
F u r t h e r d e v e l o p m e n t o f r e a c t o r d e s i g n i s hampered by a l a c k
o f a p p r o p r i a t e t h e o r y o f some e l e m e n t a r y phenomena. T h i s s i t u a
t i o n l e a d s t o e m p i r i c a l c o r r e l a t i o n s t h a t may be o f some p r a c t i
c a l u s e . Systems a p p r o a c h a i d s i n c r e a t i n g a g u i d e l i n e f o r r a t i o
n a l b i o r e a c t o r d e s i g n , e s p e c i a l l y on t h e b a s i s o f sound b i o l o g i
c a l theory.
Literature Cited
R E C E I V E D June 1, 1982
0097-6156/83/0207Ό377$06.00/0
© 1983 American Chemical Society
Polymer Entrapment
V a r i o u s methods h a v e b e e n p r o p o s e d f o r w h o l e c e l l i m m o b i l i z a
t i o n i n c l u d i n g a d s o r p t i o n and c o v a l e n t a t t a c h m e n t t o a p r e f o r m e d
c a r r i e r , c r o s s l i n k i n g , f l o c c u l a t i o n , m i c r o e n c a p s u l a t i o n , and e n
t r a p m e n t . P h y s i c a l e n t r a p m e n t i n a p o r o u s m a t r i x i s by f a r t h e most
f l e x i b l e and most commonly u s e d t e c h n i q u e . Considering the f a c t
t h a t t h e p o l y m e r n e t w o r k has t o be f o r m e d i n t h e p r e s e n c e o f t h e
f i n a l l y entrapped b i o l o g i c a l m a t e r i a l , the performance c r i t e r i a of
c h e m i c a l and p h y s i c a l n a t u r e a r e as f o l l o w s :
(1) The n e t w o r k f o r m a t i o n has t o p r o c e e d u n d e r m i l d c o n d i
t i o n s (pH and t e m p e r a t u r e ) i n an aqueous e n v i r o n m e n t ;
(2) t h e n e t w o r k has t o be c h e m i c a l l y s t a b l e u n d e r v a r i o u s r e
a c t i o n c o n d i t i o n s (pH, b u f f e r s o l u t i o n , i o n i c and n o n i o n i c s u b
strates, etc.);
(3) t h e s i z e and t h
e r a b l y as b e a d s ) has t
(4) t h e p o s s i b i l i t y f o r a l a r g e v a r i a t i o n o f b i o m a s s c o n t e n t
i n t h e c a t a l y s t s h o u l d be g i v e n ;
(5) t h e c a t a l y s t b e a d s s h o u l d be m e c h a n i c a l l y s t a b l e t o be
used i n v a r i o u s r e a c t o r c o n f i g u r a t i o n s (packed bed, f l u i d i z e d bed,
s t i r r e d tank).
A p p r o p r i a t e p o l y m e r i c c a r r i e r s c a n be o b t a i n e d f r o m p o l y m e r i c ,
o l i g o m e r i c , and monomeric p r e c u r s o r s . Due t o unwanted c h e m i c a l i n
t e r a c t i o n of such chemicals w i t h the c e l l m a t e r i a l l a r g e r s i z e of
t h e s e p r e c u r s o r s i s f a v o r a b l e . The i o n o t r o p i c g e l a t i o n , s t a r t i n g
f r o m p o l y e l e c t r o l y t e s and t h e p o l y c o n d e n s a t i o n , s t a r t i n g f r o m o l i
g o m e r i c epoxy r e s i n s , a r e t y p i c a l p r o b l e m s o l u t i o n s .
I o n o t r o p i c G e l a t i o n of P o l y e l e c t r o l y t e s
T h i s method o f n e t w o r k f o r m a t i o n i s d e f i n e d as a c r o s s l i n k i n g
r e a c t i o n of p o l y e l e c t r o l y t e s w i t h lower molecular weight m u l t i v a
lent counterions. C o n s i d e r i n g t h e p o l y m e r i c component, a n i o n i c
( e . g . , a l g i n a t e , CMC (I) o r c a t i o n i c ( c h i t o s a n (2)) substances can
be u s e d . T h i s v a r i e t y o f p o l y m e r s and t h e a p p r o p r i a t e c o u n t e r i o n s
a r e s u m m a r i z e d i n F i g u r e 1. The c h o i c e o f t h e p o l y m e r i s d e t e r
m i n e d by t h e pH r e g i o n o f t h e r e s p e c t i v e b i o c a t a l y t i c r e a c t i o n ,
s i n c e a l l i o n o t r o p i c g e l s a r e r e v e r s i b l e s t r u c t u r e s w h i c h c a n be
r e d i s s o l v e d by i n c r e a s e ( a l g i n a t e ) o r d e c r e a s e ( c h i t o s a n ) o f pH b e
yond c e r t a i n l i m i t s . A second important c r i t e r i o n i s the i o n i c
c o m p o s i t i o n o f t h e r e a c t i o n medium and t h e p o s s i b i l i t y o f i n s o l u b l e
byproduct o r complex f o r m a t i o n w i t h the network forming i o n s .
I n a t y p i c a l a l g i n a t e entrapment process the c e l l s are sus
pended i n a 3% s o d i u m a l g i n a t e s o l u t i o n and t h i s v i s c o u s s u s p e n
s i o n i s p r e c i p i t a t e d d r o p w i s e i n a 1% C a C l 2 s o l u t i o n . A f t e r 30
m i n u t e s s t a b l e C a - a l g i n a t e g e l s a r e f o r m e d where t h e c e l l s a r e i m
m o b i l i z e d i n a macroporous s t r u c t u r e . F o l l o w i n g to t h i s p r e c i p i
t a t i o n p r o c e s s a p a r t i a l d r y i n g s t e p c a n be a p p l i e d w h i c h r e s u l t s
i n a homogeneous s h r i n k i n g o f t h e p a r t i c l e s , t h u s i n c r e a s i n g c o n -
POLYANIONS
ALGINAT
CARBOXYMETHYL-
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CELLULOSE
Ca . Fe . Zn
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CARBOXY-GUAR-
Al \
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Fe 3 +
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COPOLY-STYRENE-
MALEIC ACID
POLYCATIONS
CHITOSAN Fe(CN) 6
4
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POLY-PHOSPHATE
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^ΝΗ3 Φ
Χ θ
POLY-ALDEHYDO-CARBONIC
ACID
P0LY-1-HYDR0XY-1-
SULF0NATE-PR0PEN-2
ALGINATE
s i d e r a b l y t h e m e c h a n i c a l s t a b i l i t y as w e l l a s t h e p a c k i n g d e n s i t y
of the entrapped c e l l s i t s e l f . A l l these f a c t o r s are very advanta
geous f o r t h e b i o c a t a l y t i c a p p l i c a t i o n . The f l e x i b i l i t y o f t h e a l -
g i n a t e - m e t h o d c a n be d e m o n s t r a t e d a c c o r d i n g t o t h e f o l l o w i n g p a r
a m e t e r b o u n d a r y v a l u e s : p o l y m e r c o n c e n t r a t i o n f r o m 0.5 t o 8%, CaC^
c o n c e n t r a t i o n f r o m 0.05 t o 2%, c e l l c o n c e n t r a t i o n (on wet w e i g h t
b a s i s ) f r o m 0.1 t o 100%, b e a d d i a m e t e r s f r o m 0.1 t o 5 mm, and
p r e p a r a t i o n t e m p e r a t u r e s f r o m 0 t o 80° C. Cells of different
s t r u c t u r e ; e.g., a e r o b i c (1) o r a n a e r o b i c m i c r o b e s ( 3 ) , p l a n t c e l l s
( 4 ) , mammalian c e l l s ( 5 ) , c a n be e n t r a p p e d and t h u s s t a b i l i z e d
without c o n s i d e r a b l e t o x i c i t y problems.
A p r o b l e m o f p r a c t i c a l i m p o r t a n c e i s t h e s c a l e up o f t h e i m
m o b i l i z a t i o n p r o c e s s f r o m amounts o f s e v e r a l grams t o s e v e r a l
hundred l i t e r s . W h i l e s m a l l amounts c a n e a s i l y be p r e p a r e d u s i n g
one c a p i l l a r y o r i f i c e , a b u n d l e o f s u c h c a p i l l a r y i n a s i e v e p l a t e
type c o n s t r u c t i o n w i l l g i v
i f the c a p i l l a r y c h a r a c t e r i s t i c
a r e shown i n F i g u r e 2.
I n t h i s c a s e c o v a l e n t n e t w o r k s o f h i g h m e c h a n i c a l and c h e m i
c a l s t a b i l i t y a r e o b t a i n e d as a r e s u l t o f c r o s s l i n k i n g r e a c t i o n of
e p o x i d e s w i t h m u l t i f u n c t i o n a l a m i n e s ( 6 ) . The m a i n p r o b l e m s o f
t h i s t e c h n i q u e a r e t h e t o x i c i t y o f t h e amino-component and t h e
u s u a l l y l o w p o r o s i t y o f t h e p o l y m e r i c n e t w o r k . The t o x i c i t y , mea
s u r e d by t h e v i a b i l i t y o f i m m o b i l i z e d y e a s t c e l l s , c o u l d be m i n i
m i z e d a) by p r o p e r s e l e c t i o n o f e p o x y and amino components and b)
by i n t r o d u c t i o n o f a p r e g e l l i n g t i m e i n t h e o r d e r o f 15 m i n u t e s
b e f o r e m i x i n g t h e c e l l s w i t h t h e c o n d e n s a t i n g o l i g o m e r s ( 7 ) . The
p o r o s i t y o f t h e m a t r i x i s i n t r o d u c e d by t h e i m m o b i l i z e d c e l l s i t
s e l f and by an i n t e r m e d i a t e p r e p a r a t i o n o f an i n t e r p e n e t r a t i n g
n e t w o r k w i t h an i o n o t r o p i c g e l . The i o n o t r o p i c g e l a t i o n i s a l s o
u s e d t o c o n t r o l t h e p a r t i c l e s h a p e and s i z e . A c o m p l e t e scheme o f
s u c h a n i m m o b i l i z a t i o n p r o c e s s i s shown i n F i g u r e 3. A g a i n q u i t e
h i g h c o n c e n t r a t i o n s (up t o 70% on wet w e i g h t b a s i s ) o f c e l l s c a n
be f i n a l l y i n c o r p o r a t e d i n s u c h a p o l y m e r i c s t r u c t u r e .
The v i a b i l i t y o f t h e y e a s t c e l l s , and t h u s t h e r e d u c e d t o x i c
i t y o f t h e e n t r a p m e n t m e t h o d , c a n be d e m o n s t r a t e d by c e l l g r o w t h
i n the m a t r i x , which gives r i s e to a corresponding a c t i v i t y i n
crease f o r ethanol p r o d u c t i o n from glucose ( 7 ) . This behavior i s
shown i n F i g u r e 4. The f a c t o r o f a c t i v i t y i n c r e a s e compared t o t h e
i n i t i a l v a l u e i n c r e a s e s w i t h d e c r e a s i n g i n i t i a l l o a d i n g ; however,
i t i s o b v i o u s t h a t an u p p e r l i m i t o f a c t i v i t y w i l l f i n a l l y be
reached. The r e a s o n f o r t h i s phenomenon, as w e l l as f o r t h e a c
t i v i t y d e c r e a s e w i t h i n c r e a s i n g i n c u b a t i o n t i m e , w i l l become
obvious from the d i s c u s s i o n s o f the f o l l o w i n g chapter.
PRESSURE
±
precondensotion
(15m. n.)
mixing
periodical
6g bake i*s yeast • 2ml
injection
• 20g β % alginate- sol.
, Qoooonnnnnooo 0
ο.ο
drying (24h) crosslink!ng 9r^-> ol
(20min) 2wt%CaCl2-solution
EP0XY-BEADS
alginate-dissolution (40min)
in 0.1« phosphate-buffer (pH> 6.0)
I t i s a w e l l known f a c t i n h e t e r o g e n e o u s c a t a l y s i s , t h a t t h e
c a t a l y t i c a c t i v i t y i s generally not d i r e c t l y proportional to the
c o n c e n t r a t i o n o f a c t i v e s i t e s b u t depends a l s o o n h y d r o d y n a m i c
c o n d i t i o n s i n t h e s u r r o u n d i n g o f t h e p a r t i c l e s , on p a r t i c l e s i z e
and m a t r i x p o r o s i t y . I t i s furthermore w e l l understood, that
v a r i o u s t r a n s p o r t phenomena h a v e t o be t a k e n i n t o a c c o u n t , m a i n l y
d i f f u s i o n a l transport processes which n e c e s s a r i l y a r e preceding to
the r e a c t i o n step i t s e l f .
A d i m e n s i o n l e s s number, u s u a l l y c a l l e d T h i e l e - m o d u l u s , c a n be
used t o q u a n t i t a t i v e l y account f o r t r a n s p o r t - r e a c t i o n c o u p l i n g
phenomena. A s s u m i n g t h e v a l i d i t y o f M i c h a e l i s - M e n t e n r a t e e q u a
t i o n - which i s j u s t i f i e d f o r simple enzymatic r e a c t i o n s i n whole
c e l l s t o o - t h e f o l l o w i n g e x p r e s s i o n f o r t h e T h i e l e modulus has
been d e r i v e d ( 8 ) :
f
where R i s t h e p a r t i c l e r a d i u s , v t h e r a t e o f r e a c t i o n , the
M i c h a e l i s c o n s t a n t , S t h e s u b s t r a t e c o n c e n t r a t i o n and D the e f
e
f e c t i v e s u b s t r a t e d i f f u s i v i t y i n the porous c a t a l y s t p a r t i c l e .
On t h e o t h e r hand t h e e f f e c t i v e n e s s f a c t o r η i s d e f i n e d a s t h e
r a t i o o f t h e e f f e c t i v e r e a c t i o n r a t e ν t o t h e maximum r e a c t i o n
rate ν w h i c h w o u l d be o b s e r v e d w i t h o u t t r a n s p o r t l i m i t a t i o n
max
n = ~ - (2)
max
F o r i m m o b i l i z e d c e l l c a t a l y s t s t h e r e a r e two p o s s i b i l i t i e s t o ob
t a i n t h i s f a c t o r . F i r s t l y , t h e p a r t i c l e s o f l a r g e r r a d i u s can be
g r i n d e d down t o s u c h a s m a l l s i z e t h a t p o r e d i f f u s i o n becomes neg
ligible. I n t h i s case = v ^ + .
e Q Due t o t h e s i z e and t h e
s i m p l e entrapment o f t h e c a t a l y t i c s p e c i e s l o s s from t h e m a t r i x
may b e c o n s i d e r a b l e . Therefore, secondly, the free c e l l a c t i v i t y
can be used i n t h e denominator, i f t h e e x a c t c o n c e n t r a t i o n o f c a t -
i s
a l y t i c a l l y a c t i v e immobilized c e l l s ( X )
a c t known. Since
v.-I.ilfi (3)
i s t h e s p e c i f i c r e a c t i o n r a t e o f t h e f r e e l y suspended c e l l s , t h e
equation
ν = ν ' X = v* (4)
max act
1
h o l d s , w h i c h f u r t h e r m o r e d e f i n e s v i n E q n . ( 1 ) . B a s e d o n numer
i c a l calculations t y p i c a l functions
η - f (Φ) (5)
h a v e b e e n d e v e l o p e d , w h i c h i n t e r r e l a t e t h e two d i m e n s i o n l e s s p a r
ameters and w h i c h c a n be checked e x p e r i m e n t a l l y .
To e v a l u a t e t h e a p p l i c a b i l i t y o f E q n . ( 5 ) , t h e p a r a m e t e r s i n
Eqns. (1-4) have t o be determined i n d e p e n d e n t l y . T h i s has been
done f o r t h e c l e a v a g e r e a c t i o n o f P e n i c i l l i n G t o 6APA w i t h immo
b i l i z e d E. Qoli c e l l s i m m o b i l i z e d i n e p o x i d e b e a d s ( 9 ) .
The r a d i u s R: The r a d i u s o f p a r t i c l e s o b t a i n e d f r o m i o n o t r o p -
i c g e l a t i o n i s u s u a l l y c o n t r o l l e d w i t h i n v e r y narrow l i m i t s and
t h e s i z e c a n e a s i l y b e d e t e r m i n e d by m i c r o s c o p i c m e a s u r e m e n t s .
Reaction K i n e t i c s ; The r e a c t i o n r a t e s h a v e b e e n m e a s u r e d a t
pH = 7.8 a n d Τ = 37o C, u s i n g a 5% P e n G s u b s t r a t e c o n c e n t r a t i o n .
T i t r a t i o n w i t h 0.1 m o l a r NaOH h a s b e e n u s e d t o d e t e r m i n e t h e amount
o f p r o d u c t f o r m a t i o n . The K^j v a l u e s o f f r e e a n d i m m o b i l i z e d c e l l s
have been o b t a i n e d from
e x a m p l e s i n F i g u r e 5. F o l l o w i n
enzymes d u r i n g t h e p r o c e s s o f i m m o b i l i z a t i o n , t h e i n e q u a l i t y X £ < a C
n
^ m m o b i l . °lds. f o l l o w i n g approach has been developed f o r
the determination o f X : i n a c e r t a i n experiment, i n a f i r s t ap
a c t
are l i s t e d i n Table I :
Table I . C a l c u l a t e d η- a n d Φ- v a l u e s b a s e d
on d i f f e r e n t assumptions o f r e
s i d u a l c e l l a c t i v i t y a f t e r immo
bilization.
: X
act^ imm.
% η cale. Φ cale.
10 2.36 0.59
20 1.18 1.34
30 0.79 1.44
40 0.59 1.63
50 0.47 2.11
60 0.39 2.32
70 0.34 2.50
80 0.30 2.68
90 0.26 2.84
100 0.24 3.00
C o m p a r i s o n o f s e v e r a l i m m o b i l i z e d c e l l p r e p a r a t i o n s gave p r a c t i c
a l l y i d e n t i c a l r e s u l t s , showing that a value X a c t = 0*43X i m m #
i s a t y p i c a l one f o r t h i s e p o x i d e - i m m o b i l i z a t i o n p r o c e d u r e .
E f f e c t i v e Pore D i f f u s i v i t y : The e f f e c t i v e p o r e d i f f u s i v i t y
o f p e n i c i l l i n G has been determined e x p e r i m e n t a l l y i n a b a t c h ex
p e r i m e n t ( 1 0 ) . M i x i n g a c e r t a i n number o f c a t a l y s t b e a d s h a v i n g a
homogeneous s u b s t r a t e c o n c e n t r a t i o n S ( g / 1 ) , w i t h a c e r t a i n v o l u m e
a
o f f r e s h , n o n - c o n d u c t i n g w a t e r , t h e d i f f u s i o n p r o c e s s c a n be f o l
l o w e d by t h e c o n d u c t i v i t y a n d t h e c o n c e n t r a t i o n i n c r e a s e i n t h e
supernatent l i q u i d . I f S i s the s u b s t r a t e c o n c e n t r a t i o n i n the
e
p a r t i c l e a t t + «> a n d S t h e c o n c e n t r a t i o n a t t i m e t , R t h e p a r t i
2
c l e r a d i u s (cm) a n d D t h e e f f e c t i v e d i f f u s i v i t y ( c m / s e c ) t h e
e
d i f f u s i o n c o e f f i c i e n t ca
In (S-S )/(S -S ) vs. t
e a e
(6)
Such a p l o t i s shown i n F i g u r e 7.
Comparison o f Theory and E x p e r i m e n t : I f t h e s i m p l e model o f
t r a n s p o r t l i m i t a t i o n due t o p o r e d i f f u s i o n h o l d s , a l l e x p e r i m e n t
a l l y d e t e r m i n e d p a i r s o f η and Φ s h o u l d f a l l o n t h e n o n - d o t t e d
η = ί(Φ) - f u n c t i o n shown i n F i g u r e 6. U s i n g t h e i n d e p e n d e n t l y
d e t e r m i n e d parameters as d e s c r i b e d b e f o r e such v a l u e s have been
d e t e r m i n e d f o r a number o f c a t a l y s t p r e p a r a t i o n s u s e d i n t h e p e n
i c i l l i n G c l e a v a g e r e a c t i o n . The e x c e l l e n t agreement b e t w e e n c a l
c u l a t i o n o f Eqn. (5) a n d e x p e r i m e n t a l d e t e r m i n a t i o n c a n b e s e e n
f r o m F i g u r e 8. I n t h i s s e r i e s o f e x p e r i m e n t s two d i f f e r e n t
s t r a i n s o f E. ooli c e l l s h a v e b e e n u s e d , t h e one o f them h a v i n g
been o b t a i n e d by g e n e t i c e n g i n e e r i n g l e a d i n g t o a t e n f o l d a c t i v i t y
i n c r e a s e compared t o t h e c o n v e n t i o n a l s t r a i n ATCC 11 105 (1)·
C a t a l y s t O p t i m i z a t i o n : I n a n o t h e r r e a c t i o n example t h e a p
p l i c a t i o n o f the Thiele-modulus concept f o r the o p t i m i z a t i o n o f
c a t a l y s t c o m p o s i t i o n c o u l d be demonstrated. Here t h e o x i d a t i o n o f
g l u c o s e t o g l u c o n i c a c i d , c a t a l y z e d by A c e t o b a c t e r s i m p l e x c e l l s
has been s t u d i e d , where oxygen i s t h e r a t e l i m i t i n g s u b s t r a t e .
I n t h i s case a C a - a l g i n a t e m a t r i x has been used f o r c e l l immobil
ization. U s i n g s i m p l i f i e d e q u a t i o n s f o r t h e c a l c u l a t i o n o f D ande
furthermore assuming X a c t = Xi
m m # ( 1 2 ) , t h e r e l a t i o n between r e l a
t i v e a c t i v i t y (η i n %) a n d p a r t i c l e d i a m e t e r c o u l d be c a l c u l a t e d
for different c e l l concentrations. As c a n be shown i n F i g u r e 9,
a good agreement w i t h e x p e r i m e n t a l d a t a i s o b t a i n e d .
The a b s o l u t e a c t i v i t y f o r g l u c o n i c a c i d p r o d u c t i o n i s o b
t a i n e d i f the s p e c i f i c a c t i v i t y o f the d i f f e r e n t preparations i s
m u l t i p l i e d w i t h t h e c e l l c o n c e n t r a t i o n . The n o n - d o t t e d c u r v e i n
F i g u r e 10 i s t h e r e s u l t o f t h i s c a l c u l a t i o n , w h i c h g i v e s a good
Ο Γ
Î ln[(S - S ) / (S
e a - Se)]
•
-2 -
-4 h
i t i i i l » 1
1.0
0.5
0.2h
0.1
-I ι ι 1—
1 2 3 4
P a r t i c l e Diameter (mm)
C a t a l y s t D e a c t i v a t i o n and O p e r a t i o n a l Stability
Coming b a c k t o t h e p e n i c i l l i n G c l e a v a g e r e a c t i o n , l o n g t i m e
e x p e r i m e n t s w i t h e p o x i d e - i m m o b i l i z e d E. ooli c e l l s h a v e b e e n r e
p o r t e d e a r l i e r (6) . W i t h o u t any c o r r e c t i o n an e f f e c t i v e h a l f - l i f e
v a l u e t , i 2 o f c a t a l y t i c a c t i v i t y o f 43 d a y s h a s b e e n d e t e r m i n e d .
R e p e a t e d e x p e r i m e n t s c o n f i r m e d t h e a b r u p t change o f s l o p e i n t h e
a c t i v i t y - t i m e curve of consecutive batch r e a c t i o n s . In another
e x p e r i m e n t , where t h e c e l l
t r a t i o n a t the s u r f a c e o
l i n e a c t i v i t y - t i m e f u n c t i o n has b e e n f o u n d w i t h a much s m a l l e r
v a l u e t j / 2 H d a y s (9).
=
I t t h e r e f o r e becomes o b v i o u s , t h a t
t r a n s p o r t l i m i t a t i o n due t o much h i g h e r c e l l l o a d i n g and l o n g e r
d i f f u s i o n p a t h w a y s i n a b e a d - t y p e c a r r i e r m i g h t be r e s p o n s i b l e f o r
t h e two d i f f e r e n t s l o p e s .
The e x p e r i m e n t a l d a t a a r e shown i n F i g u r e 11a, t o g e t h e r w i t h
a d o t t e d l i n e , e x t r a p o l a t i n g the r e a c t i o n c o n t r o l l e d regime to
z e r o t i m e . On t h e o t h e r h a n d some m o d e l c a l c u l a t i o n s h a v e b e e n
performed under the assumption t h a t , a t the b e g i n n i n g of the ex
periment, the r e a c t i o n r a t e i s i n the d i f f u s i o n c o n t r o l l e d regime.
Then o n l y a r e s t r i c t e d s p h e r i c a l s h e l l c l o s e t o t h e b e a d s u r f a c e
i s p a r t i c i p a t i n g i n t h e s u b s t r a t e c o n v e r s i o n . I f s u p e r i m p o s e d on
the r e a c t i o n a time dependent c a t a l y s t d e a c t i v a t i o n o c c u r s , the
reactive s h e l l s h o u l d move t o t h e c e n t e r o f t h e c a t a l y s t p a r t i
c l e where a c t i v e c e l l s a r e a v a i l a b l e t o s u b s t i t u t e f o r the d e a c t i
vated c e l l s c l o s e r to the s u r f a c e .
Assuming d i f f e r e n t r a t e e q u a t i o n s f o r the time dependent
d e a c t i v a t i o n r e a c t i o n the graphs i n F i g u r e l i b are obtained.
W i t h o u t a c h i e v i n g q u a n t i t a t i v e agreement i n such a s i m p l i f i e d c a l
c u l a t i o n , we b e l i e v e t h a t t h e good q u a l i t a t i v e agreement o f t h e
non-dotted l i n e s i n F i g u r e l i b w i t h the experimental curve i n
F i g u r e 11a s u p p o r t s o u r c o n s i d e r a t i o n s i n p r i n c i p l e . The g e n e r a l
c o n c l u s i o n i s t h a t one s h o u l d be v e r y c a r e f u l i n d e r i v i n g i n t r i n s i c
h a l f - l i f e values of immobilized c e l l s from o v e r a l l o p e r a t i o n a l
s t a b i l i t y data of b i o c a t a l y t i c conversion. D i f f u s i o n a l l i m i t a t i o n
w i l l always l e a d to a r t i c i a l l y higher v a l u e s .
Acknowledgements
5+
4+ experimental v
-
observation \^ 30 d
3t Λ . . .
6-ΑΡΑ 43 d
• 2
1 +
—r—
10 20 30 40
t
days « cycles
_ 100
Literature cited
0097-6156/83/0207-0395$06.00/0
© 1983 American Chemical Society
The a p p a r a t u s h a s b e e n r e p o r t e d p r e v i o u s l y ( 1 ) . The m i c r o
m a n i p u l a t o r had c o a r s e c o n t r o l i n t h e X and Y a x e s and a p r e c i s e
v e r n i e r f o r the Ζ a x i s . A r e f e r e n c e p l a n e was e s t a b l i s h e d by
l o w e r i n g t h e m i c r o p r o b e u n t i l e l e c t r i c a l c o n t a c t was e s t a b l i s h e d
a t p o i n t s s e v e r a l m i l l i m e t e r s away f r o m t h e c o l o n y i n a l l d i r e c
tions. The c o l o n y was t r a v e r s e d by t h e m i c r o p r o b e a t t h e c e n t e r
l i n e , a t s e v e r a l l o c a t i o n s p r o g r e s s i n g t o w a r d t h e e d g e , and a d j a
c e n t a g a r was a l s o p r o b e d . S m a l l , medium, and l a r g e c o l o n i e s
were s e l e c t e d .
R e s u l t s and Discussion
F i r s t e l e c t r i c a l c o n t a c t w i t h t h e c o l o n y was n o t r e p r o d u c
i b l e , and an u n c e r t a i n t y o f a b o u t 5 m i c r o m e t e r s e x i s t s . Further
more, t h e s i g n a l f r o m t h
e r r a t i c near the p o i n t
agar remote from the c o l o n y
proof i s o f f e r e d , i t i s thought t h a t o u t e r c e l l s or a t h i n f i l m
o f m o i s t u r e c a n d r a w away f r o m t h e e l e c t r o d e a s a r e s u l t o f
e l e c t r i c a l s t i m u l a t i o n . When a d v a n c e d a few m i c r o n s p a s t t h e
p o i n t of f i r s t e l e c t r i c a l c o n t a c t , the m i c r o e l e c t r o d e d e l i v e r e d
a stable signal.
P r o f i l e s f r o m t y p i c a l t r a v e r s e s a r e i n F i g u r e 1. From s u c h
p r o f i l e s , l o c a t i o n s o f a g i v e n o x y g e n c o n c e n t r a t i o n were t a k e n
t o c o n s t r u c t t h e c o n t o u r l i n e s f o r F i g u r e 2. C o l o n i e s were
assumed t o be s y m m e t r i c a l , so o n l y one s i d e was p r o b e d . Note
t h a t t h e a b s c i s s a c h a n g e s , and t h e c o l o n i e s f o r F i g u r e 2C and
2D a r e much l a r g e r t h a n t h o s e f o r 2A and 2B. The c o l o n i e s d i d
n o t t e n d t o s i t e x a c t l y on t h e p l a n e o f t h e a g a r ; t h i s may be
e r r o r because the p r e c i s i o n of p o s i t i o n i n g the m i c r o e l e c t r o d e i s
g r e a t compared t o p o s s i b l e bumps i n t h e a g a r s u r f a c e . However,
a s t h e shape o f t h e c u r v e f o r f i r s t e l e c t r i c a l c o n t a c t was
u s u a l l y p e c u l i a r a t the edges o f the c o l o n y , i t i s l i k e l y t h a t
g r o w t h and m e t a b o l i s m d i s t o r t t h e a g a r .
No s u p p l e m e n t a l g l u c o s e was i n t h e a g a r f o r t h e c o l o n y f o r
F i g u r e 2A. Compared t o a c o l o n y o f s i m i l a r s i z e on a g a r p l u s
g l u c o s e ( F i g u r e 2 B ) , t h e c o l o n y w i t h l i t t l e demand f o r o x y g e n
i s r i c h i n oxygen throughout. Shape o f t h e c o l o n y c h a n g e s a s i t
grows; h e i g h t remains f a i r l y c o n s t a n t as the c o l o n y spreads.
O u t e r p o r t i o n s o f a c o l o n y a r e r i c h i n o x y g e n , and s t e e p g r a d i e n t s
e x i s t t o w a r d t h e a g a r s u r f a c e . Oxygen c a n d i f f u s e t h r o u g h t h e
a i r i n t e r f a c e o f the c o l o n y o r can e n t e r the s u r r o u n d i n g agar
and d i f f u s e t o w a r d t h e c o l o n y a s t h e c o n t o u r s i n d i c a t e . Glucose
i s a l a r g e r m o l e c u l e t h a n o x y g e n and d i f f u s e s more s l o w l y . The
s h a p e o f t h e c o n t o u r l i n e s and t h e i r s t e e p n e s s n e a r t h e a g a r
i n d i c a t e regions of r a p i d metabolic a c t i v i t y . I t s h o u l d be
p o s s i b l e to r e l a t e the contours to r a t e of glucose d i f f u s i o n
through the colony.
T h i s new method f o r i n v e s t i g a t i n g c o l o n i a l g r o w t h and mass
t r a n s f e r c a n be e x t e n d e d t o v a r i o u s o r g a n i s m s and d i f f e r e n t m e d i a .
-
UJ
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' Il \ L *\ *
5
- j\ ' 7 III' ι I \ \\\\ Ν
' 'IV : !
CL ·'/'' '' · '··\
// Μ \ / t II
/ I !
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J ;\ \ο ' : ι ι
6 ν
— 1 ! ί \ £ y : ! 1
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o o -60
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1
1
1 I » / ι
6! 1
1 1 ! \ ' ·
y -80 1 1 51 \ ' ι
LU , 1 1
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ο 1 1
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Figure 2A and B. Contours of dissolved oxygen in colonies of P. ovalis of different sizes. Note
that the colonies are much smaller than those shown in Figures 2C and 2D. Continued on next page.
^
Contours of dissolved oxygen in a colony of P. ovalis. Continued on next page.
Figure 2C.
The r e l e a s e o f o x y g e n f r o m c o l o n i e s o f p h o t o s y n t h e t i c o r g a n i s m
w o u l d be p a r t i c u l a r l y i n t e r e s t i n g . Measurement o f d i s s o l v e d
o x y g e n c o n t o u r s p r o v i d e s new p e r s p e c t i v e s t o t h e a n a l y s i s o f
c o l o n i a l g r o w t h ( 2 ) . F u r t h e r m o r e , b e t t e r u n d e r s t a n d i n g o f mass
t r a n s f e r t o c o l o n i e s h a s economic v a l u e f o r b e t t e r o p e r a t i o n o f
p r o c e s s e s dependent on a t t a c h e d o r g a n i s m s . F o r example, t r i c k
l i n g f i l t e r s f o r b i o l o g i c a l waste treatment, p u r i f i c a t i o n o f
streams by s l i m e s , and f o u l i n g o f c o o l i n g towers by s l i m e s i n
v o l v e c o l o n i a l growth.
Acknowledgement
T h i s p r o j e c t was s u p p o r t e d b y t h e N a t i o n a l S c i e n c e F o u n d a t i o n
t h r o u g h G r a n t //CPE-7920121.
Literature Cited
R E C E I V E D June 1, 1982
Phycomyces Sporangiophores
D u r i n g t h e p a s t 100 y e a r s a number o f l a b o r a t o r i e s h a v e
s t u d i e d the growing " a n t i c s " of the g i a n t , s i n g l e - c e l l e d sporan
g i o p h o r e o f t h e f i l a m e n t o u s f u n g u s , Phycomyces b l a k e s l e e a n u s .
" A n t i c s " i s a p p r o p r i a t e h e r e s i n c e , i n terms o f d e s c r i b i n g t h e
v a r i e t y o f growth p a t t e r n s t h a t t h e mature s p o r a n g i o p h o r e p r o
d u c e s , i t c e r t a i n l y a p p e a r s t o be t h e " c l o w n " o f t h e p l a n t w o r l d .
Even l e f t t o i t s own d e v i c e s and n o t s u b j e c t e d t o any s e n s o r y
i n p u t s , i t i s happy i n a d a r k , damp room a t a b o u t 22°C; i t s b e
h a v i o r i s s t i l l q u i t e e x t r a o r d i n a r y b e c a u s e o f i t s phenomenal
g r o w t h r a t e o f t h r e e m i l l i m e t e r s p e r h o u r (50 ym/min) s i m u l t a n e
o u s l y c o u p l e d w i t h a r o t a t i o n r a t e o f 12 t o 15 d e g r e e s p e r m i n .
Shown on t h e TV m o n i t o r i n F i g u r e 1 i s t h e u p p e r p a r t o f a m a t u r e
Phycomyces s p o r a n g i o p h o r e c o n s i s t i n g o f a c y l i n d r i c a l s t a l k , a b o u t
100 ym i n d i a m e t e r crowned w i t h a s p h e r i c a l s p o r e s a c k , a b o u t 500
pm i n d i a m e t e r .
B i o p h y s i c i s t s have l o n g b e e n f a s c i n a t e d w i t h t h i s o r g a n i s m
because of i t s unique sensory a p p a r a t u s . Both the d i r e c t i o n o f
g r o w t h and t h e r a t e o f g r o w t h a r e q u a n t i t a t i v e l y c o n t r o l l e d by a
v a r i e t y of w e l l - d e f i n e d s t i m u l i : they respond t o both the i n t e n
s i t y and t h e d i r e c t i o n o f e i t h e r v i s i b l e o r UV l i g h t , t o a v a r i e t y
o f w i n d g r a d i e n t s , and t o t o u c h . The o r g a n i s m e l i c i t s c o m p l e x
0097-6156/83/0207-0403$06.00/0
© 1983 American Chemical Society
M a t e r i a l s and M e t h o d s
W i l d - t y p e Phycomyces b l a k e s l e e a n u s s p o r a n g i o p h o r e s ,
N R R L 1 5 5 5 ( - ) , o r i g i n a l l y o b t a i n e d f r o m M. D e l b r i i c k , w e r e grown i n
s h e l l v i a l s c o n t a i n i n g 5.0% p o t a t o d e x t r o s e a g a r (PDA) w i t h 1.0%
yeast extract. The s h e l l v i a l s were i n c u b a t e d u n d e r d i f f u s e i n
c a n d e s c e n t l i g h t i n a h i g h - h u m i d i t y room w i t h a t e m p e r a t u r e r a n g e
E x p e r i m e n t a l R e s u l t s and S t o c h a s t i c M o d e l i n g . M e a s u r e m e n t s
o f r o t a t i o n a l and e l o n g a t i o n a l g r o w t h r a t e s f o r a t y p i c a l e x p e r i
ment a r e shown i n F i g u r e 2. The s a m p l e i n t e r v a l i s one m i n u t e ,
and t h e r e a r e 110 m e a s u r e m e n t s . I t c e r t a i n l y a p p e a r s t h a t b o t h
the g r o w t h and r o t a t i o n s e r i e s a r e h i g h l y i r r e g u l a r i n t e r m s o f
t h e i r growth r a t e s . I t i s d i f f i c u l t t o s e e any s y s t e m a t i c b e h a v
i o r and t h e r e i s l i t t l e a p p a r e n t c o r r e l a t i o n b e t w e e n t h e two.
These q u a l i t a t i v e o b s e r v a t i o n s must be q u a n t i f i e d .
E s t i m a t e s o f t h e a u t o c o r r e l a t i o n f u n c t i o n and t h e p a r t i a l
a u t o c o r r e l a t i o n s f o r the r o t a t i o n a l growth curve i n F i g u r e 2 a r e
p r e s e n t e d i n F i g u r e s 3 and 4. The a u t o c o r r e l a t i o n f u n c t i o n o f t e n
d e p i c t s d e p e n d e n c e o f a measurement v a l u e on r e c e n t p r e v i o u s
v a l u e s w h e r e a s t h e p a r t i a l a u t o c o r r e l a t i o n s show t h e s i g n i f i c a n c e
ί
ISô
s:
ο
&
ο*
s:
c:
2.5
S 55
•β «
i l
II
s: ο
ο
s:
"a
.1
Figure 3. A utocorrelation function of a rotational growth series. Figure 4. Partial autocorrelation of a rotational growth series.
410 BIOCHEMICAL ENGINEERING
g + a + a = e
k l*k-l 2*k-2 k
t h
where g£ i s t h e i element o f t h e s e r i e s ,
a ^ , a£ a r e m o d e l p a r a m e t e r s , and
e± i s t h e i t n
model e r r o r o r r e s i d u a l .
L e a s t s q u a r e s e s t i m a t i o n was a p p l i e d t o t h i s s e r i e s f o r t h e
s e c o n d - o r d e r m o d e l a b o v e , and t h e f o l l o w i n g p a r a m e t e r v a l u e s w e r e
determined:
a x = 0.570
and
a 2 = 0.412
Such a m o d e l d e s c r i b e s a n underdamped o s c i l l a t o r y b e h a v i o r w i t h a
s i n u s o i d a l p e r i o d o f 11 m i n u t e s and damping c o e f f i c i e n t o f 0.44.
This p e r i o d o f o s c i l l a t i o n corresponds t o the "hunting" swings o f
the sporangiophore; t h e r e f o r e , i t appears t h a t o u r model accounts
f o r t h e g r o s s m e c h a n i c a l b e h a v i o r o f Phycomyces and i t s i n t e r
a c t i o n w i t h o u r measurement s y s t e m .
I t i s p o s s i b l e t o remove t h i s s y s t e m a t i c b e h a v i o r f r o m t h e
d a t a and examine l o c a l v a r i a t i o n s i n e l o n g a t i o n a l growth r a t e by
s t u d y i n g t h e r e s i d u a l s e r i e s o f t h e above model. T h i s r e s i d u a l
s e r i e s was a n a l y z e d and f o u n d t o be e s s e n t i a l l y w h i t e : t h e cumu
l a t i v e p e r i o d o g r a m i s p r e s e n t e d i n F i g u r e 8. T h i s i s i n c o n t r a s t
to t h e p e r i o d o g r a m o f t h e o r i g i n a l e l o n g a t i o n a l s e r i e s , s e e F i g
u r e 9, w h i c h shows s i g n i f i c a n t d e v i a t i o n f r o m t h e 45° l i n e . We
c a n summarize t h e a n a l y s i s o f b o t h g r o w t h s e r i e s b y s t a t i n g t h a t
l o c a l g r o w t h b e h a v i o r e x h i b i t e d b y t h e s p o r a n g i o p h o r e i s random
FREQUENCY
Conclusions
We h a v e shown t h a
and e l o n g a t i o n a l g r o w t
correlated. S y s t e m a t i c b e h a v i o r i n e l o n g a t i o n a l g r o w t h was
a t t r i b u t e d to the slower " h u n t i n g " motion of the sporangiophore.
The i r r e g u l a r p a t t e r n s i n r o t a t i o n a l and e l o n g a t i o n a l g r o w t h do
n o t a p p e a r t o be i n t e r d e p e n d e n t n o r r e l a t e d t o t h e same m a j o r
source or stimulus.
Discussion
A l l o r g a n i s m s t h a t p o s s e s s a r i g i d e x o s k e l e t o n such as the
a r t h r o p o d s , i n s e c t s and c r u s t a c e a n s must h a v e e v o l v e d one o f many
p o s s i b l e s t r a t e g i e s i n order to increase i n s i z e , i . e . to uniform
l y i n c r e a s e i n v o l u m e . The a r t h r o p o d s s i m p l y m o l t t h e i r o l d e x o
s k e l e t o n and t h e n r e d e p o s i t a new one a f t e r a s h o r t b u r s t o f i n
t e n s i v e growth. P l a n t s i n g e n e r a l , but f u n g i i n p a r t i c u l a r , have
d e v i s e d a s l i g h t l y d i f f e r e n t s t r a t e g y t o e n a b l e them t o grow e v e n
though they are a l s o encased i n a r a t h e r r i g i d c e l l w a l l . The
new s y n t h e s i z e d c e l l w a l l , t e r m e d t h e p r i m a r y w a l l , a p p e a r s as an
end r e s u l t o f g i a n t c a r b o h y d r a t e m i c r o f i b r i l s b e i n g d e p o s i t e d on
the i n n e r s u r f a c e of the e x i s t i n g p r i m a r y c e l l w a l l . In a d d i
t i o n , i t has b e e n shown t h a t t h e l o n g i t u d i n a l a x i s o f t h e s e n e w l y
l a i d down p o l y m e r s i s p e r p e n d i c u l a r t o t h e n e t d i r e c t i o n o f
growth of the c e l l w a l l i t s e l f . Cell wall thickening resulting
from newly deposited m i c r o f i b r i l s i s c o n s t a n t l y b e i n g counter
b a l a n c e d by c e l l w a l l e x t e n s i o n . The d r i v i n g mechanism o f c e l l
w a l l e x t e n s i o n i s the l a r g e pressure d i f f e r e n c e , the c e l l ' s
t u r g o r p r e s s u r e , b e t w e e n t h e i n s i d e o f t h e c e l l w a l l and i t s
e x t e r n a l e n v i r o n m e n t . B e c a u s e t h e p o l y m e r s a r e l a i d down p e r p e n
d i c u l a r l y to i t s net d i r e c t i o n of growth, during c e l l w a l l exten-
t i o n they are p a s s i v e l y r e o r i e n t e d towards the c e l l ' s l o n g i t u d i
n a l a x e s , and i n a d d i t i o n , t r a n s p o r t e d t o w a r d t h e o u t e r s u r f a c e
o f t h e c e l l w a l l . The m o d e l d e s c r i b e d a b o v e i s b a s i c a l l y known
as t h e m u l t i n e t t h e o r y o f c e l l w a l l g r o w t h f i r s t p r o p o s e d by
R o e l o f s e n and Houwink (12) and e x p e r i m e n t a l l y v e r i f i e d i n N i t e l l a
by G e r t e l and G r e e n ( 1 3 ) .
-.5
-3 -2 -1 θ 1 2 3 4 5 6 7 8 9 18 11 12 13 14 15 16 17
S i n c e t h e s e n e w l y l a i d down m i c r o f i b r i l s a r e i n c o n s t a n t
m o t i o n , t h e p r i m a r y c e l l w a l l b e h a v e s much l i k e a v i s c o - e l a s t i c
fluid. B a r t n i c k i - G a r c i a (14) i n 1970 p r o p o s e d t h a t t h e n e w l y
s y n t h e s i z e d c e l l w a l l i s i n a constant s t a t e of f l u x i n terms of
s y n t h e s i s and d e g r a d a t i o n r e s u l t i n g f r o m t h e p r e s e n c e o f s y n t h e
s i z i n g and l y t i c enzymes, r e s p e c t i v e l y . I n 1974 O r t e g a and Gamow
(15) p r o p o s e d t h a t t h e o r i e n t a t i o n o f m i c r o f i b r i l s , o r b u n d l e s o f
m i c r o f i b r i l s , c o u l d a c c o u n t f o r b o t h c e l l e l o n g a t i o n and c e l l
rotation. They a l s o d e d u c e d t h a t t h e m a g n i t u d e o f t h e r a t i o o f
r o t a t i o n t o e l o n g a t i o n must be a f u n c t i o n o f t h e m i c r o f i b r i l
a n g l e , the a n g l e b e i n g measured i n r e s p e c t to the l o n g i t u d i n a l
a x i s of the growing c e l l w a l l . T h i s model p r e d i c t e d t h a t the
f i b r i l s l o c a t e d i n the lower r e g i o n of the growing zone, those
which would have a l r e a d y m a x i m a l l y r e o r i e n t a t e d towards the l o n g
i t u d i n a l a x i s , w o u l d show maximum r o t a t i o n t o e l o n g a t i o n r a t i o s ,
and t h i s p r e d i c t i o n was
Our p r e s e n t r e s u l t
t h e o r y as p r e s e n t e d a b o v e s i n c e i t c l e a r l y p r e d i c t s t h a t any
i r r e g u l a r i t y i n t h e g r o w t h r a t e w o u l d be d i r e c t l y c o u p l e d w i t h
an i r r e g u l a r i t y i n t h e r o t a t i o n r a t e . Our p r e s e n t d a t a w o u l d be
c o n s i s t e n t w i t h a r e o r i e n t a t i o n m o d e l i n w h i c h t h e maximum r o t a
t i o n and e l o n g a t i o n o c c u r i n d i f f e r e n t r e g i o n s o f t h e GZ. Recent
f i n e s t r u c t u r e s t u d i e s i n our l a b o r a t o r y have c o n f i r m e d the 1958
r e p o r t by Cohen and D e l b r u c k t h a t t h e maximun e l o n g a t i o n r a t e
o c c u r r i n g i n the r e g i o n of the growing zone i s d i s t i n c t l y d i f f e r
e n t f r o m t h a t o c c u r r i n g i n t h e r e g i o n o f maximum r o t a t i o n r a t e .
Our p r e s e n t a n a l y s i s i s c o n s i s t e n t w i t h t h i s f i n d i n g b e c a u s e i f
b o t h r o t a t i o n and e l o n g a t i o n a r e s t o c h a s t i c i n t e r m s o f g r o w t h
i r r e g u l a r i t i e s , we w o u l d n o t e x p e c t t o f i n d a c o r r e l a t i o n i f t h e
r o t a t i o n and e l o n g a t i o n o c c u r r e d i n d i f f e r e n t and i n d e p e n d e n t
r e g i o n s of the growing zone.
L a s t l y , we a r e i n t r i g u e d by t h e s i n u s o i d a l b e h a v i o r , h a v i n g
a p e r i o d o f 11 m i n u t e s , i n t h e e l o n g a t i o n r a t e . A t t h i s t i m e we
h a v e no way o f d e t e r m i n i n g w h e t h e r t h i s o s c i l l a t i o n i s r e a l i n
the sense t h a t the net r a t e of growth o s c i l l a t e s w i t h t h i s p e r i o d
o r w h e t h e r we a r e o b s e r v i n g t h e w e l l - k n o w n h u n t i n g b e h a v i o r ( 1 0 ) ;
Phycomyces s p o r a n g i o p h o r e s show b o t h a f a s t , 5 t o 7.5 m i n , and a
s l o w , 30 t o 60 m i n , o s c i l l a t i o n . Our g u e s s w o u l d be t h a t t h e
1 1 - m i n u t e p e r i o d we h a v e o b s e r v e d i s a d i r e c t r e s u l t o f t h e mea
1
s u r e m e n t e r r o r c a u s e d by t h e s p o r a n g i o p h o r e s h u n t i n g behavior.
I n o r d e r t o c o n f i r m t h i s s p e c u l a t i o n , we w o u l d r e q u i r e f a s t e r
s a m p l i n g r a t e s f o r b o t h e l o n g a t i o n and r o t a t i o n a l g r o w t h . Such
an i n c r e a s e i n s a m p l i n g r a t e w o u l d a l s o f a c i l i t a t e more d e t a i l e d
study of the s t o c h a s t i c growth b e h a v i o r . We b e l i e v e an e x t e n s i o n
o f o u r p r e s e n t measurement t e c h n i q u e w o u l d a l l o w f o r t h i s . This
w o u l d be t o c o u p l e a c o m p u t e r - b a s e d p a t t e r n r e c o g n i t i o n s y s t e m t o
o u r m i c r o s c o p i c v i d e o e q u i p m e n t . By h a v i n g t h e computer f o l l o w
motion of s e v e r a l t r a c k i n g p a r t i c l e s , a high r e s o l u t i o n p r o f i l e
o f b o t h g r o w t h r a t e s i s f e a s i b l e . Such an e x t e n s i o n t o o u r p r e s
e n t a p p a r a t u s w o u l d r e q u i r e l e s s t h a n $100,000 i n c a p i t a l f u n d i n g .
A p p e n d i x : A B r i e f D e s c r i p t i o n o f Time S e r i e s A n a l y s i s
The t e c h n i q u e s o f t i m e s e r i e s a n a l y s i s h a v e b e e n a p p l i e d
a c r o s s a b r o a d s p e c t r u m o f f i e l d s , n o t a b l y b u s i n e s s and e c o n o m i c s ,
e n g i n e e r i n g , and m e t e o r o l o g y . They h a v e g a i n e d w i d e a c c e p t a n c e
p a r t l y due t o t h e h i g h y i e l d o f m o d e l i n g i n f o r m a t i o n one a c h i e v e s
t h r o u g h t h e a p p l i c a t i o n o f a s t r a i g h t - f o r w a r d , e a s y - t o - u s e method
ology. T h e r e a r e two common s c e n a r i o s :
1) a n a l y s i s o f a s i n g l e t i m e s e r i e s , u s u a l l y a s a m p l e d mea
s u r e m e n t s i g n a l , t o d e t e r m i n e i t s s y s t e m a t i c and random
character
2) a n a l y s i s o f two o r more t i m e s e r i e s t o a s s e s s and q u a n
t i f y c o n n e c t i v e r e l a t i o n s h i p s (one must be c a r e f u l w i t h
claims of "cause-and-effect" h e r e — a d d i t i o n a l informa
t i o n on s t r u c t u r
I t i s d e s i r a b l e , from a
i n as s i m p l e a form as p o s s i b l e . Such a r e l i n e a r models w i t h a
m i n i m a l number o f p a r a m e t e r s . T h e r e a r e two p r i m a r y s t e p s t o t h e
m o d e l i n g p r o c e s s : i d e n t i f i c a t i o n o f t h e m o d e l f o r m and e s t i m a t i o n
of the model's parameters. These s t e p s a r e a p p r o p r i a t e l y f o l l o w e d
by t e s t i n g o f t h e m o d e l ' s a b i l i t y t o f i t o r p r e d i c t new d a t a .
I t i s t y p i c a l o f a s i n g l e t i m e s e r i e s t h a t measurements w i l l
show some dependence on t h e i r r e c e n t p a s t h i s t o r y — m a n y p r o c e s s e s
are i n e r t i a l i n behavior. The a u t o c o r r e l a t i o n f u n c t i o n ( o r i t s
e s t i m a t e f r o m t h e s e r i e s ' d a t a ) w i l l r e v e a l s u c h d e p e n d e n c e , and
t h e p a r t i a l a u t o c o r r e l a t i o n s w i l l h e l p show how f a r i n t o t h e p a s t
t h i s " a u t o r e g r e s s i v e " m o d e l must e x t e n d . The m o d e l t h u s s e l e c t e d
c a n be f i t t o t h e d a t a by one o f many r e g r e s s i o n t e c h n i q u e s , e.g.
l i n e a r l e a s t squares. The r e s i d u a l s , t h o s e components o f t h e
d a t a w h i c h a r e n o t e x p l a i n e d by t h e a u t o r e g r e s s i v e m o d e l , f o r m
another s e r i e s . I f the r e s i d u a l s e r i e s i s u n c o r r e l a t e d ( t o i t
s e l f ) , t h i s i n d i c a t e s a random b e h a v i o r w h i c h we c a n n e i t h e r p r e
d i c t n o r r e g u l a t e — w e j u s t have t o l i v e w i t h i t as u n c e r t a i n be
havior. O f t e n , however, i t i s p o s s i b l e t o model t h e r e s i d u a l s
a l s o i n a s i m i l a r , a u t o r e g r e s s i v e manner—which would i n d i c a t e
t h a t a l t h o u g h we c a n n o t r e g u l a t e t h e s e q u a n t i t i e s , we c a n do some
prediction. This s t r u c t u r e o f the r e s i d u a l s e r i e s i s u s u a l l y
b u i l t b a c k i n t o t h e o r i g i n a l m o d e l , and t h e t e r m i n o l o g y u s e d f o r
t h e r e s i d u a l d e s c r i p t i o n i s "moving a v e r a g e . " I t i s then best t o
r e f i t t h e e n t i r e m o d e l by r e g r e s s i o n . A g e n e r a l f o r m f o r t h e
a u t o r e g r e s s i v e - m o v i n g average model i s
g. + a-g. -+...+ a g.
&
= e. + c-e. - +...+
ô
c e.
k l k-l n k-n k 1 k-1 ρ k-p
g, + a- g, -+...+ a g,
6 ô
= b χ, , + b-x. ,-+...+ b χ. ,
&
k l k-l n k-n ο k-d 1 k-d-1 m k-d-m
+ e. + c-e. ,+...+ c e.
k 1 k-1 ρ k-p
may b e p r o p o s e d w h i c h i n c l u d e s t h e a u t o r e g r e s s i v e d e p e n d e n c e o f
the o u t p u t v a r i a b l e ( g ^ ) , a " c a u s e " dependence o n v a l u e s o f t h e
i n p u t v a r i a b l e (x^) and a d e s c r i p t i o n o f r e s i d u a l (e^) b e h a v i o r
as b e f o r e . S e l e c t i o n o f m o d e l f o r m ( w h i c h may i n c l u d e a d e l a y o f
d sample i n t e r v a l s b e t w e e n t h e i n p u t a n d o u t p u t v a r i a b l e s ) i s
f a c i l i t a t e d b y s t u d y o f t h e c r o s s - c o r r e l a t i o n f u n c t i o n and i m p u l s e
response f u n c t i o n estimates i n a d d i t i o n t o the a u t o c o r r e l a t i o n
f u n c t i o n and p a r t i a l a u t o c o r r e l a t i o n s
selected, regression technique
Two m o d e l - c h e c k i n g methods a r e common, and t h e y a r e o f t e n
a p p l i e d t o a "new" s e t o f d a t a . F i r s t , t h e r e s i d u a l s a r e a n a
l y z e d f o r l a c k o f c o r r e l a t i o n o r " w h i t e n e s s . " White n o i s e has
the c h a r a c t e r i s t i c t h a t a l l f r e q u e n c i e s c o n t a i n e d i n i t s h o u l d be
present i n equal strength. The c u m u l a t i v e p e r i o d o g r a m i s t h e
i n t e g r a l o f a spectrum e s t i m a t e ; t h e r e f o r e , f o r w h i t e n o i s e , i t
s h o u l d b e t h e i n t e g r a l o f a c o n s t a n t o r a l i n e a r ramp f u n c t i o n .
The " w h i t e n e s s " o f t h e r e s i d u a l s c a n b e a n a l y z e d b y p l o t t i n g
t h e i r c u m u l a t i v e p e r i o d o g r a m and o b s e r v i n g c l o s e n e s s t o t h e i d e a l
ramp l i n e . A n o t h e r common d i a g n o s t i c t e c h n i q u e i s t o d e t e r m i n e
the s t a t i s t i c a l e f f i c i e n c y o f a d d i n g one o r two p a r a m e t e r s ( a n d
terms) t o t h e model. T h i s w i l l check i f t h e minimal p a r a m e t r i c
m o d e l c h o s e n i s t r u l y p a r s i m o n i o u s . The s t a t i s t i c a l F - t e s t b a s e d
on t h e e x t r a - s u m - o f - s q u a r e s p r i n c i p l e i s u s e f u l f o r t h i s e f f i
ciency test.
Literature Cited
R E C E I V E D June 1, 1 9 8 2
0097-6156/83/0207-0421$06.50/0
© 1983 American Chemical Society
c o n c e n t r a t i o n s o f 20-30% from h y d r o l y s i s o f c e l l u l o s e i n a p r o c e s s
for ethanol p r o d u c t i o n f r o m c e l l u l o s i c m a t e r i a l s . I t has a l s o
been c o n f i r m e d ( 6 ) t h a t c e l l u l a s e a c t i v i t y per u n i t volume can be
i n c r e a s e d by i n c r e a s i n g t h e c e l l u l o s e c o n c e n t r a t i o n i n t h e medium.
But i t i s n o t p o s s i b l e to handle more t h a n 6% cellulose in
c o n v e n t i o n a l fermenter because o f r h e o l o g i c a l problems. In o r d e r ,
t h e r e f o r e , t o i n c r e a s e t h e c e l l u l o s e c o n c e n t r a t i o n h i g h e r t h a n 6%,
SSF seems t o be t h e most a t t r a c t i v e a l t e r n a t i v e (5)· To yam a (_7)
has a l r e a d y shown t h a t s u g a r s y r u p o f 22% c o n c e n t r a t i o n can be
o b t a i n e d by h y d r o l y z i n g d e l i g n i f i e d r i c e s t r a w o r b a g a s s e w i t h t h e
c o n c e n t r a t e d c e l l u l a s e s o b t a i n e d by T r i c h o d e r m a r e e s e i i n SSF.
R e c e n t l y , i n t e r e s t i n t h e p r o d u c t i o n o f a m y l a s e s and c e l l u l a s e s b y
SSF i s i n c r e a s i n g b e c a u s e a g r e a t demand o f t h e s e enzymes i s
envisaged i n t h e n e a r f u t u r e . These enzymes a r e r e q u i r e d t o
c o n v e r t s t a r c h and c e l l u l o s e i n t o g l u c o s e f o r f u r t h e r f e r m e n t a t i o n
i n t o e t h a n o l to a l l e v i a t
0 5 ) . But t h e s u r v e y o
is known a b o u t t h e g r o w t h c h a r a c t e r i s t i c s and behavior of
m i c r o o r g a n i s m s i n SSF.
O r i g i n of S o l i d S t a t e Fermentation
L i g n i n i n the p l a n t c e l l w a l l n o t o n l y e n c r u s t s t h e c e l l u l o s e
m i c r o f i b r i l s i n a s h e a t h - l i k e manner, b u t i s bonded p h y s i c a l l y and
c h e m i c a l l y t o t h e p l a n t p o l y s a c c h a r i d e s (16)« Lignin-carbohydrate
bonds form m e t a b o l i c b l o c k s that greatly limit the a c t i o n o f
m i c r o b i a l h e m i c e l l u l a s e s and c e l l u l a s e s . P h y s i c a l l y , l i g n i n f o r m s
a b a r r i e r s u p p r e s s i n g t h e p e n e t r a t i o n by p o l y s a c c h a r i d e - d i g e s t i n g
enzymes ( 17 ) . U n l e s s t h e l i g n i n i s d e p o l y m e r i z e d , s o l u b i l i z e d , o r
removed, t h e cellulose and the hemicelluiοses cannot be
metabolized by most microorganisms· As m e n t i o n e d e a r l i e r , o n l y
w h i t e - r o t and b r o w n - r o t f u n g i ( B a s i d i o m y c e t e s ) a r e a b l e t o u t i l i z e
carbohydrates from l i g n o c e l l u l o s e s but t h e y a r e v e r y s l o w growing
(4,14,15,18,19). Therefore, these f u n g i are not v e r y promising,
from the economic p o i n t o f v i e w as candidates to ferment
l i g n o c e l l u l o s e s f o r the p r o d u c t i o n o f a n i m a l feed or cellulases.
The case is s i m i l a r with other basidiomycetes especially
mushrooms. A g a r i c u s bisporus
v o l v a c e a , and P l e u r o t u
p r o d u c e f r u i t i n g b o d i e s ( m u s h r o o m s ) . Mushroom g r o w i n g , however,
i s j u s t i f i e d b e c a u s e o f t h e i r economic v a l u e a s f o o d d e l i c a c i e s .
Pretreatment of L i g n o c e l l u l o s e s
c h l o r i t e t r e a t e d s u b s t r a t e , on t h e other h a n d , i s composed of
h e m i c e l l u l o s e s and c e l l u l o s e , w h i c h w o u l d be a good s u b s t r a t e f o r
the production of hemicellulases ( e s p e c i a l l y xylanases) and
cellulases s i m u l t a n e o u s l y i n m o n o c u l t u r e or mixed c u l t u r e . There
is a great future f o r s u c h an enzyme s y s t e m ( m i x t u r e of
h e m i c e l l u l a s e s and c e l l u l a s e s ) f o r the complete h y d r o l y s i s o f
l i g n o c e l l u l o s e s i n t o monomer s u g a r s .
Choice of Microorganism
In n a t u r e , b a c t e r i a g r o w b e s t o n l y when i n a l i q u i d p h a s e , o r
at least when t h e n u t r i e n t s are i n free water. Likewise,
s i n g l e - c e l l e d f u n g i , t h e y e a s t s , g r o w w e l l when t h e n u t r i e n t s are
in a s o l u b l e f o r m . Such m i c r o o r g a n i s m s may n o t be a b l e t o g r o w
s u c c e s s f u l l y i n SSF w h e r e s u b s t r a t e c a r b o n i s n o t available in
soluble form. A limite
m a t e r i a l s i n t o animal fee
Alcaligenes faecalis) or yeasts ( A u r e o b a s i d i u m p u l l u l a n s and
C a n d i d a u t i l i s ) has b e e n r e p o r t e d i n s e m i - s o l i d s t a t e f e r m e n t a t i o n
( 3 , 3a) . Low protein yields i n the final product m i g h t be
a t t r i b u t e d to the fact that i n such system the unicellular
o r g a n i s m s ( b a c t e r i a and y e a s t s ) were u n a b l e t o p e n e t r a t e d e e p i n t o
the t i s s u e f o r complete u t i l i z a t i o n o f the s u b s t r a t e .
Streptomycetes have not been t r i e d f o r SSF so f a r but
H e s s e l t i n e (_1) has r e p o r t e d t h a t he has seen a preparation in
w h i c h S t r e p t o m y c e s a u r e o f a c i e n s was grown on m i l l e t s e e d s , t o be
used i n s w i n e f e e d m i x e s i n t h e U.S.S.R.
On the o t h e r h a n d , f i l a m e n t o u s f u n g i t y p i c a l l y g r o w i n n a t u r e
on s o l i d s u b s t r a t e s , s u c h a s , wood, s e e d s , s t e m s , r o o t s and l e a v e s
of p l a n t s , w i t h o u t t h e p r e s e n c e o f f r e e w a t e r (_1) . H i g h p r o t e i n
c o n t e n t s o f 20-24% i n t h e f i n i s h e d p r o d u c t h a v e b e e n r e c o r d e d by
Chahal et a l . (2) i n SSF of corn stover with Chaetomium
cellulolyticum. High p r o t e i n content i n the final product has
b e e n a t t r i b u t e d t o t h e f a c t t h a t t h e hyphae o f C. c e l l u l o l y t i c u m
h a v e t h e power t o p e n e t r a t e deep i n t o the intercellular and
i n t r a c e l l u l a r spaces f o r b e t t e r u t i l i z a t i o n o f the s u b s t r a t e ( 4 0 ) .
Most f i l a m e n t o u s f u n g i h a v e s u c h i n t r u s i o n power. A i s t (41) has
given a good r e v i e w o f t h e p e n e t r a t i o n o f f u n g a l h y p h a l t i p s i n t o
p l a n t c e l l w a l l s . He concluded that t h e mechanism o f fungal
penetration i n t o s u b s t r a t e s c o u l d be m e c h a n i c a l a n d / o r e n z y m a t i c .
I t i s , t h e r e f o r e , e v i d e n t t h a t the c h o i c e of the microorganisms
f o r s u c c e s s f u l SSF i s l i m i t e d t o f i l a m e n t o u s o r g a n i s m s - f u n g i and
actinomycetes.
F u n g a l hyphae a r e s e p t a t e , t u b u l a r , u n i s e r i a t e f i l a m e n t s , on
a v e r a g e 10-15 ym i n d i a m e t e r . The g r o w t h i n l e n g t h o c c u r s a t t h e
t i p and i s c o n f i n e d t o t h i s a r e a , so t h a t i n s e p t a t e hyphae when a
cell i s cut o f f from the apex i t i s no l o n g e r c a p a b l e o f a n y
Figure 1. Bore holes in spruce tracheid made by the hyphae of white rot fungus
P o l y p o r u s versicolor (X 20,250). Reproduced, with permission, from Ref. 43.
Copyright 1965, Syracuse University Press.
Figure 2. Advanced stage of decay around bore hole and inner side of secondary
wall of two fiber tracheids of sweetgum sapwood, X 2700. Reproduced, with
permission, from Ref. 43. Copyright 1965, Syracuse University Press.
a s w e l l as on t h e e n d s o f t h e s u b s t r a t e p a r t i c l e . Such b r o k e n and
exposed c e l l s were t h e f i r s t s i t e s t o be a t t a c k e d by t h e o r g a n i s m .
F i g u r e 6 shows m y c e l i a l g r o w t h on s u c h b r o k e n e x p o s e d c e l l s o n t h e
l a t e r a l s i d e o f the s u b s t r a t e p a r t i c l e .
When s u c h s u b s t r a t e p a r t i c l e s , c o l o n i z e d by the organism,
were p r e s s e d slightly under a cover s l i p , they d i s i n t e g r a t e d
easily. The d i s i n t e g r a t e d m a t e r i a l c o n t a i n e d some s u b s t r a t e c e l l s
s h o w i n g p e n e t r a t i o n by hyphae and d e v e l o p m e n t o f m y c e l i a l g r o w t h i n
the c e l l lumina a t v a r i o u s s t a g e s . In F i g u r e 7 p e n e t r a t i o n of a
s i n g l e hypha i n t o the l u m e n o f a t h i n l o n g i t u d i n a l f i b e r c e l l i s
c l e a r l y s e e n . As s o o n a s t h e hypha e s t a b l i s h e s i t s e l f in the
lumen a t h i c k m y c e l i a l mass i s d e v e l o p e d w i t h i n t h e c e l l ( F i g u r e
8). I t was c o n c l u d e d f r o m t h e s e o b s e r v a t i o n s t h a t t h e hyphae of
C. c e l l u l o l y t i c u m entered i n t o the c e l l lumen t h r o u g h n a t u r a l
openings, mechanical breaks, or spaces ( c r e a t e d by s o l u b i l i z a t i o n
o f hemic e l l u i ο se s and l i g n i
the c e l l w a l l of the substrat
lumen, the hypha d i g e s t e d the c e l l w a l l s t a r t i n g f r o m the i n s i d e
towards the o u t s i d e . U l t i m a t e l y , the cell collapsed leaving
behind m y c e l i a l biomass r i c h i n p r o t e i n . Reese ( 4 8 ) has a l s o
r e p o r t e d t h a t i n a t t a c k i n g c o t t o n , many f u n g i p e n e t r a t e d through
t h e f i b e r w a l l i n t o t h e lumen and d i d most o f t h e i r d i g e s t i n g f r o m
within (Figure 9). S i m i l a r l y B r a v e r y ( 4 6 ) has r e p o r t e d the g r o w t h
o f P o l y s t i c t u s v e r s i c o l o r i n t h e wood c e l l lumen and has shown
t h a t d i g e s t i o n of the c e l l i s i n i t i a t e d from i n s i d e to outside
(Figure 3).
P o s t u l a t e d Mechanism o f F u n g a l G r o w t h i n S o l i d State
1. P l a u s i b l y p h y s i c a l s i g n a l s a r e t r a n s m i t t e d from c e l l u l o s e to
the f u n g a l c e l l n u c l e u s t o t r i g g e r the s y n t h e s i s o f the f i r s t
enzyme ( E ) , w h i c h
1 causes splitting of fibrils and
microfibrils f r o m the c r y s t a l l i n e p o r t i o n o f c e l l u l o s e f i b e r .
The a c t i o n o f E- h e r e i s t h e same a s e x p l a i n e d by Reese (48)
for C^. The is still a c o n t r o v e r s i a l enzyme and i s
c o n f u s e d w i t h e x o - 1 , 4 - $ - D - g l u c a n c e l l o b i o h y d r o l a s e . The most
important r o l e of C^ to s p l i t g l u c o s e polymer c h a i n s from
c r y s t a l l i n e c e l l u l o s e p r o p o s e d i n 1950 ( 5 9 ) i s s t i l l retained
here as w e l l as b y Reese ( 6 0 ) . T h e r e f o r e , t h e r e i s e v e r y
p o s s i b i l i t y t h a t c l o s e c o n t a c t o f c e l l u l o s e and the organism
i s most i m p o r t a n t t o p r o d u c e C^(E^) enzyme, i f n o t , f o r o t h e r
components o f c e l l u l a s e s ( e x o - and e n d o - g l u c o n a s e s ) .
Figure 6. Hyphal growth on sites like those shown in Figure 5. From Ref. 40.
Figure 7. Penetration by a single hyphae into cell lumen through the broken end
(X 94.5). From Ref. 40.
Figure 8. Mycelial biomass developed in the cell lumen (X 94.5). From Ref. 40.
Figure 10. Utilization of secondary wall from the inside of the lumen of wood
cell, as explained in the text.
3. C e l l o b i o s e i s a b s o r b e d b y t h e f u n g a l c e l l s where i t t r i g g e r s
t h e s y n t h e s i s o f t h e t h i r d enzyme, 3 - g l u c o s i d a s e ( E ~ ) t o b r e a k
c e l l o b i o s e into glucose u n i t s . The E^ i s a n i n t r a c e l l u l a r
enzyme b u t a s m a l l q u a n t i t y i s a l s o s e c r e t e d o u t s i d e o f t h e
fungal c e l l . Larg
the older cells o
Thus hydrolyzes c e l l o b i o s e into glucose u n i t s i n s i d e as
w e l l as o u t s i d e the fungal c e l l s .
Physiological Aspects
T e m p e r a t u r e . D u r i n g SSF t h e t e m p e r a t u r e i n t h e s u b s t r a t e
rises due to heat generated b y m e t a b o l i s m . The r i s e i n
t e m p e r a t u r e i s d i r e c t l y r e l a t e d t o t h e d e p t h o f t h e s u b s t r a t e and
the m e t a b o l i c a c t i v i t i e s o f t h e organisms. I t has been recorded
(61) that d u r i n g composting o f straw f o r mushroom g r o w i n g , t h e
temperature o f the outer l a y e r o f t h e compost h e a p ( o n e m e t e r
h i g h ) i s a r o u n d 37 C, w h i l e i n t h e i n n e r p o r t i o n t h e t e m p e r a t u r e
rises as high a s 60-77 C. On t h e o t h e r h a n d , i n t h e i n n e r m o s t
region the temperature reaches o n l y 32-49 C b e c a u s e o f l o w
m i c r o b i a l a c t i v i t y , due t o a n a e r o b i c c o n d i t i o n s i n t h a t r e g i o n .
Edwards ( 6 2 ) had r e p o r t e d t h a t o n e K g ( d r y w e i g h t ) o f o r g a n i c
matter ( c e l l u l o s e ) when consumed b y m i c r o o r g a n i s m p r o d u c e d 0.597
K g o f w a t e r , 1.465 K g o f C 0 a n d 14,960 ΒTU. Some o f t h e h e a t
2
A e r a t i o n . A e r a t i o n has t h e f o l l o w i n g f u n c t i o n s i n SSF:
( i ) To s u p p l y 0^ f o r a e r o b i c m e t a b o l i s m .
( i i ) To c o n t r o l the t e m p e r a t u r e .
(iii) To remove CO^, w a t e r v a p o u r s , and v o l a t i l e m e t a b o l i t e s
produced d u r i n g metabolism.
The problem of a e r a t i o n w i l l i n c r e a s e c o r r e s p o n d i n g l y w i t h
the i n c r e a s e i n the thickness of the substrate layer. Proper
aeration i n the SSF a l s o d e p e n d s o n t h e a i r s p a c e s a v a i l a b l e i n
t h e s u b s t r a t e . F i b r o u s m a t e r i a l s c a n p r o v i d e more a i r s p a c e s t h a n
finely powdered s u b s t r a t e . A e r a t i o n seems t o be a v e r y c r i t i c a l
requirement. O c h r a t o x i n p r o d u c t i o n by A s p e r g i l l u s o c h r a c e u s i n a
r o t a t i n g drum f e r m e n t e r s t o p p e d when t h e a i r f l o w r a t e was g r e a t e r
t h a n 0.1 l i t e r / K g / m i n u t e ( 6 3 ) . I n c o n t r a s t , t h e increase in air
flow rate through a column of c o r n i n o c u l a t e d w i t h A s p e r g i l l u s
f l a v u s i n c r e a s e d the r a t e o f production of a f l a t o x i n (64-65).
S i m i l a r l y i t was reporte
p r o d u c t i o n by A s p e r g i l l u
c o n s i d e r a b l y when t h e a e r a t i o n was i n c r e a s e d .
Moisture. In n a t u r e , vegetables and fruits contain about
60-80% m o i s t u r e . T h i s much m o i s t u r e i s s u f f i c i e n t t o e n c o u r a g e
t h e g r o w t h o f m i c r o o r g a n i s m s (66,67 ) . But Christensen (68)
reported t h a t E u r o t i u m h a l o p h i l i c u m c a n g r o w on wheat g r a i n s a t
13-14% m o i s t u r e . M o r t o n and E g g i n s ( 6 9 ) have a l s o r e p o r t e d that
some fungi like A b s i d i a corymbifera, Fusarium s o l a n i and
Chaetomium trilatérale can g r o w and p e n e t r a t e i n wood a t a 14%
m o i s t u r e l e v e l a t 35°C.
Growth o f s o f t - r o t f u n g i i n s i t u a t i o n s o f e x t r e m e d r y n e s s has
b e e n r e c o r d e d by Duneun and E s l y n ( 7 0 ) . A b a s i d i o m y c e t e , S e r p u l a
l a c r y m a n s i s w e l l known t o c o l o n i z e d r y timber i n buildings
causing the c o n d i t i o n s known as " d r y r o t " . T h i s i s a c h i e v e d by
the fungus i n i t i a t i n g growth i n timber w i t h a h i g h water content
and then using the e n e r g y and n u t r i e n t s gained to produce
r h i z o m o r p h s w h i c h c a n e x p l o r e d r y t i m b e r many m e t e r s away f r o m the
initial colony. Though s u c h r h i z o m o r p h s c a n transport water,
f u r t h e r water i s produced a t the s i t e o f a c t i o n by the u t i l i z a t i o n
o f t h e t i m b e r w i t h t h e e v o l u t i o n o f CO- ( 7 1 ) .
V a r i o u s l e v e l s o f m o i s t u r e i n SSF have been r e p o r t e d for
different products. About 7 5 % m o i s t u r e i n s t r a w was used f o r SCP
p r o d u c t i o n (2 ,3,3a) w h e r e a s f o r a f l a t o x i n p r o d u c t i o n i t was 33.3%
in rice and 48.4% i n s t r a w (1). Thus m o i s t u r e l e v e l i n t h e SSF
d e p e n d s on t h e n a t u r e o f t h e s u b s t r a t e , o r g a n i s m and the type of
end product.
pH. M o s t f u n g i a r e a b l e t o g r o w i n a w i d e pH r a n g e o f 5-8.
A d j u s t m e n t o f pH d u r i n g t h e g r o w t h i n SSF i s v e r y d i f f i c u l t . Some
o f t h e s u b s t r a t e ( s t r a w s , g r a i n s ) have good b u f f e r i n g c a p a c i t y and
t h e r e may not be any need t o a d j u s t t h e pH d u r i n g SSF. But
maintenance of pH a r o u n d 4.5 is very crucial for cellulase
p r o d u c t i o n w i t h T. r e e s e i (_5 ) .
Osmotic P r e s s u r e . Raising the osmotic potential of a
material by a d d i t i o n of s a l t or sugar or both to a l e v e l higher
t h a n t h a t o c c u r i n g i n the c y t o p l a s m i s a w e l l t r i e d method of
microbial i n h i b i t i o n and i s w i d e l y u s e d i n p r e s e r v a t i o n o f f o o d s ,
s u c h a s meats and f r u i t s . Sugar c o n c e n t r a t i o n s i n t h e r e g i o n s of
50-70% a r e u s u a l l y a d e q u a t e ; s a l t i s added t o 2 0 - 2 5 % l e v e l ( 7 1 ) .
However, t h e r e are some o s m o p h i l i c and halophyllic organisms
( S a c c h a r o m y c e s r o u x i i , S. m e l l i s and A s p e r g i l l u s g l a u c u s s e r i e s )
which can grow i n c o n c e n t r a t e d sugar solutions (71 ) . High
concentrations of salts ( n u t r i e n t s ) are used i n SSF f o r SCP
p r o d u c t i o n from s t r a w ( 2 ) . T h e r e f o r e , t h e m i c r o o r g a n i s m s capable
o f w i t h s t a n d i n g h i g h o s m o t i c p r e s s u r e w i l l be more s u i t a b l e under
s u c h c o n d i t i o n s , o t h e r w i s e , t h e r e q u i r e d n u t r i e n t s a r e t o be added
in frequent s m a l l doses to a v o i d h i g h osmotic p o t e n t i a l i n the
substrate.
C h a r a c t e r i s t i c s o f an O r g a n i s m f o r SCP and C e l l u l a s e P r o d u c t i o n
P r o d u c t i o n o f SCP an
depend on t h e t h o r o u g h u n d e r s t a n d i n
b e h a v i o r o f SCP and cellulase producing organisms under such
conditions before the a p p l i c a t i o n o f b i o c h e m i c a l e n g i n e e r i n g to
s c a l e up t h e p r o c e s s e s . The o r g a n i s m f o r s u c c e s s f u l SSF should
have t h e f o l l o w i n g p r i m a r y q u a l i t i e s :
1. I t i s a b l e to u t i l i z e mixtures o f v a r i o u s p o l y s a c c h a r i d e s .
2. I t s h o u l d have c o m p l e t e enzyme s y s t e m s t o s w i t c h over i t s
metabolism f r o m one p o l y s a c c h a r i d e t o a n o t h e r p o l y s a c c h a r i d e
( f o u n d i n c o m p l e x s u b s t r a t e s ) w i t h o u t any l a g p h a s e .
3. It i s able to penetrate deep i n t o the t h i c k l a y e r o f the
s u b s t r a t e as w e l l as i n t o t h e c e l l s o f t h e s u b s t r a t e .
4. I t i s a b l e to grow i n h i g h c o n c e n t r a t i o n s o f n u t r i e n t s .
5. I t grows i n a v e g e t a t i v e f o r m and d o e s n o t sporulate during
the f e r m e n t a t i o n t i m e .
6. I t s h o u l d be f a s t g r o w i n g t o a v o i d c h a n c e s o f c o n t a m i n a t i o n by
other fast-growing organisms.
7. I t i s a b l e to grow i n low m o i s t u r e content o f the s u b s t r a t e .
8. It i s able t o g r o w i n t h e p r e s e n c e o f p h e n o l i c and t o x i c
compounds p r o d u c e d d u r i n g t h e p r e t r e a t m e n t o f t h e s u b s t r a t e .
Acknowledgement
The a u t h o r i s v e r y g r a t e f u l t o D r . D.J. K u s h n e r , p r o f e s s o r o f
Microbiology, University of O t t a w a , O t t a w a , O n t a r i o , Canada f o r
h i s i n v a l u a b l e s u g g e s t i o n s and f o r e d i t i n g t h e m a n u s c r i p t .
Literature Cited
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American Soc. Animal S c i . . 1976, 27, 189-191.
4. Z a d r a z i l , F. European J. A p p l . M i c r o b i o l . 1977, 4, 273-281.
5. Chahal, D.S. "Enzymatic Hydrolysis of Cellulose -
State-of-the A r t " . N.R.C. Canada, Report. 1982, pp. 101.
6. Nystrom, J.M. and DiLuca, P.H. Ρroc. of B i o c o n v e r s i o n Symp.
I.I.T., New D e l h i , 1977, 293-304.
7. Toyama, N. B i o t e c h o l . Bioeng. Symp. No. 6, 1976, 207-219.
8. Chang, S.T. "The Chinese Mushroom". The Chinese U n i v e r s i t y
o f Hong Kong, Shatin, N.T. 1972.
9. M i a l l , L.M. i n "The Filamentous Fungi" vol. 1, ed. Smith,
J.E.; Berry, D.R. Edward A r n o l d . 1975; 104-121.
10. Hayes, W.A.; N a i r , N.G. i n "The Filamentous Fungi"; v o l . 1,
ed. Smith, J.E.; B e r r y , D.R. Edward A r n o l d . 1975; 212-248.
11. Takamine, J . J . Indust Eng Chem 1914 6, 824-828
12. Wood, B.J.B.; Min
ed. Smith, J.E.; Berry
13. Thorn,C. "The Penicillia" Pailliere, T i n d a l & Cox, London,
1930.
14. Cochrane, V.W. "Physiology o f Fungi" John Wiley and Sons,
Inc. New-York. 1958; p. 524.
15. Detroy,R.W.; L i n d e n f e l s e r , L . A . ; S t . J u l i a n J r . , G.;0rton,
W.L. B i o t e c h o l . Bioeng. Symp. No. 10. 1980, 135-148.
16. H i g u c h i , T. Adv. Enzymology. 1971, 34, 207-277.
17. K i r k , T.; Haskin, J.M. ACS Symp. S e r . 69, 1973, 124-126.
18. Chahal, D.S.; Gray, W.D. in " B i o d e t e r i o r a t i o n o f M a t e r i a l s -
Microbiological and A l l i e d Aspects", e d s . Walter, A.H.;
E l p h i c , J.S. E l s e v e i r Publ. Co. Barking, Essex, England.
1968, p. 584-593.
19. Kuhlman, E.G. Canadian J. Botany, 1970, 48, 1787-1793.
20. Chahal, D.S. i n "Advances in A g r i c u l t u r a l Microbiology" ed.
Subba Rao, N.S. Oxford and IBH P u b l . Co., New D e l h i 1982 ( i n
Press).
21. M i l l e t t , M.A.; Baker, A.J.; S a t t a r , L.D. B i o t e c h o l . Βioeng.
Symp. No. 6 1976, 125-153.
22. Tarkow, H.; Feist, W.D. Adv. Chem. S e r . 95, 1969, 197-218.
23. Fan, L.T.; Lee, Y.-H.; Beardmore, D.H. B i o t e c h o l . Bioeng.
1980, 22, 177-199.
24. F i e s t , W.C.; Baker, A.J.; Tarkow, H. J. Animal S c i . , 1970,
30, 832-835.
25. M i l l e t , M.A.; Baker, A.J.; Fiest, W.C.; Mellenberger, R.W.;
S a t t a r , L.D. J. Animal S c i . , 1970, 31, 781-788.
26. Chahal, D.S.; Moo-Young, M. Develop. Indust. M i c r o b i o l . 1981,
22, 143-159.
27. Bender, R. U.S. Patent No. 4, 136, 207, 1979.
28. Casebier, R.L.; Hamilton, J.K.; Hergert, H.L. T a p p i , 1969,
52, (12) 2369-2377.
29. Lora, J.H.; Wayman, M., T a p p i , 1978, 61, ( 6 ) 47-50.
30. Noble, G. " F i n a l Report submitted to U.S. Dept. Energy,
Fuels from Biomass Program", Iotech Corporation L t d . Ottawa,
R E C E I V E D June 1, 1982
1
J. E. ROLLINGS , M. R. OKOS, and G. T. TSAO
Purdue University, West Lafayette, IN 47907
1
Current address: Worcester Polytechnic Institute, Worcester, MA 01609
0097-6156/83/0207-0443$06.00/0
© 1983 American Chemical Society
Experimental
P u r i f i e d c o r n s t a r c h p r e p a r e d by wet m i l l i n g
was o b t a i n e d f r o m Sigma C h e m i c a l Company, S t .
L o u i s , MO ( l o t no. 68C-0244) and used as s u b s t r a t e .
A c o m m e r c i a l g r a d e , e n d o - a c t i n g α-amylase used t o
h y d r o l y s e the s t a r c h s u b s t r a t e was a g i f t o f Novo
L a b o r a t o r i e s , W i l t o n , CT (Thermamy 1 - h i g h temper
a t u r e s t a b l e enzyme). A s t o c k enzyme s o l u t i o n
was p r e p a r e d by d i l u t i n g th e c o m m e r c i a l g r a d e
l i q u i d enzyme ( 1 : 1 9 ) w i t h 0.01 M a c e t a t e b u f f e r
pH 6.0 and s t o r e d a t 40°C. T h i s enzyme s o l u t i o n
was u s e d f o r t h e h y d r o l y s i s e x p e r i m e n t s as
d e s c r i b e d below. A l l c h e m i c a l s used i n t h i s s t u d y
were r e a g e n t g r a d
The r e a c t o
s t a n d a r d two l i t e r Ace G l a s s w a r e , f o u r - p o r t b a t c h
reactor. The c e n t r a l p o r t was u s e d t o h o u s e t h e
a g i t a t o r s h a f t i n a j a c k e t e d column. The a g i t a t o r
was powered by a v a r i a b l e speed m o t o r o p e r a t i n g a t
140 rpm. The t h r e e s i d e p o r t s w e r e u s e d t o h o u s e
t h e i n t e r n a l t h e r m i s t o r , a NBS t h e r m o m e t e r , and a
t e f l o n p l u g f o r the sampling p o r t . The r e a c t o r
was t h e r m o s t a t e d b o t h i n t e r n a l l y and e x t e r n a l l y i n
a well-mixed o i l bath. T h i s thermostated system
was f o u n d t o a c h i e v e a d e s i r e d t e m p e r a t u r e w i t h i n
t h r e e h o u r s and t o m a i n t a i n t h e t e m p e r a t u r e w i t h i n
± 0.3°C c o n t i n u o u s l y t h e r e a f t e r . A schematic
r e p r e s e n t a t i o n o f t h e a p p a r a t u s i s shown i n F i g u r e 1
S t a r c h h y d r o l y s a t e s a m p l e s were e x t r a c t e d f r o m
t h e b a t c h r e a c t o r a t t i m e d i n t e r v a l s and a s s a y e d by
e i t h e r t r a d i t i o n a l end g r o u p a n a l y s i s as d e s c r i b e d
p r e v i o u s l y (JLl) o r by SEC. The SEC a p p a r a t u s u s e d
i n t h e s e s t u d i e s i s shown i n F i g u r e 2. No s i n g l e
c h r o m a t o g r a p h i c r e s i n column i s c a p a b l e o f r e s o l v
i n g more t h a n two and one h a l f d e c a d e s i n m o l e c u l a r
w e i g h t f o r random c o i l p o l y m e r s (_12). During
l i q u e f a c t i o n , s t a r c h m o l e c u l e s range i n m o l e c u l a r
s i z e f r o m a p p r o x i m a t e l y 10** dJl/g t o a p p r o x i m a t e l y
10^ dA/g (9). I n o r d e r t o extend the m o l e c u l a r s i z e
r a n g e and t h e r e b y o b t a i n a m a x i m a l amount o f
m o l e c u l a r i n f o r m a t i o n f r o m each e x p e r i m e n t , a two-
column SEC s y s t e m was d e v e l o p e d w h i c h i s c a p a b l e o f
s e p a r a t i n g 4.5 - 5.0 d e c a d e s i n m o l e c u l a r s i z e f o r
water s o l u b l e polymers ( 1 0 ) . T h i s system c o n s i s t s
o f a 25.6 cm l o n g S e p h a r o s e CL-6B column c o u p l e d
w i t h a 17.85 cm l o n g S e p h a r o s e CL-2B c o l u m n . Each
column was 8 mm i n i n s i d e d i a m e t e r . The c h r o m a t o
g r a p h i c r e s i n s p a c k e d i n t h e s e columns were f r a c t i o n
a t e d by a wet s i e v i n g t e c h n i q u e t o o b t a i n b e a d s o f
Motor
Positive
Displacement w
Solvent
Filter
Conical Flask
Solvent Reservoir
Rheodyne Loop
Injector
Chromatographic
Column
American Chemical
Society Library
1155 16th St., N-W.
In Foundations of Biochemical Engineering; Blanch, H., et al.;
Washington,
ACS Symposium Series; U.C. 20036
American Chemical Society: Washington, DC, 1983.
448 BIOCHEMICAL ENGINEERING
V - V
K U
AV V - V
t ο
t o t a l i n t e r s t i t i a l volume o f t h e c o l u m n .
R e s u l t s and Discussion
In o r d e r t o d e s c r i b e the k i n e t i c s o f enzymatic
s t a r c h d e p o l y m e r i z a t i o n , i n f o r m a t i o n on r e a c t i o n
r a t e , r e a c t i o n e x t e n t , and p r o d u c t d i s t r i b u t i o n
p r o f i l e s are r e q u i r e d . T r a d i t i o n a l end-group ana
l y s i s can be u s e d t o a l i m i t e d e x t e n t i n t h e f i r s t
two a r e a s , b u t w i l l n o t p r o v i d e i n f o r m a t i o n a b o u t
the l a s t important s u b j e c t . H e n c e , SEC p r o f i l e s
can p r o v i d e s u f f i c i e n t i n s i g h t i n t o t h e mechanism
of starch degradation.
E f f e c t o f T e m p e r a t u r e on R e a c t i o n R a t e . The
i n i t i a l r a t e o f s t a r c h h y d r o l y s i s was i n v e s t i g a t e d
a t f i v e t e m p e r a t u r e s (40°C, 57.5°C, 67.5°C, 80°C
and 95°C) u s i n g t h r e e d i f f e r e n t s u b s t r a t e c o n c e n
t r a t i o n s ( 0 . 4 4 % , 0.88%, and 1.76% w t / v o l ) by
τ 1
r—— 1
r
m o n i t o r i n g the p r o d u c t i o n of r e d u c i n g sugars
( d e x t r o s e e q u i v a l e n c e , D.E.). At each i s o t h e r m ,
the i n i t i a l r a t e d a t a were shown t o f o l l o w
M i c h a e l i s - M e n t e n k i n e t i c s (11) c o n s i s t e n t w i t h t h e
o b s e r v a t i o n s o f p r e v i o u s r e p o r t s (.16,12)· Hence,
L i n e w e a v e r - B u r k p l o t s c o u l d be g e n e r a t e d f r o m the
d a t a y i e l d i n g v a l u e s o f a p p a r e n t maximum i n i t i a l
v e l o c i t i e s a t e a c h o f the t e m p e r a t u r e s . This
i n f o r m a t i o n i s p l o t t e d i n F i g u r e 4 w i t h an assumed
A r r h e n i u s dependency, which f o r t h i s system c l e a r l y
does n o t e x i s t . The s a l i e n t f e a t u r e s o f t h i s
study are that there e x i s t s n e a r l y a three f o l d
o r d e r o f m a g n i t u d e i n c r e a s e i n t h e a p p a r e n t maximum
v e l o c i t y as t h e s t a r c h s u b s t r a t e i s m o d i f i e d due t o
the g e l a t i n i z a t i o phenomena A t t h h i g h e s t tem
perature tested,
T h i s i s due t o t h e r m a enzyme
T h i s l a r g e i n c r e a s e i n the r e a c t i o n r a t e i n d i c a t e s
t h a t t h e o v e r r i d i n g e f f e c t i n v o l v e s the p h y s i c a l
s t a t e o f the s t a r c h g r a n u l e . Non-gelatinized
s t a r c h i s h i g h l y c r y s t a l l i n e (18) and t h u s t h e
s u s c e p t i b i l i t y o f s t a r c h t o be e n z y m a t i c a l l y
h y d r o l y z e d u n d e r t h e s e c o n d i t i o n s i s much l o w e r
t h a n f o r s t a r c h g r a n u l e s t h a t have been d i s r u p t e d
by h e a t i n g i n aqueous s u s p e n s i o n . Through t h e
p r o c e s s o f g e l a t i n i z a t i o n , the o r i g i n a l i n t e r n a l
o r d e r i n g of the s t a r c h m o l e c u l e s w i t h i n the g r a n u l e s
i s d i s r u p t e d , the g r a n u l e i m b i b e s l a r g e amounts o f
w a t e r and s w e l l s . E v e n t u a l l y due t o combined
a c t i o n o f s h e a r i n g w i t h i n t h e r e a c t o r and o s m o t i c
s w e l l i n g , granule i n t e g r i t y i s completely lost.
L a r g e s t a r c h m o l e c u l e s may t h e n r e a s s o c i a t e ( r e t r o
grade) producing a g e l m a t r i x . I t c a n n o t be
assumed t h a t t h e p r o g e s s o f t h e r e a c t i o n w i l l f o l l o w
the same s e q u e n c e i f t h e s u b s t r a t e i s i n v a r i o u s
physical states. I t i s therefore of i n t e r e s t to
f o l l o w the m o l e c u l a r w e i g h t d i s t r i b u t i o n p r o f i l e s
d u r i n g the c o u r s e of the r e a c t i o n at v a r i o u s s t a t e s
of macromolecular aggregation. The aqueous SEC
a p p a r a t u s d e s c r i b e d above was s p e c i f i c a l l y d e v e l o p e d
for t h i s purpose. T h i s t e c h n i q u e was shown t o be
a p p r o x i m a t e l y 10^ t i m e s more a c c u r a t e t h a n r e d u c i n g
s u g a r measurements f o r low d e g r e e o f c o n v e r s i o n
starch h y d r o l y s i s reactions (10).
E f f e c t o f T e m p e r a t u r e on H y d r o l y s i s E x t e n t and
P r o d u c t D i s t r i b u t i o n . I t was shown above t h a t s l o w
i n i t i a l r a t e of r e a c t i o n i s c h a r a c t e r i s t i c of opera
t i n g a t low t e m p e r a t u r e s ( b e l o w t h e g e l a t i n i z a t i o n
range of s t a r c h ) whereas r a p i d i n i t i a l r a t e s are
TEMPERATURE ( Ο β
95° 80 e
72 e
62° 50° 40°
1 1 1 J
20 i 1
10
! ι
• η
ABOVE Ι \ 1 BELOW
GELATINIZATION \, GELATINIZATION
1.0 _RANGE 1 y RANGE
.1
.03
1 1 II 1
2.7 2.8 2.9 3.0 3.1 3.2
l/T K"'xlO s
o b s e r v e d a t h i g h t e m p e r a t u r e s ( a b o v e the g e l a t i n i z a
t i o n of starch). However, t h e d i s t r i b u t i o n s o f
m a c r o m o l e c u l a r components d u r i n g h y d r o l y s i s a t
v a r i o u s t e m p e r a t u r e s have n o t b e e n r e p o r t e d and a r e
thus o f i n t e r e s t .
SEC p r o f i l e s o f h y d r o l y s i s p r o d u c t s o f enzyme-
t r e a t e d s t a r c h as a f u n c t i o n o f r e a c t i o n t i m e a t
60°C and 80°C a r e shown i n F i g u r e s 5 and 6 r e s p e c
tively. I n b o t h c a s e s , a common enzyme dose o f
one ml o f t h e 1:19 s t o c k Novo Thermamyl s o l u t i o n was
m i x e d w i t h a s l u r r y o f 15 gms s t a r c h d r y b a s i s i n
75 ml w a t e r and added t o t h e r e a c t o r . The f i n a l
volume was 1500 m l .
A t 60°C, b e l o w t h e g e l a t i n i z a t i o n ( m e l t i n g )
range o f the g r a n u l e s distributio i
e x t r e m e l y non-rando
the i n i t i a l stage y significan
amount o f i n t e r m e d i a t e m o l e c u l a r s i z e p r o d u c t s
( i . e . , b e t w e e n h y d r o d y n a m i c volume 10^ and 10^
dl/mole) detected. Throughout the time course of
l i q u e f a c t i o n , the m a j o r i t y o f t h e m o l e c u l a r s p e c i e s
present are e i t h e r very h i g h i n molecular weight
( e x c l u d e d from t h e SEC column) o r low m o l e c u l a r
w e i g h t end p r o d u c t s . Low m o l e c u l a r w e i g h t p r o d u c t s
c o n t i n u o u s l y accumulate w h i l e h i g h m o l e c u l a r weight
m a t e r i a l s d i s a p p e a r w i t h o u t d e t e c t i o n o f any s i g n i
f i c a n t amount o f i n t e r m e d i a t e m o l e c u l a r w e i g h t
material. T h i s s i t u a t i o n c o u l d r e s u l t i f t h e enzyme
i s a l l o w e d t o a c t o n l y on r e l a t i v e l y s h o r t , e x p o s e d
c h a i n s of s t a r c h present i n the c r y s t a l l i n e m a t r i x
o f the g r a n u l e , o r i f l o n g e r c h a i n m o l e c u l e s f r e e d
f r o m t h e g r a n u l e a r e more s u s c e p t i b l e t o enzyme
h y d r o l y s i s than the molecules a s s o c i a t e d w i t h the
g r a n u l e and a r e t h e r e f o r e p r e f e r e n t i a l l y d e g r a d e d .
A l t e r n a t i v e l y t h i s s i t u a t i o n c o u l d a l s o a r i s e i f the
a v a i l a b l e s t a r c h i s s i m p l y o v e r l o a d e d w i t h enzyme.
Other authors suggest that p r e f e r e n t i a l " p i t t i n g "
may o c c u r due t o d i f f e r i n g d e g r e e s i n s u b s t r a t e
susceptibility. The r e a c t i o n u n d e r t h e s e c o n d i t i o n s
does procède t o c o m p l e t i o n a l t h o u g h r e q u i r i n g a p p r o x
i m a t e l y ten hours f o r the c o n v e r s i o n .
Above t h e g e l a t i n i z a t i o n r a n g e (80°C, see
F i g u r e 6) t h e p r o d u c t d i s t r i b u t i o n d u r i n g r e a c t i o n
i s c o n s i d e r a b l y d i f f e r e n t t h a n 60°C. A g a i n , the
d i s t r i b u t i o n o f m o l e c u l a r s i z e s i s n o t random.
D u r i n g t h e e a r l y s t a g e s o f the r e a c t i o n , i n a d d i t i o n
t o t h e v e r y h i g h m o l e c u l a r w e i g h t and low m o l e c u l a r
weight products, a large f r a c t i o n of intermediate
m o l e c u l a r weight d e x t r i n s (between hydrodynamic
v a l u e 10^ and 10·* d l / m o l e , see F i g u r e 5) a r e p r e s e n t .
1 I I I I I
E f f e c t o f S t a r c h R e c r y s t a l l i z a t i o n on H y d r o
l y s i s E x t e n t , P r o d u c t D i s t r i b u t i o n , and R e a c t i o n
Rates. Since retrograded ( r e c r y s t a l l i z e d ) starch
i s t h o u g h t t o be more r e s i s t a n t t o e n z y m a t i c d e p o l y
m e r i z a t i o n t h a n u n r e t r o g r a d e d s t a r c h (.22,22), i t i s
o f i n t e r e s t t o compare t h e h y d r o l y s i s o f r e t r o g r a d e d
starch to non-retrograded starch. Extremely r e t r o
g r a d e d s t a r c h was p r e p a r e d by f r e e z e - d r y i n g p r e g e l a -
t i n i z e d s t a r c h , and b a l l - m i l l i n g t h i s m a t e r i a l t o a
f i n e powder. T h i s m a t e r i a l was e n z y m a t i c a l l y
h y d r o l y z e d a t 80°C, and t h e m o l e c u l a r s i z e d i s t r i
b u t i o n was d e t e r m i n e d by s i z e e x c l u s i o n c h r o m a t o
graphy. The c h r o m a t o g r a p h i c p r o f i l e s a r e shown
i n F i g u r e 8. The c h r o m a t o g r a p h i c p r o f i l e o f
unreacted, p r e g e l a t i n i z e d , f r e e z e - d r i e d , b a l l -
m i l l e d s t a r c h ("0" t i m e c h r o m a t o g r a m ) shows t h a t
s i g n i f i c a n t d e p o l y m e r i z a t i o n o c c u r r e d d u r i n g the
b a l l - m i l l i n g operation. D u r i n g the course o f
enzymatic d e p o l y m e r i z a t i o n of t h i s r e c r y s t a l l i z e d
s t a r c h , f o u r d i s t i n c t p e a k s a r e p r e s e n t : one n e a r
t h e e x c l u s i o n l i m i t o f t h e column s e t , one a t the
end p r o d u c t s , and two i n t e r m e d i a t e p e a k s . These
i n t e r m e d i a t e p e a k s a r e d e t e c t a b l e as e a r l y as two
and o n e - h a l f m i n u t e s i n t o t h e r e a c t i o n The p e a k s
appeared at K y v a l u e
A
0.6, t h e same v a l u e
s t a r c h was r e a c t e d a t 80°C. These values for
t h e two i n t e r m e d i a t e p e a k s i n c r e a s e d s l i g h t l y w i t h
i n c r e a s i n g r e a c t i o n time, a g a i n c o n s i s t e n t w i t h the
r e s u l t s shown a t 80°C f o r n o n - p r e g e l a t i n i z e d s t a r c h .
Thus t h e r e s i s t a n t , i n t e r m e d i a t e m o l e c u l a r s i z e
s t a r c h p r o d u c t s formed a f t e r s e v e r e rétrogradation
d u r i n g f r e e z i n g are q u i t e s i m i l a r t o the groups
formed when a n o n - p r e g e l a t i n i z e d s t a r c h i s enzyma
t i c a l l y d e p o l y m e r i z e d u n d e r t h e same c o n d i t i o n s .
No change i n t h e c h r o m a t o g r a p h i c p r o f i l e s i s
d e t e c t e d a f t e r e l e v e n m i n u t e s and the r e a c t i o n does
not proceed to completion.
Conclusion
z a t i o n p r o c e s s w i l l o c c u r even a t h i g h t e m p e r a t u r e
operation.
Although the i n i t i a l r a t e o f the r e a c t i o n i s
g r e a t e s t a t t e m p e r a t u r e s above t h e g e l a t i n i z a t i o n
range o f the s u b s t r a t e , the f i n a l e x t e n t o f con
v e r s i o n i s g r e a t e s t a t low t e m p e r a t u r e o p e r a t i o n
f o r a common amount o f enzyme l o a d i n g . This
s i t u a t i o n e x i s t s due t o t h e r m a l d e a c t i v a t i o n o f t h e
catalyst. Optimal operation of a starch l i q u e
f a c t i o n o p e r a t i o n must a c c o u n t f o r b o t h t h e r a t e o f
i n c r e a s e d s u b s t r a t e s u s c e p t i b i l i t y c a u s e d by t h e
d i s r u p t i o n o f the i n t e r n a l macromolecular o r d e r i n g
w i t h i n t h e g r a n u l e and t h e l o s s o f e n z y m a t i c
a c t i v i t y due t o t h e r m a l d e a c t i v a t i o n .
A p h y s i c a l model f starch gelatinization
rétrogradation, an
from these r e s u l t s
r e c r y s t a l l i z a t i o n v i a rétrogradation p r o c e s s w i l l
occur quite r a p i d l y f o r p r e g e l a t i n i z e d starch. I t
i s t h e r e f o r e not p o s s i b l e to study the enzymatic
hydrolysis of starch without considering r e t r o -
gradation e f f e c t s . The p h y s i c a l p i c t u r e f o r t h e
o v e r a l l p r o c e s s i s summarized i n F i g u r e 9. The
i n i t i a l p o p u l a t i o n o f s t a r c h g r a n u l e s i s suspended
i n aqueous s o l u t i o n . When s u b j e c t e d f o r a p e r i o d
o f t i m e a t o r above t h e g e l a t i n i z a t i o n t e m p e r a t u r e
r a n g e o f t h e m a t e r i a l , some o f t h e s t a r c h g r a n u l e s
melt. The m o l e c u l a r components o f t h e g r a n u l e
w i l l then r e a s s o c i a t e t o form c r y s t a l l i n e b o d i e s ,
some o f w h i c h may be more o r l e s s r e s i s t a n t t o
enzymatic h y d r o l y s i s than other c r y s t a l types. The
enzyme w i l l h y d r o l y z e s u s c e p t i b l e bond l i n k a g e s t o
low m o l e c u l a r w e i g h t o l i g o s a c c h a r i d e s as a f u n c t i o n
o f t i m e and c o n c e n t r a t i o n .
,E(t),t
Literature Cited
ALICIAN V. QUINLAN
Massachusetts Institute of Technology, Department of Mechanical Engineering,
Cambridge, MA 02139
The temperatur
many m i c r o b i a l processe
composition. As a r e s u l t , the o p e r a t i n g temperature
of b i o l o g i c a l r e a c t o r s should be v a r i e d to keep such
process r a t e s maximal as t h e i r chemical m i l i e u x vary.
The laws r e l a t i n g optimum operating temperature to
medium composition are not w e l l known. T h i s r e l a t i o n
was t h e o r e t i c a l l y i n v e s t i g a t e d f o r processes whose
r a t e s s a t u r a t e in s u b s t r a t e c o n c e n t r a t i o n . The
Michaelis-Menten r e a c t i o n mechanism was modified to
describe m i c r o b i a l biomass production and metabolite
e x c r e t i o n i n both batch and continuous r e a c t o r s .
Monod r a t e laws whose m a c r o - c o e f f i c i e n t s depend on
d i l u t i o n r a t e as w e l l as temperature were d e r i v e d .
Conditions f o r which these c o e f f i c i e n t s may fall,
fall, or show a minimum with rising temperature and
d i l u t i o n r a t e were enumerated. The optimum tempera-
ture was shown to vary with both s u b s t r a t e concentra-
t i o n and d i l u t i o n r a t e . The p a t t e r n of v a r i a t i o n was
explained in terms of the thermal sensitivities of
the c o e f f i c i e n t s . The validity of the t h e o r e t i c a l
r e s u l t s was discussed r e l a t i v e to published data and
found promising but i n need o f experimental t e s t i n g .
The r a t e s o f m i c r o b i a l l y m e d i a t e d p r o c e s s e s t y p i c a l l y show
a s i n g l e r a t e maximum a s t e m p e r a t u r e r i s e s ( 1 - 7 ) . Changes i n a
microorganism's chemical environment can s h i f t the temperature
a
a t w h i c h t h e t h e r m a l r a t e maximum o c c u r s (3-7)· As c o n s e q u e n c e ,
given the chemically changing m i l i e u x o f b i o l o g i c a l r e a c t o r s , t h e i r
o p e r a t i n g t e m p e r a t u r e s w o u l d h a v e t o be v a r i e d t o o p t i m i z e p r o c e s s
rates (8,9). In b i o l o g i c a l reactors, substrate concentration i s
t h e most i m p o r t a n t c h e m i c a l v a r i a b l e . So, i t ' s most d e s i r a b l e
t o know how t h e optimum o p e r a t i n g t e m p e r a t u r e depends on s u b s t r a t e
concentration.
0097-6156/83/0207-0463$07.25/0
© 1983 American Chemical Society
Reaction Mechanism
Ε + S ^ ES • Ε + Ρ
( k ) p r o d u c t (P) w i t h a c o n c o m i t a n t r e c y c l i n g o f f r e e enzyme.
3
I n b i o l o g i c a l r e a c t o r s , m i c r o b i a l biomass p l a y s a r o l e a n a l o
gous t o t h e enzyme's r o l e . L e t Y be t h e a v e r a g e number o f s u b
s t r a t e consumption s i t e s per microorganism. Then,
Ύ Ξ C/N (1)
where C i s t h e c o n c e n t r a t i o n o f c o n s u m p t i o n s i t e s i n t h e m i c r o b i a l
p o p u l a t i o n and Ν i s t h e c o n c e n t r a t i o n o f m i c r o o r g a n i s m s i n t h e
population. S i m i l a r l y , l e t β be t h e a v e r a g e b i o m a s s p e r m i c r o
organism, w i t h
β Ξ Β/Ν (2)
Β = (0/Y)C (3)
E q u a t i o n (3) s t a t e s t h a t t h e b i o m a s s c o n c e n t r a t i o n o f a m i c r o b i a l
p o p u l a t i o n i s d i r e c t l y p r o p o r t i o n a l t o t h e c o n c e n t r a t i o n o f sub
s t r a t e consumption s i t e s i n the p o p u l a t i o n .
As f o r t h e enzyme, t h e s u b s t r a t e c o n s u m p t i o n s i t e s may be
p a r t i t i o n e d i n t o two p o r t i o n s : t h o s e c a p a b l e o f c o n s u m i n g s u b
s t r a t e ( C ) a n d t h o s e n o t c a p a b l e ( C ) . An e q u i v a l e n t b i o m a s s
c n
may b e a s s i g n e d t o e a c h p o r t i o n t h r o u g h e q u a t i o n ( 3 ) :
B = (6/Y)C (4a)
B = (B/Y)C (4b)
The t o t a l b i o m a s s o f a m i c r o b i a l p o p u l a t i o n c a n t h e n a l s o be
p a r t i t i o n e d i n t o two p o r t i o n s s o t h a t
Β = B c + B n (5)
I n t h i s c o n t e x t , B p l a y s a r o l e a n a l o g o u s t o f r e e enzyme, a n d
c
B , t o enzyme-substrate complex.
n
I n conformance w i t h t h i s i n t e r p r e t a t i o n o f B , s u b s t r a t e i s
c
assumed t o be consumed a t a r a t e j o i n t l y p r o p o r t i o n a l t o i t s c o n
c e n t r a t i o n S a n d B , i . e . , a t a mass f l o w r a t e c S B .
c c Similarly,
t h e m e t a b o l i t e o f i n t e r e s t i s assumed t o be e x c r e t e d a t a r a t e
p r o p o r t i o n a l o n l y t o B , i . e . , a t a mass f l o w r a t e e B . I n t h i s
n n
case, t h e m i c r o s c o p i c r a t e c o e f f i c i e n t s c and e p l a y r o l e s a n a l o
gous t o t h o s e o f k a n d k . r e s p e c t i v e l y .
x 2 >
F u r t h e r m o r e , s a t u r a t e d and u n s a t u r a t e d consumption s i t e s a r e
assumed t o c y c l e a s c o m p l e x e d a n d f r e e enzyme d o , b u t w i t h no
c o n c o m i t a n t r e l e a s e o f s u b s t r a t e o r m e t a b o l i t e i n t o t h e medium.
S a t u r a t e d s i t e s a r e assumed t o u n s a t u r a t e a t a r a t e p r o p o r t i o n a l
o n l y t o t h e i r own c o n c e n t r a t i o n , i . e . , a t a mass f l o w r a t e u B . n
On t h e o t h e r h a n d , u n s a t u r a t e d s i t e s a r e assumed t o s a t u r a t e a t
a r a t e j o i n t l y p r o p o r t i o n a l t o s u b s t r a t e c o n c e n t r a t i o n and t h e i r
own, i . e . , a t a mass f l o w r a t e s S B . c I f each consumption s i t e
becomes s a t u r a t e d when o n e s u b s t r a t e m o l e c u l e o c c u p i e s i t , t h e n
the m i c r o s c o p i c s a t u r a t i o n and consumption c o e f f i c i e n t s a r e e q u a l .
T h i s i s t h e c a s e assumed by t h e M i c h a e l i s - M e n t e n r e a c t i o n mech
a n i s m . However, i f η m o l e c u l e s a r e r e q u i r e d f o r s a t u r a t i o n o f
one s i t e , t h e n s = ( l / n ) c .
The m o d i f i e d r e a c t i o n mechanism must a l s o a c c o u n t f o r b i o m a s s
w a s t a g e (and t h e c o n c o m i t a n t l o s s o f c o n s u m p t i o n s i t e s ) t h r o u g h
r e s p i r a t i o n , e x c r e t i o n , d e a t h , e t c . B i o m a s s w a s t a g e i s assumed
to take p l a c e a t a r a t e p r o p o r t i o n a l t o t h e biomass c o n c e n t r a t i o n
o f t h e p o p u l a t i o n , i . e . , a t a mass f l o w r a t e wB = w B + wB . c n
F i n a l l y , s u b s t r a t e i s added t o a c o n t i n u o u s r e a c t o r a t a
mass f l o w r a t e d S , w h e r e S i s t h e c o n c e n t r a t i o n o f i n f l o w i n g
Q 0
s u b s t r a t e and d i s a d i l u t i o n r a t e e q u a l t o t h e r a t i o o f t h e
v o l u m e t r i c i n f l o w r a t e t o t h e volume o f f l u i d i n t h e r e a c t o r .
For a constant f l u i d volume, i n f l o w i n g s u b s t r a t e a l s o d i s p l a c e s
s u b s t r a t e , b i o m a s s , a n d m e t a b o l i t e a t mass f l o w r a t e s e q u a l ,
r e s p e c t i v e l y , t o d S , dB - d B + d B , a n d dM.
c n
A t t h i s p o i n t , a l l t h e n e c e s s a r y mass c o n c e n t r a t i o n s a n d
f l o w s h a v e b e e n d e f i n e d . The r e s u l t i n g r e a c t i o n mechanism i s
s c h e m a t i z e d i n F i g u r e 1. The s o l i d l i n e s r e p r e s e n t t h e mass
f l o w s , a n d t h e d a s h e d l i n e s show w h i c h c o n c e n t r a t i o n s i n f l u e n c e
t h e i r r a t e s . A y i e l d c o e f f i c i e n t Y must be i n t r o d u c e d t o make
t h e u n i t s o f S a n d M c o m p a t i b l e w i t h t h o s e o f B a n d Bn. E a c h c
u n i t o f S consumed i s assumed t o p r o d u c e Y u n i t s o f B. S i m i l a r
l y , each u n i t o f M excrete i unit
R a t e Laws
The r a t e o f c h a n g e o f e a c h c o n c e n t r a t i o n shown i n F i g u r e 1
may be o b t a i n e d b y summing a l l i t s mass i n f l o w s a n d o u t f l o w s :
•
dS/dt Ξ S = d ( S - S) - c S B / Y
0 c (6)
dB /dt = B
c c
- (c - s ) S B c + uB n - (w + d ) B c
(7)
dB / d t = sSB c - (u + w + d ) B n
(8)
η
dM/dt = M = e B / Y - dM.
n
(9)
The r a t e s o f b i o m a s s p r o d u c t i o n a n d m e t a b o l i t e e x c r e t i o n
c a n be shown t o s a t u r a t e i n s u b s t r a t e c o n c e n t r a t i o n i f B i s c
So, o n t h e t i m e s c a l e o f c h a n g e s i n B , B 2 0, a n d f r o m e q u a t i o n
c n
(8):
Β s SB /K C (Ha)
η
where Κ = (u + w + d ) / s (lib)
B c Z B K / ( K + S) (12a)
B n ζ B S / ( K + S) (12b)
S Ζ d ( S - S) - ^ ( B / Y j S / f K
Q + S) (13a)
where y = cK (13b)
m
The s e c o n d t e r m i n e q u a t i o n (13a) r e p r e s e n t s t h e r a t e o f s u b s t r a t e
consumption, o r n u t r i e n t uptake. I t s a t u r a t e s i n s u b s t r a t e con
c e n t r a t i o n a c c o r d i n g t o a Monod r a t e l a w where b o t h Κ a n d y / Y m
a r e known t o v a r y w i t h t e m p e r a t u r e a n d d i l u t i o n r a t e ( 1 1 - 2 1 ) .
Biomass P r o d u c t i o n . S i m i l a r l y , s u b s t i t u t i o n o f equation
(12a) i n t o ( 1 0 ) y i e l d s t h
l y used f o r continuous c u l t u r
The f i r s t t e r m i n e q u a t i o n ( 1 4 ) , r e p r e s e n t i n g t h e g r o s s r a t e o f
b i o m a s s p r o d u c t i o n , i s i d e n t i c a l w i t h t h e f u n c t i o n Monod ( 2 5 )
o r i g i n a l l y adopted " t o express c o n v e n i e n t l y t h e r e l a t i o n between
e x p o n e n t i a l growth r a t e and c o n c e n t r a t i o n o f an e s s e n t i a l n u t r i e n t .
Such a r e c t a n g u l a r h y p e r b o l i c f u n c t i o n h a s b e e n d e r i v e d many t i m e s
f r o m v a r i o u s r e a c t i o n mechanisms ( 2 6 - 3 0 ) , b u t none h a s a d d r e s s e d
the p r e s e n t case o f c o n t i n u o u s c u l t u r e systems where y and Κ m
c,s,u,w, and t h e i r v a r i a t i o n w i t h d i l u t i o n r a t e i s e x p l i c i t .
To e s t i m a t e t h e v a l u e s o f t h e m i c r o - c o e f f i c i e n t s c,s,u,w a s
f u n c t i o n s o f t e m p e r a t u r e , e q u a t i o n ( 1 4 ) c a n be r e w r i t t e n a s
Β/Β Ζ V ( S - S ) / ( S + K)
B T (15a)
where V. Ξ y - (w + d) (15b)
Β m
= (c - s) Κ + u (15c)
S T Ξ (w + d ) K / V B (15d)
E q u a t i o n (15a) r e p r e s e n t s a Monod r a t e l a w w i t h a t h r e s h o l d s u b
s t r a t e c o n c e n t r a t i o n S-j ( F i g u r e 2 ) . I t c a n be l i n e a r i z e d t o a
generalized Eadie-Augustinsson equation (31-33):
ν ζ V B - K(v/S) - V S ( 1 / S )
B T (16)
τ 1 1 1 1 1
GENERIC MONOD RATE - SATURATION LAW
WITH THRESHOLD SUBSTRATE CONCENTRATION
I ι ι ι ι ι ι I
Ο 10 20 30 40 50 60 70
S (ppm ELEMENT)
Figure 2. Properties of the rate law for net biomass production in batch (a = 0)
and continuous (d > 0) reactors. Symbols defined in text and in legend of
symbols.
where ν i s t h e s p e c i f i c n e t b i o m a s s p r o d u c t i o n r a t e B/B m e a s u r e d
i n the reactor a t p a r t i c u l a r values o f substrate concentration,
t e m p e r a t u r e , and d i l u t i o n r a t e . A l i n e a r r e g r e s s i o n o f ν v e r s u s
v/S and 1/S w i l l y i e l d v a l u e s f o r t h e m a c r o - c o e f f i c i e n t s V g , K,
and S- a s f u n c t i o n s o f t e m p e r a t u r e and d i l u t i o n r a t e . The v a l u e s
o f t h e m i c r o - c o e f f i c i e n t s a s f u n c t i o n s o f t e m p e r a t u r e c a n t h e n be
e s t i m a t e d f r o m e q u a t i o n s ( l i b ) , ( 1 5 c ) , a n d (15d) a s s u m i n g c = s .
(M + dM)/(B/Y) Ζ V S / ( S + K) M (17a)
where V = e (17b)
m
Thermal Sensitivity of
S.j0 t h r o u g h t h e t e m p e r a t u r e d e p e n d e n c i e s o f t h e m i c r o - c o e f f i c i e n t s
( c , e , s , u , w ) . E a c h o f t h e m i c r o - c o e f f i c i e n t s i s assumed t o obey
an A r r h e n i u s temperature l a w o f t h e form ( 3 4 ) :
k = A exp ( - E / R T )
A K (18)
A = a positively-valued coefficient
that i s p r a c t i c a l l y independent
of temperature
E^ Ξ a p o s i t i v e l y - v a l u e d apparent
l
a c t i v a t i o n energy ( c a l . mol )
R Ξ t h e e a s c o n s t a n t (1.987 c a l .
Y 1
mol- K- )
T R = degrees Kelvin
W i t h i n t h e t e m p e r a t u r e r a n g e 0 - 50°C, e q u a t i o n ( 1 8 ) may be
approximated as f o l l o w s ( 1 0 ) :
α Ξ A exp (-1.843 χ 1 0 * 3
E^) (19b)
Τ = degrees Celsius
θ „ Ξ 1.481 χ 1 0 / E 5
A (19c)
α A
H a l f - S a t u r a t i o n C o e f f i c i e n t K. For batch c u l t u r e , e q u a t i o n
( l i b ) reduces t o :
K|
I f s r i s e s more r a p i d l y w i t h t e m p e r a t u r e t h a n b o t h n u m e r a t o r t e r m s ,
then the b a t c h - c u l t u r e h a l f - s a t u r a t i o n c o e f f i c i e n t should always
d e c r e a s e as t e m p e r a t u r e i n c r e a s e s . I f s r i s e s more r a p i d l y t h a n
t h e n u m e r a t o r t e r m d o m i n a n t a t l o w t e m p e r a t u r e s b u t more s l o w l y
t h a n t h e t e r m d o m i n a n t a t h i g h t e m p e r a t u r e s , i t s h o u l d show a
t e m p e r a t u r e minimum. I f s r i s e s more s l o w l y t h a n b o t h n u m e r a t o r
t e r m s ( F i g u r e 3 ) , i t s h o u l d a l w a y s r i s e w i t h t e m p e r a t u r e ( F i g u r e 4,
d = 0)
The t h e r m a l s e n s i t i v i t i e s o f t h e b a t c h - c u l t u r e h a l f - s a t u r a t i o n
c o e f f i c i e n t s f o r s e v e r a l m i c r o b i a l p r o c e s s e s (11-14) h a v e b e e n
observed to resemble the l i n e a r p o r t i o n o f the d = 0 curve i n
F i g u r e 4. However, a few d e v i a t i o n s f r o m t h i s p a t t e r n h a v e b e e n
s e e n where t h e h a l f - s a t u r a t i o n c o e f f i c i e n t s showed e i t h e r a
n e g a t i v e s l o p e o r a maximum ( 1 3 ) . O n l y t h e t h e r m a l maximum
i s i n c o n s i s t e n t w i t h e q u a t i o n s (18) - ( 2 0 ) .
For continuous c u l t u r e ,
K
|d > ο - K
|d = ο + ( 1 / s ) d ( 2 1 )
Figure 3. Temperature dependencies assumed for the five microscopic rate coeffi-
cients defined in Figure 1. The microcoefficients c and s have units (ppm element
Khr' , e, u, and w have units Khr'K
1
1 1 1 1 1 1 1—
VARIATION OF
HALF - SATURATION COE FFICIENT
WITH
TEMPERATURE AND DILUTION RATE
4
~d « 100 Khr -ι /
LU /
ΙΟ « 5 0
1
/
Lu
_J
LU
«25/
ε -Ο "
CL /
CL /
/
/
BATCH
/ d «Ο
u • w*d
w « 0.5 exp(T/5.l)
u « 10 exp(T/l6.9)
s « 5 0 exp (T/20)
I I I I L
10 20 30 40
TTC)
Figure 4. Theoretical thermal sensitivity of the half-saturation coefficient in batch
(a = 0) and continuous (a > 0) culture with the parameter values specified in
Figure 3. Dashed line, locus of thermal minima ( O).
M a x i m u m - S p e c i f i c - G r o w t h - R a t e C o e f f i c i e n t V^. In batch c u l t u r e ,
e q u a t i o n s ( l i b ) and (13b) c o m b i n e t o y i e l d :
w
«|d = 0 = c u / s + c w / s ( 2 2 )
The b a t c h - c u l t u r e m a x i m u m - s p e c i f i c - g r o w t h - r a t e c o e f f i c i e n t s h o u l d
f a l l w i t h i n c r e a s i n g t e m p e r a t u r e i f s r i s e s more r a p i d l y t h a n b o t h
c u and cw. I t s h o u l d show a minimum i f s r i s e s more r a p i d l y t h a n
the n u m e r a t o r o f t h e t e r m d o m i n a n t a t l o w t e m p e r a t u r e s b u t l e s s
r a p i d l y than the o t h e r term. I t should always i n c r e a s e i f s r i s e s
l e s s r a p i d l y t h a n t h e n u m e r a t o r s o f b o t h t e r m s . When c = s , i t
a l s o s h o u l d a l w a y s i n c r e a s e ( F i g u r e 5, d = 0 ) .
The d a t a (11-14) a v a i l a b l e t o t e s t t h e p r e d i c t e d t h e r m a l s e n
s i t i v i t y of the b a t c h - c u l t u r e maximum-specific-growth-rate co
e f f i c i e n t were o b t a i n e d from s u b s t r a t e consumption e x p e r i m e n t s .
From e q u a t i o n ( 1 3 a ) , t h e m a x i m u m - v e l o c i t y c o e f f i c i e n t f o r s u b
s t r a t e c o n s u m p t i o n i s V^/Y. D a t a p r e s e n t e d i n t h i s form must be
m u l t i p l i e d by a y i e l d c o e f f i c i e n t Y t o c o n v e r t t o y « I f the m
y i e l d c o e f f i c i e n t i s assumed t o be p r a c t i c a l l y c o n s t a n t , t h e n
y / Y s h o u l d show t h e same t h e r m a l s e n s i t i v i t y p a t t e r n as y .
m m
The t h e r m a l s e n s i t i v i t y o f y / Y i n b a t c h c u l t u r e i s w e l l d e s c r i b e d
m
by t h e l i n e a r p o r t i o n o f t h e d = 0 c u r v e i n F i g u r e 5, and s o i s
t h a t o f y as l o n g as Y i s n e a r l y c o n s t a n t .
m
The c o n t i n u o u s - c u l t u r e y s h o u l d i n c r e a s e l i n e a r l y w i t h d i l u t i o n
m
r a t e , and t h e i n f l u e n c e o f t h e d i l u t i o n t e r m s h o u l d grow w i t h
r i s i n g t e m p e r a t u r e i f s r i s e s more s l o w l y t h a n c . I f c = s , t h e
c o n t i n u o u s - c u l t u r e y should a s y m p t o t i c a l l y approach the d i l u t i o n
m
0 10 20 30 40
T(°C)
r a t e a s t e m p e r a t u r e f a l l s and t h e b a t c h c u l t u r e p a s t e m p e r a t u r e
m
r i s e s ( F i g u r e 5, d > 0 ) .
The l i n e a r p o r t i o n s o f t h e d > 0 c u r v e s i n F i g u r e 5 r e s e m b l e
the t h e r m a l s e n s i t i v i t i e s o b s e r v e d f o r y (17,18) and y / Y
m (15,16,
m
19) i n c o n t i n u o u s c u l t u r e . M o r e o v e r , i n agreement w i t h e q u a t i o n
(23) y / Y has b e e n o b s e r v e d t o i n c r e a s e l i n e a r l y w i t h d i l u t i o n
m
r a t e ( 2 0 ) . I t has a l s o b e e n s e e n t o d e c r e a s e s l i g h t l y w i t h i n
c r e a s i n g d i l u t i o n r a t e (21), which d i s a g r e e s w i t h equation (23).
M a x i m u m - V e l o c i t y C o e f f i c i e n t s Vft V^.
t According to equations
( l i b ) and ( 1 5 c ) , t h e m a x i m u m - v e l o c i t y c o e f f i c i e n t f o r b i o m a s s p r o
d u c t i o n depends on t h e m i c r o s c o p i c r a t e c o e f f i c i e n t s a s f o l l o w s :
In g e n e r a l , depending e s p e c i a l l
t h i s m a x i m u m - v e l o c i t y c o e f f i c i e n t may v a r y i n a c o m p l i c a t e d f a s h i o n
w i t h b o t h t e m p e r a t u r e and d i l u t i o n r a t e . However, a s c a p p r o a c h e s
s i n v a l u e , the i n f l u e n c e o f a l l the m i c r o - c o e f f i c i e n t s but u
v a n i s h e s , and i n t h e l i m i t o f c = s ,
V B = u (25)
T h r e s h o l d C o e f f i c i e n t S T * F o r b a t c h c u l t u r e and t h e s p e c i a l
case c = s, the t h e r m a l s e n s i t i v i t y o f t h e t h r e s h o l d c o e f f i c i e n t
i s d e t e r m i n e d a s f o l l o w s by e q u a t i o n s ( 1 5 d ) , ( 2 0 ) , and ( 2 5 ) :
S |
T d m 0 = (w/s) + (w/u) (w/s) (26)
A t t e m p e r a t u r e s where u d o m i n a t e s w, t h e b a t c h - c u l t u r e t h r e s h o l d
c o e f f i c i e n t should f a l l e x p o n e n t i a l l y w i t h temperature i f s r i s e s
more r a p i d l y w i t h t e m p e r a t u r e t h a n w. A t t e m p e r a t u r e s where w
d o m i n a t e s u, i t s h o u l d a l s o f a l l e x p o n e n t i a l l y w i t h r i s i n g tem
2
p e r a t u r e i f s u r i s e s more r a p i d l y t h a n w . I f b o t h terms d e
c r e a s e as t e m p e r a t u r e r i s e s , i t s h o u l d a l w a y s d i m i n i s h as tem
perature r i s e s . I f the term dominant a t low temperatures f a l l s
2
S | T d > 0 - S |T d m 0 + (1/s) [ 1 + 2 (w/u)] d + ( l / s u ) d (27)
R a t e I s o t h e r m s . D e p e n d i n g on s u b s t r a t e c o n c e n t r a t i o n , t h r e e
t h e r m a l s e n s i t i v i t y p a t t e r n s may be s e e n i n b o t h b a t c h a n d c o n
t i n u o u s r e a c t o r s when t h e n e t b i o m a s s p r o d u c t i o n r a t e ( a s s p e c i f i e d
by e q u a t i o n s ( l i b ) and ( 1 5 ) and F i g u r e s 3, 4, and 6) i s p l o t t e d
1 1 1 1 1 1 1
VARIATION OF
T H R E S H O L D S U B S T R A T E CONCENTRATION
WITH
_ T E M P E R A T U R E AND DILUTION R A T E _
T(°C)
Figure 6. Theoretical thermal sensitivity of the threshold coefficient in batch (à
= 0) and continuous (à > 0) culture with the parameter values specified in Figure
3. Dashed line, locus of thermal minima (O).
v e r s u s s u b s t r a t e c o n c e n t r a t i o n w i t h t e m p e r a t u r e as a p a r a m e t e r
( F i g u r e 7 ) . A t l o w s u b s t r a t e c o n c e n t r a t i o n s (S < 1 . 2 ) , t h e r a t e
d i m i n i s h e s a s t e m p e r a t u r e r i s e s and h e n c e shows a n e g a t i v e t h e r m a l
s e n s i t i v i t y ; w h e r e a s , a t h i g h s u b s t r a t e c o n c e n t r a t i o n s (S > 4·4),
t h e r a t e i n c r e a s e s w i t h t e m p e r a t u r e and t h u s has a p o s i t i v e t h e r m a l
sensitivity. A t i n t e r m e d i a t e s u b s t r a t e c o n c e n t r a t i o n s (1.2 < S <
4 . 4 ) , t h e r a t e f i r s t r i s e s and t h e n f a l l s w i t h i n c r e a s i n g tem
perature. I n o t h e r words, a t moderate s u b s t r a t e c o n c e n t r a t i o n s ,
the thermal s e n s i t i v i t y s w i t c h e s s i g n from p o s i t i v e to n e g a t i v e ,
w h i c h means t h e r a t e goes t h r o u g h a maximum a s t e m p e r a t u r e r i s e s .
M o r e o v e r , t h e maximum r a t e i s d e t e r m i n e d by a h i g h e r t e m p e r a t u r e
i s o t h e r m a s s u b s t r a t e c o n c e n t r a t i o n i n c r e a s e s . As a c o n s e q u e n c e ,
t h e optimum t e m p e r a t u r e s h o u l d r i s e when s u b s t r a t e c o n c e n t r a t i o n
increases.
The r a t e i s o t h e r m s f o m e t a b o l i t excretio (a s p e c i f i e d b
e q u a t i o n s ( l i b ) and (17
thermal s e n s i t i v i t y pattern
isotherms. T h u s , t h e r a t e o f m e t a b o l i t e e x c r e t i o n may a l s o h a v e
an optimum t e m p e r a t u r e t h a t s h i f t s t o h i g h e r v a l u e s a s s u b s t r a t e
concentration r i s e s .
1 1 1 1 1 Γ
NET BIOMASS PRODUCTION
S (ppm ELEMENT)
τ 1 1 1 τ—
NET BIOMASS PRODUCTION
THEORETICAL RATE ISOCONCENTRATES
Τ CO
— ι 1 1 1 1 1 1
METABOLITE EXCRETION
T H E O R E T I C A L RATE ISOCONCENTRATES
CONTINUOUS CULTURE ! d = 2 5 Khr" 1
0.5
M • dM
(B/Y)
(Khr )1
0.1
0.05
S s
0 . 0 2 5 ppm
ELEMENT!
0.02
0.01
Figure 10. Predicted influence of substrate concentration and dilution rate on the
temperature that maximizes the rates of net biomass production and metabolite
excretion in batch (a = 0) and continuous (a > 0) culture.
becomes v e r y l o w , t h e optimum t e m p e r a t u r e a s y m p t o t i c a l l y a p p r o a c h
es a minimum v a l u e c h a r a c t e r i s t i c o f e a c h d i l u t i o n r a t e . The
minimum optimum t e m p e r a t u r e i s t h e t e m p e r a t u r e a t w h i c h t h e s l o p e
o f t h e h a l f - s a t u r a t i o n c o e f f i c i e n t ( F i g u r e 4) e q u a l s t h a t o f t h e
maximum-velocity c o e f f i c i e n t , namely, the m i c r o s c o p i c e x c r e t i o n
r a t e c o e f f i c i e n t e. As s u b s t r a t e c o n c e n t r a t i o n i n c r e a s e s , t h e
i n f l u e n c e o f t h e d i l u t i o n r a t e p r o g r e s s i v e l y d i m i n i s h e s , and t h e
optimum t e m p e r a t u r e c u r v e s c o n v e r g e . A t h i g h s u b s t r a t e c o n
c e n t r a t i o n s , t h e optimum t e m p e r a t u r e i n c r e a s e s a l m o s t l i n e a r l y
w i t h the logarithm of substrate concentration.
Thermo-Chemical O p t i m i z a t i o n o f P r o c e s s Rates
A t t h i s p o i n t , t h e g o a l o f t h i s p a p e r has b e e n a c h i e v e d .
The two f a m i l i e s o f c u r v e s shown i n F i g u r e 10 c o n s t i t u t e t h e r m o
chemical rate-optimizatio
ture, substrate concentration
o f t h e s e t h r e e v a r i a b l e s a r e f i x e d , e a c h f a m i l y s p e c i f i e s what
v a l u e t h e t h i r d must h a v e t o m a x i m i z e i t s r a t e . The s e p a r a t i o n
o f t h e two f a m i l i e s means t h a t b o t h r a t e s c a n n o t be s i m u l t a n e
o u s l y maximized. As a r e s u l t , an o p t i m i z a t i o n s t r a t e g y may be
n e e d e d , s u c h a s o p e r a t i n g a t t h e optimum t e m p e r a t u r e o f t h e
p r o c e s s whose r a t e i s most s e n s i t i v e t o t e m p e r a t u r e .
Summary and C o n c l u s i o n s
T h i s p a p e r showed how c h a n g e s i n s u b s t r a t e c o n c e n t r a t i o n
and d i l u t i o n r a t e may s h i f t t h e optimum t e m p e r a t u r e o f m i c r o b i a l
p r o c e s s e s whose r a t e s s a t u r a t e i n s u b s t r a t e c o n c e n t r a t i o n a c c o r d
i n g t o Monod r a t e l a w s . The optimum t e m p e r a t u r e s h i f t was a t t r i b u
t e d t o t h e e f f e c t s o f t e m p e r a t u r e and d i l u t i o n r a t e on t h e m a c r o -
c o e f f i c i e n t s i n t h e Monod r a t e l a w s . E q u a t i o n s d e s c r i b i n g t h e s e
e f f e c t s were d e r i v e d from a s u i t a b l y m o d i f i e d M i c h a e l i s - M e n t e n
r e a c t i o n mechanism. W i t h o n l y a few e x c e p t i o n s , t h e s e e q u a t i o n s
a g r e e d w i t h p u b l i s h e d d a t a when A r r h e n i u s t e m p e r a t u r e d e p e n d e n c i e s
were assumed f o r t h e m i c r o - c o e f f i c i e n t s i n t h e r e a c t i o n mechanism.
As a c o n s e q u e n c e , t h e s e e q u a t i o n s and t h e optimum t e m p e r a t u r e
c u r v e s may be c o n s i d e r e d a s p l a u s i b l e , e x p e r i m e n t a l l y t e s t a b l e
hypotheses. They d e s e r v e c a r e f u l q u a n t i t a t i v e t e s t i n g b e c a u s e
t h e y p r o m i s e t o u n i f y h e r e t o f o r e f r a g m e n t a r y and s e e m i n g l y c o n
t r a d i c t o r y b a t c h and c o n t i n u o u s c u l t u r e d a t a . S u c h u n i f i c a t i o n
i s needed t o d e v e l o p a r a t i o n a l m e t h o d o l o g y f o r o p t i m i z i n g t h e
d e s i g n and o p e r a t i o n o f b i o l o g i c a l r e a c t o r s i n w h i c h s u b s t r a t e
c o n c e n t r a t i o n , t e m p e r a t u r e , and d i l u t i o n r a t e a r e e c o n o m i c a l l y
important v a r i a b l e s .
Legend o f Symbols
Β Ξ t o t a l b i o m a s s c o n c e n t r a t i o n i n r e a c t o r ; e q u a l s sum o f
B p l u s B ; mg d r y w e i g h t p e r l i t e r .
c n
B c = biomass c o n c e n t r a t i o n a s s o c i a t e d w i t h s u b s t r a t e con
s u m p t i o n s i t e s c a p a b l e o f c o n s u m i n g s u b s t r a t e ; mg
dry weight per l i t e r .
B n = biomass c o n c e n t r a t i o n a s s o c i a t e d w i t h s u b s t r a t e con
sumption s i t e s n o t c a p a b l e o f consuming s u b s t r a t e ;
mg d r y w e i g h t p e liter
C Ξ t o t a l concentratio consumptio
i n m i c r o b i a l p o p u l a t i o n ; e q u a l s sum o f C p l u s C^;
c
number o f s i t e s p e r l i t e r .
C c = c o n c e n t r a t i o n o f s u b s t r a t e consumption s i t e s capable
o f consuming s u b s t r a t e ; number o f s i t e s p e r l i t e r .
C n = c o n c e n t r a t i o n o f s u b s t r a t e consumption s i t e s n o t
c a p a b l e o f c o n s u m i n g s u b s t r a t e ; number o f s i t e s
per l i t e r .
Ε = c o n c e n t r a t i o n o f f r e e enzyme.
Ν Ξ c o n c e n t r a t i o n o f m i c r o o r g a n i s m s i n r e a c t o r ; number o f
organisms per l i t e r .
Ρ = concentration of product(s).
1 1
R Ξ g a s c o n s t a n t = 1.987 c a l . m o l " Κ" .
S = s u b s t r a t e c o n c e n t r a t i o n i n r e a c t o r ; ppm o f a n e l e m e n t .
S Q = i n l e t s u b s t r a t e c o n c e n t r a t i o n ; same u n i t s a s S.
C o n t i n u e d on n e x t page
t e m p e r a t u r e ; °C.
t e m p e r a t u r e ; °K.
maximum p o s s i b l e s p e c i f i c r a t e o f n e t b i o m a s s p r o d u c t i o n
1 1
i n the reactor; time" (e.g., K h r " ) .
maximum p o s s i b l e r a t e o f g r o s s m e t a b o l i t e formation;
1 1
time" (e.g., K h r " ) .
y i e l d c o e f f i c i e n t ; ppm d r y w e i g h t p r o d u c e d p e r ppm
e l e m e n t consumed
d i l u t i o n r a t e (flow/volume); r a t e a t which s u b s t r a t e ,
b i o m a s s , and m e t a b o l i t e d i s p l a c e d f r o m c u l t u r e v e s s e l ;
1
time" .
1
microscopic metabolite-excretion rate c o e f f i c i e n t ; time" .
g e n e r i c m i c r o s c o p i c r a t e c o e f f i c i e n t , e.g., c,f,s,u,w.
m i c r o s c o p i c u n s a t u r a t i o n r a t e c o e f f i c i e n t ; t i m e *.
1
m i c r o s c o p i c wastage r a t e c o e f f i c i e n t ; time" .
3 = a v e r a g e b i o m a s s p e r m i c r o o r g a n i s m ; mg d r y w e i g h t p e r
organism.
Ύ = a v e r a g e number o f s u b s t r a t e c o n s u m p t i o n s i t e s p e r
o r g a n i s m ; number o f s i t e s p e r o r g a n i s m .
PJJJ = 1
maximum s p e c i f i c g r o w t h r a t e c o e f f i c i e n t ; t i m e " " .
θ = r e t a r d a t i o n t e m p e r a t u r e ; °C.
Acknowledgments
The r e s e a r c h r e p o r t e d i n t h i s p a p e r was s u p p o r t e d i n p a r t
by t h e D e p a r t m e n t o f M e c h a n i c a
a g r a n t f r o m t h e Therm
Literature Cited
R E C E I V E D July 7, 1 9 8 2
e s s e n t i a l l y p r o p o r t i o n a l to the t o t a l r a t e o f sugar
uptake both during the growth and f l o c c u l a t i o n phase.
The increase with temperature o f s u b s t r a t e uptake,
yeast growth and f l o c c u l a t i o n , and metabolite produc-
t i o n r a t e is simply described by Arrhenius type o f
relationships.
0097-6156/83/0207Ό489$06.00/0
© 1983 American Chemical Society
EXPERIMENTAL
d(X ) T
d(TFS)
= - R (1)
dt X/S dt
(Xy) being the t o t a l yeast concentration
(TFS) t h e t o t a l fermentable sugar c o n c e n t r a t i o n
R the yeast t o sugar y i e l d
X/S
The e x p e r i m e n t a l l y o b s e r v e d d e c r e a s e o f R x / s d u r i n g t h e f e r
mentation has p r e v i o u s l
c e l l u l a r sterol concentratio
a b s e n c e o f o x y g e n . We f o u n d i t most c o n v e n i e n t t o m a t h e m a t i c a l l y
r e l a t e t h e d e c r e a s e o f Rx/s t o t h e i n c r e a s e o f t h e e t h a n o l c o n c e n
t r a t i o n i n t h e medium, ( E t h ) , a s :
R = Y
(2)
X/S X/S 4
(Eth)
1 +
Y e a s t f l o c c u l a t i o n , an e s s e n t i a l phenomenon i n b e e r f e r m e n t a
t i o n , i s i n f l u e n c e d by t h e medium c o m p o s i t i o n , e s p e c i a l l y by t h e
g l u c o s e c o n c e n t r a t i o n , and i s d e l a y e d by t h e m i x i n g e f f e c t o f CO2
p r o d u c t i o n . The t i m e v a r i a t i o n o f t h e s u s p e n d e d y e a s t c o n c e n t r a
t i o n i s t h u s t a k e n a s t h e d i f f e r e n c e between t h e g r o w t h and f l o c
c u l a t i o n r a t e as :
d(X ) T
dt dt
- k
CO,
(x )
s
(3)
K
G1
w i t h ( X ) t h e suspended y e a s t c o n c e n t r a t i o n
5
k f
the yeast f l o c c u l a t i o n constant
(Gl) the glucose c o n c e n t r a t i o n
K
the i n h i b i t i o n constant o f f l o c c u l a t i o n by g l u c o s e
G1
k t h e m i x i n g c o n s t a n t by CO^
m
d
t h e r a t e o f CO^ d e s o r p t i o n
r
co 2
d(Mal) _ (Mal) 1 , χ , n
V
dt " " Mal K + (Mal M a l
K
G1
d(Sac)
dt " * sac k (
S a c ) (
V ( 6 )
d(Mlt) . (Mit) 1 , γ χ m
"dt - - Mit v
KMit
Z T W- I V , , J W
:
1—+ (cry (
v ( 7 )
1
K K
G1
~dF~~ " * F r K
V
r + (Fr) ~ (GÎT V " 342 "dt ( W
Fr 1 + -pr-
K
G1
d(AA) (AA) 1 d (
V n n )
dt AA Κ Δ + (AA) ~ TThrT dt v J
Thr
Φ* - - Vs <">
The ethanol to sugar y i e l d , R£/s, was found to s l i g h t l y i n
crease during the fermentation as a r e s u l t of the decrease of the
growth y i e l d . Based on a carbon balance of the sugar conversion
i n t o yeast, ethanol and CO2, the v a r i a t i o n of R^/s can be r e l a t e d
to the p r e v i o u s l y expressed v a r i a t i o n of Rx/s as
1 , °> 4 3 ( Y
x/s - R
x/s }
(12)
R = Y
E/S E/S +
0,52 Y E / s + 0,27 Y C Q 2 / S
a s n e
w i t h Yçyg and Yfjg^/s ^ i n i t i a l ethanol to sugar and CO2 to
sugar y i e l d , r e s p e c t i v e l y .
Μ . γ ÎÎV . k ( M ) ( 1 3 )
Y k W
dt " M/X dt M
d ( C t
W ρ
= — Κ
diTFS)
d(TFS) qd
< HH CU U
^ °3") ;
Π ) ( wld(TFS) ( .
dt
c o y s ~6t~ " St + k
d[ ( C 0
2d - ) ( C 0
2 )
j^t^ ( }
w i t h (CO^çj) the d i s s o l v e
(CO2)* the s a t u r a t i o n CO2 concentration
(HCO^") the bicarbonate concentration
k^ the d e s o r p t i o n constant
w a s
The CO2 to sugar y i e l d , Rc02/S> observed to s l i g h t l y i n c r e a s e
d u r i n g the fermentation. As p r e v i o u s l y f o r the v a r i a b l e ethanol to
c a n
sugar y i e l d , the value of Rc02/S be r e l a t e d to the i n i t i a l CO2
anc
to sugar y i e l d , Yc02/S * the v a r i a b l e yeast to sugar y i e l d as
R
C0 /S = Y
C02/S
1 + °> 4 3 (Y
x/s - X/S R }
(15)
2
0,52 Y E / 5 + 0,27 Y C 0 2 / S
ν =
-^[ ν- ν*]^- (ϋ ε (16)
Θ η
^CÛ2 ^ * 9 the CO2 flow r a t e l e a v i n g the fermentor.
The r e s u l t i n g s e t of d i f f e r e n t i a l equations i s n u m e r i c a l l y
i n t e g r a t e d by s t a n d a r t Runge Kutta procedure. The d i f f e r e n t para
meters of the model are determined t o o b t a i n the best agreement
between the theory and the experimental r e s u l t s .
CONCLUSIONS
TIME, h
Figure 1. Time variation of total yeast, suspended yeast, and total fermentable
sugar concentrations in a laboratory scale fermentation. Key: O , total yeast; ·,
suspended yeast; +, total fermentable sugar.
τ Γ τ Γ
ο^ 8h
g Ι" € + * ^ ι ο γ ϊ : ·. ·
^ 4h/ saturation^
ο
t a t i o n . An e s s e n t i a l f i n d i n g o f b o t h l a b o r a t o r y and p i l o t p l a n t
experiments i s the decrease i n c e l l u l a r growth a c t i v i t y d u r i n g the
c o u r s e o f the f e r m e n t a t i o n , the growth phase ending a f t e r about
100 h o u r s o f f e r m e n t a t i o n . T h i s f a l l i n c e l l u l a r v i a b i l i t y i s n o t
c a u s e d by s u g a r o r amino a c i d s l i m i t a t i o n s . I t may p a r t l y r e s u l t
f r o m e t h a n o l a c c u m u l a t i o n i n t h e medium, o r as s u g g e s t e d by o t h e r
s t u d i e s ( 6 ) , from l i m i t a t i o n s i n i n t r a c e l l u l a r s t e r o l s which are
no l o n g e r s y n t h e s i z e d u n d e r a n a e r o b i c c o n d i t i o n s . A c c o r d i n g t o
our r e s u l t s , the r a t e o f sugar uptake i s d i r e c t l y p r o p o r t i o n a l t o
t h e s u s p e n d e d y e a s t c o n c e n t r a t i o n , t h e r a t e o f amino a c i d s u p t a k e
and o f some a r o m a t i c m e t a b o l i t e s p r o d u c t i o n i s d i r e c t l y r e l a t e d
t o t h e y e a s t g r o w t h r a t e , w h e r e a s e t h a n o l and C02 p r o d u c t i o n i s
e s s e n t i a l l y p r o p o r t i o n a l to the t o t a l r a t e o f sugar consumption.
As d e m o n s t r a t e d by t h e good agreement between e x p e r i m e n t s
and t h e o r y , t h e t i m e v a r i a t i o f th d i f f e r e n t specie i th
f e r m e n t a t i o n medium c a
s i m p l e model b a s e d on y e a s physica processe
o f y e a s t f l o c c u l a t i o n , CO2 d e s o r p t i o n and CO2 m i x i n g . E s s e n t i a l
i n t h e model i s t h e i n t r o d u c t i o n o f a y e a s t t o s u g a r y i e l d d e p e n
d i n g on t h e m e a s u r a b l e e t h a n o l c o n c e n t r a t i o n t o r e p r e s e n t t h e l o s s
of c e l l u l a r v i a b i l i t y .
F u r t h e r e x p e r i m e n t a l and t h e o r e t i c a l i n v e s t i g a t i o n s a r e b e i n g
done t o s t u d y i n g r e a t e r d e t a i l t h e p o s s i b l e i n f l u e n c e o f o t h e r
key p r o c e s s p a r a m e t e r s , p i t c h i n g r a t e , p r e s s u r e , w o r t a e r a t i o n , on
t h e f e r m e n t a t i o n p r o c e s s and t h e f i n a l b e e r q u a l i t y .
LITERATURE CITED
1
JEANINE M. COSTA and ANTONIO R. MOREIRA
Colorado State University, Department of Agricultural and Chemical Engineering,
Fort Collins, CO 80523
Due t o t h e r i s i n g c o s t s a s s o c i a t e d w i t h p r o d u c i n g c h e m i c a l s
f r o m f o s s i l f u e l s , a l c o h o l f e r m e n t a t i o n s b a s e d on r e n e w a b l e
r e s o u r c e s a r e b e c o m i n g more and more a t t r a c t i v e a s an a l t e r n a t i v e
r o u t e f o r o b t a i n i n g commodity c h e m i c a l s . However, a p r o b l e m
common t o many o f t h e s e f e r m e n t a t i o n s i s t h e r e l a t i v e l y l o w c o n -
c e n t r a t i o n of the d e s i r e d product i n the f i n a l fermented b r o t h .
B a c t e r i a l ethanol fermentations stop a t a l c o h o l l e v e l s i n the
r a n g e o f 5-8 w e i g h t p e r c e n t C l ) . A more d r a m a t i c t o x i c e f f e c t i s
o b s e r v e d i n t h e a c e t o n e - b u t a n o l f e r m e n t a t i o n . The t o t a l s o l v e n t s
c o n c e n t r a t i o n i s 2-3 w e i g h t p e r c e n t a t t h e p o i n t where s o l v e n t
l e v e l s a r e t o x i c t o t h e p r o d u c i n g C l o s t r i d i u m (2). Butanol,
b u t y r i c a c i d , and a c e t i c a c i d a r e t h e m o s t t o x i c p r o d u c t s o f t h e
acetone-butanol fermentation. I t has b e e n p r e v i o u s l y shown (3)
t h a t a t b u t a n o l c o n c e n t r a t i o n s i n t h e r a n g e o f 0.10-0.15 M, 50%
i n h i b i t i o n i s o b s e r v e d s i m u l t a n e o u s l y f o r t h e maximum s p e c i f i c
g r o w t h r a t e , t h e n u t r i e n t u p t a k e r a t e , and t h e membrane-bound
ATPase a c t i v i t y . This end-product t o x i c i t y r e s u l t s i n l a r g e
1
Current address: International Flavors and Fragrances, Inc., Union Beach, N J 07735
0097-6156/83/0207-0501$06.00/0
© 1983 American Chemical Society
energy e x p e n d i t u r e s f o r p r o d u c t r e c o v e r y as w e l l as i n l a r g e
c a p i t a l i n v e s t m e n t s due t o t h e l a r g e s i z e e q u i p m e n t r e q u i r e d t o
r e a c h an e c o n o m i c a l l y a t t r a c t i v e s c a l e o f p r o d u c t i o n . Conse
q u e n t l y , k i n e t i c d a t a i s needed t o d e v e l o p a b a s i c u n d e r s t a n d i n g
of t h e s e f e r m e n t a t i o n p r o c e s s e s and t o p e r m i t an o p t i m a l d e s i g n
f o r t h e f e r m e n t o r s y s t e m s t o be u s e d f o r t h e p r o d u c t i o n o f t h e s e
chemicals. T h i s paper addresses t h e k i n e t i c s o f end-product
i n h i b i t i o n o f c e l l growth i n t h e acetone-butanol f e r m e n t a t i o n .
M a t e r i a l s a n d Methods
M i c r o o r g a n i s m and C u l t u r e C o n d i t i o n s . C l o s t r i d i u m a c e t o -
b u t y l i c u m s t r a i n ATCC 824 was u s e d i n t h i s s t u d y . Maintenance
c u l t u r e s were grown i n c o r n mash medium f o r 72 h o u r s a t 37 C a n d
then r e f r i g e r a t e d a t 4 t 5 C Th h mediu c o n s i s t e d f
5% (w/v) c o r n m e a l w i t
anaerobic conditions. Th appropriat
volume o f d i s t i l l e d w a t e r f o r one h o u r . The mash was t h e n d i s
t r i b u t e d i n t o s c r e w c a p t u b e s (16 χ 125 mm) a n d a u t o c l a v e d f o r 15
m i n u t e s a t 121 C.
The m i c r o o r g a n i s m was r o u t i n e l y t r a n s f e r r e d a n a e r o b i c a l l y i n
s c r e w c a p t u b e s w i t h 10 m l o f medium c o m p r i s e d o f 5 0 % ( v / v ) t h i o -
g l y c o l l a t e 135C medium ( D i f c o L a b o r a t o r i e s , D e t r o i t , M i c h i g a n ) a n d
50% ( v / v ) s o l u b l e medium c o n t a i n i n g t h e f o l l o w i n g components i n
g/1: K H P 0 , 0.75; Κ 2 Η Ρ Ο 4 , 0.75; MgS04, 0.20; M n S 0 . H 0 , 0.01;
2 4 4 2
G r o w t h C h a l l e n g e S t u d i e s . The e f f e c t o f t h e f e r m e n t a t i o n
p r o d u c t s o n t h e g r o w t h r a t e o f C I . a c e t o b u t y l i c u m was d e t e r m i n e d
by t h e f o l l o w i n g p r o c e d u r e . A 24-hour o l d c u l t u r e was u s e d a s a
5% i n o c u l u m t o f l a s k s c o n t a i n i n g 200 m l o f 2% (w/v) g l u c o s e
s o l u b l e medium. A f t e r 10-12 h o u r s , t h e s e c e l l s w e r e u s e d a s a 2 0 %
(v/v) i n o c u l u m f o r f l a s k s c o n t a i n i n g s o l u b l e m e d i a w i t h 2% (w/v)
g l u c o s e . A f t e r a l a g p h a s e o f 30-45 m i n u t e s , t h e c u l t u r e s were
c h a l l e n g e d w i t h v a r i o u s c o n c e n t r a t i o n s o f e t h a n o l , b u t a n o l , and
acetone. G r o w t h was m o n i t o r e d b y h o u r l y measurements o f o p t i c a l
d e n s i t y a t 560 nm.
The p r o c e d u r e f o r d e t e r m i n a t i o n o f g r o w t h r a t e s i n t h e
p r e s e n c e o f a c e t i c a n d b u t y r i c a c i d was m o d i f i e d a s f o l l o w s . The
i n o c u l u m was p r e p a r e d b y u s i n g c e l l s f r o m a c o r n t u b e a s a 1 0 %
(v/v) i n o c u l u m f o r t h i o g l y c o l l a t e / s o l u b l e medium c o n t a i n i n g 20 g/1
MES b u f f e r ( 2 - [ N - m o r p h o l i n o ] e t h a n e s u l f o n i c a c i d ) , (Sigma Chemi
c a l Co., S t . L o u i s , MO). A f t e r 10-12 h o u r s , t h e s e c e l l s w e r e
used as a 20% (v/v) inoculum f o r f l a s k s c o n t a i n i n g s o l u b l e media
(20 g/1 g l u c o s e + 20 g/1 MES b u f f e r ) . A f t e r a l a g p h a s e o f 30-45
m i n u t e s , t h e c e l l s were c h a l l e n g e d w i t h v a r i o u s c o n c e n t r a t i o n s o f
a c e t i c and b u t y r i c a c i d .
pH C o n t r o l l e d F e r m e n t a t i o n . A 7 - l i t e r New B r u n s w i c k M i c r o -
f e r m f e r m e n t o r (New B r u n s w i c k s c i e n t i f i c , E d i s o n , NJ) w i t h a
w o r k i n g volume o f 4 l i t e r s was u s e d i n t h i s s t u d y . The a g i t a t i o n
s p e e d was m a i n t a i n e d a t 2 0 0 rpm d u r i n g t h e f e r m e n t a t i o n . The pH
o f t h e f e r m e n t a t i o n s t a r t e d a t 6.2 and was a l l o w e d t o f a l l t o pH
5.0. The pH was t h e r e a f t e r m a i n t a i n e d a t 5.0 by t h e a d d i t i o n o f
1 Ν NaOH on a demand b a s i s . The t e m p e r a t u r e was c o n t r o l l e d a t
37 C
R e s u l t s and Discussion
Product C h a l l e n g e d Growth S t u d i e s . To s t u d y t h e i n h i b i t o r y
f a c t o r s of the acetone-butanol fermentation, the growth r a t e s of
CI. acetobutylicum i n the presence of each fermentation product
were d e t e r m i n e d . The end p r o d u c t s u s e d i n t h i s s t u d y i n c l u d e d
e t h a n o l , b u t a n o l , a c e t o n e , a c e t i c a c i d , and b u t y r i c a c i d . From
the slopes of the l e a s t squares r e g r e s s i o n l i n e s of o p t i c a l
d e n s i t y v s . t i m e d a t a , t h e maximum s p e c i f i c g r o w t h r a t e s i n t h e
p r e s e n c e o f v a r y i n g c o n c e n t r a t i o n s o f e a c h i n h i b i t o r ( y ) were
m
d e t e r m i n e d . The r e s u l t s f o r e a c h f e r m e n t a t i o n p r o d u c t a r e shown
in Figures 1 - 3. T h e r e a p p e a r s t o be a t h r e s h o l d c o n c e n t r a
t i o n w h i c h must be r e a c h e d b e f o r e g r o w t h i n h i b i t i o n o c c u r s . This
c o n c e n t r a t i o n was f o u n d t o v a r y w i t h e a c h i n h i b i t o r s t u d i e d .
Above t h e t h r e s h o l d c o n c e n t r a t i o n , t h e g r o w t h i n h i b i t i o n c a n be
d e s c r i b e d by a l i n e a r r e l a t i o n s h i p o f t h e f o r m :
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μ 1
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m m κ ο = ο
Ρ
C o n c e n t r a t i o n s c a u s i n g a 5 0 % r e d u c t i o n i n g r o w t h r a t e were a l s o
determined. A summary o f t h e i n h i b i t i o n d a t a o b t a i n e d i s g i v e n
i n Table I .
Table I. Summary o f G r o w t h I n h i b i t i o n D a t a
M 2/1
Butyric Acid 0.07
Butanol 0.15 0.06 0.15 11.0
Acetic Acid 0.19 0.05 0.13 8.0
Acetone 0.98 0.26 0.88 43.5
Ethanol 1.01 0.26 1.10 51.0
The i n h i b i t i o n c o n s t a n t Kp f o r e a c h p r o d u c t was c a l c u l a t e d
from t h e s l o p e o f the l e a s t squares r e g r e s s i o n l i n e o f y ^ / V ^ v s .
Ρ data. The i n h i b i t i o n c o n s t a n t s f o r e t h a n o l a n d a c e t o n e a r e
approximately t e n times g r e a t e r than t h a t f o r b u t a n o l , a c e t i c
a c i d , and b u t y r i c a c i d . T h i s i s i n d i c a t i v e o f t h e r e l a t i v e l y low
t o x i c i t y o f a c e t o n e a n d e t h a n o l a s compared w i t h t h e o t h e r f e r
mentation products.
F o r e a c h f e r m e n t a t i o n p r o d u c t , t h e P v a l u e was p l o t t e d
Q
a g a i n s t t h e Kp v a l u e a s shown i n F i g u r e 4. The t h r e s h o l d c o n
c e n t r a t i o n was f o u n d t o i n c r e a s e l i n e a r l y w i t h a n i n c r e a s i n g Kp
value. The e q u a t i o n o f t h i s l i n e was d e t e r m i n e d t o b e :
t h e s t r a i g h t l i n e shown i n F i g u r e 4. L i n d e n e t a l . (3) h a v e
shown t h a t t h e e n d p r o d u c t t o x i c i t y i n t h e a c e t o n e - b u t a n o l f e r
m e n t a t i o n o c c u r s b y a l t e r i n g membrane f u n c t i o n a l i t y . The l i n e a r
r e l a t i o n s h i p b e t w e e n Kp a n d P may i n d i c a t e t h a t t h e i n h i b i t i o n
Q
f o u n d t o be n o n i n h i b i t o r y t o c e l l g r o w t h . The h i g h e s t c o n c e n t r a
t i o n s o f b u t y r i c a c i d and a c e t i c a c i d w h i c h h a v e b e e n o b s e r v e d
d u r i n g f e r m e n t a t i o n a r e i n t h e r a n g e o f 3.5 t o 4.0 g/1. These
l e v e l s o f a c i d s a p p r o a c h t h o s e c a u s i n g 50% i n h i b i t i o n o f g r o w t h .
T y p i c a l butanol concentrations observed d u r i n g a fermentation are
11-12 g/1. Butanol i s the only product t h a t reaches l e v e l s
a c t u a l l y c a u s i n g 50% g r o w t h i n h i b i t i o n .
R e c e n t r e s u l t s r e p o r t e d by L e u n g and Wang (5) show a c e t i c
and b u t y r i c a c i d c o n c e n t r a t i o n s c a u s i n g 50% i n h i b i t i o n o f g r o w t h ,
w h i c h a r e about t w i c e t h e l e v e l s o b t a i n e d i n t h i s work. The
a p p a r e n t d i s a g r e e m e n t b e t w e e n t h e two s e t s o f d a t a c o u l d be due
t o t h e d i f f e r e n t methods u s e d i n e a c h c a s e t o s t a b i l i z e t h e pH o f
t h e f e r m e n t a t i o n m e d i a d u r i n g an a c i d c h a l l e n g e .
The m e t h o d o l o g y u s e d i n t h i s s t u d y i n v o l v e d t h e use o f a
b i o l o g i c a l b u f f e r , MES ( 2 - [ N - m o r p h o l i n o ] e t h a n e s u l f o n i a c i d )
s t a b i l i z e t h e pH. Cell
t a i n i n g MES b u f f e r and t h
d i f f e r e n c e i n g r o w t h r a t e s . T h i s i n d i c a t e s t h a t MES has no
e f f e c t on t h e g r o w t h r a t e o f t h e o r g a n i s m .
The m e t h o d o l o g y t o s t a b i l i z e t h e pH o f t h e f e r m e n t a t i o n
m e d i a r e p o r t e d by L e u n g and Wang (5) i n v o l v e d t h e p r e p a r a t i o n o f
200 g/1 s o l u t i o n s o f a c e t a t e and b u t y r a t e by t i t r a t i o n w i t h NaOH.
I n t h i s manner, t h e c e l l s were e x p o s e d t o Na+ c o n c e n t r a t i o n s i n
t h e r a n g e o f 2-7 g/1. The h i g h c o n c e n t r a t i o n s o f Na+ may h a v e an
o s m o t i c o r membrane s t a b i l i z i n g e f f e c t w h i c h may a c c o u n t f o r t h e
h i g h e r t o l e r a n c e t o a c i d s . However, i n s p i t e o f t h e s e d i s c r e p a n
c i e s , t h i s i n v e s t i g a t i o n supports the c o n c l u s i o n t h a t the con
c e n t r a t i o n o f b u t a n o l i s an i m p o r t a n t p a r a m e t e r i n t h e a c e t o n e -
butanol fermentation.
Attempts t o model the c e l l growth curve d u r i n g a b a t c h f e r
m e n t a t i o n by c o n s i d e r i n g t h e i n h i b i t i o n due t o e a c h p r o d u c t
s e p a r a t e l y r e s u l t e d i n much h i g h e r g r o w t h r a t e s t h a n were a c t u a l l y
observed. Two f a c t o r s c o u l d c o n t r i b u t e t o t h i s d i s c r e p a n c y . On
t h e one h a n d , t h e p r o d u c t i n h i b i t i o n e x p e r i m e n t s were c a r r i e d o u t
u s i n g e a c h f e r m e n t a t i o n p r o d u c t s e p a r a t e l y . However, i n an a c t u a l
fermentation the products are present together. T h i r t e e n hours
i n t o t h e f e r m e n t a t i o n , as shown i n F i g u r e 5 , t h e c o n c e n t r a t i o n o f
e a c h p r o d u c t i s b e l o w t h e t h r e s h o l d c o n c e n t r a t i o n when g r o w t h
i n h i b i t i o n was d e t e r m i n e d t o o c c u r . T h i s i n d i c a t e s t h a t no g r o w t h
i n h i b i t i o n s h o u l d be o b s e r v e d . However, t h e c e l l g r o w t h r a t e a t
t h i s t i m e was d e t e r m i n e d t o be o n e - h a l f o f t h e maximum s p e c i f i c
c e l l g r o w t h r a t e . T h i s i n d i c a t e s t h a t some s y n e r g i s m o c c u r s
among t h e s e v e r a l f e r m e n t a t i o n p r o d u c t s and i t i s p r o b a b l y t h e
t o t a l concentration of products which i s important i n determining
t h e t o x i c e f f e c t on g r o w t h r a t e .
The o t h e r f a c t o r c o u l d be t h e d i f f e r e n c e i n t o x i c e f f e c t s o f
p r o d u c e d s o l v e n t s as o p p o s e d t o a d d e d s o l v e n t s . Novak e t a l . (6)
have shown t h a t e t h a n o l p r o d u c e d d u r i n g a b a t c h f e r m e n t a t i o n o f
S a c c h a r o m y c e s c e r e v i s i a e i s more i n h i b i t o r y t h a n e t h a n o l added t o
the fermentation. Therefore, i t i s p o s s i b l e t h a t the i n h i b i t i o n
s sa
δ·
ο
-Ci
ε §
JD . -s:
s «ο
ci
>>«*§
1*1
Η<
Ο Ο
£ ,3
^ ©Ο
«ο
la
"Ι
s:
§δ
p a r a m e t e r s d e t e r m i n e d i n t h i s s t u d y , based on added s o l v e n t s ,
m i g h t be l e s s s e v e r e t h a n t h e a c t u a l v a l u e s due t o t h e s o l v e n t s
produced during the fermentation.
Conclusions
From t h e s e s t u d i e s o f g r o w t h i n h i b i t i o n a n d f e r m e n t a t i o n
k i n e t i c s i n the acetone-butanol fermentation, the f o l l o w i n g
c o n c l u s i o n s may be made:
2. F o r each f e r m e n t a t i o
t i o n below which n growt
a l i n e a r decrease i n growth r a t e i s observed w i t h an i n c r e a s e
i n product concentration.
fermentation product.
4. P r e l i m i n a r y o b s e r v a t i o n s seem t o i n d i c a t e t h a t t h e g r o w t h
i n h i b i t i o n c a u s e d by s o l v e n t s p r o d u c e d d u r i n g f e r m e n t a t i o n
i s d i f f e r e n t from t h e i n h i b i t i o n caused by e x t e r n a l l y added
solvents.
Legend o f Symbols
Κ i n h i b i t i o n constant (M)
Ρ —
Ρ p r o d u c t c o n c e n t r a t i o n (M)
P^ threshold product concentration (M)
μ maximum s p e c i f i c g r o w t h r a t e (hr~l)
m
μί maximum s p e c i f i c g r o w t h r a t e i n p r e s e n c e o f
1
i n h i b i t o r (hr" )
Acknowledgments
P a r t i a l s u p p o r t f o r t h i s r e s e a r c h was p r o v i d e d b y t h e U.S.
D e p a r t m e n t o f E n e r g y u n d e r S u b c o n t r a c t No. ΧΚ-φ-9059-l. The
a u t h o r s a l s o w i s h t o a c k n o w l e d g e D r . James C. L i n d e n f o r h e l p f u l
discussions.
Literature Cited
R E C E I V E D August 2, 1982
515
Chemotaxis— Ε
Continued
and competition in mixed cultures 276/ Eccrinolysis in mixed cultures 214
Chromatography, size exclusion, Ecology, and demand theory of
procedure 445-449 gene regulation 15
Clostridium acetobutylicim, acetone- Ecosystems, effects of cell motility
butanol fermentation 501-512 on populations 265-293
Clumping, mass transfer in viscous Efficiency
systems with microbial network energetic, of ion pumps 326
structure 351 ion pumps 323, 326
Colpoda steinii, feeding behavior thermodynamic, for non-
in mixed cultures 215, 216, 219 equilibrium 300-304
Commensalism, in mixed cultures 210-212 Electrodes, enzyme 46, 48, 50/
Competition in mixed cultures 205-209 Electrogenic pumps—See Ion pumps
effect of cell motility 286-289 Enteric bacteria
mathematical models 206-209 demand theory 15
Continuous culture gene regulation 15, 18/-21/
Hansenula polymorpha 187, 188/ and demand theory of gene
single-cell protein productio
Continuous reactors
rate laws 466, 468 Enzymatic hydrolysis of starch 443-461
reaction mechanism 464-467/ effect of starch recrystallization 457, 458/
Control effect of temperature 448
See also Regulation Enzyme activity
of reactor in reactor design 370-373/ mechanisms of regulation 73/
Cooper-Helmstetter model 137, 138, 140 physiological patterns affecting 71,72/
Cornell model 100-102/, 114-121/ Enzyme amount
predictions 116-121/ mechanisms of regulation 77-83
Crowding in mixed cultures 222, 223 physiological mechanisms of
Cybernetic approach, diauxic growth 164 regulation 75
Cybernetic perspective physiological patterns of
cell control system 165-176 regulation 76, 77
microbial growth 161-178 Enzyme electrodes 46, 48, 50/
Cytometry, flow 135, 139/ Enzyme inactivation in vivo 55, 57
Enzvme systems, kinetics 27-52
Henri equation 27-32
D
Enzyme-catalyzed reactions, kinetics
Demand theory immobilized enzymes 38
Agrobacterium tumefaciens as particulate substrates 32-37
example 22 soluble substrates 27-32
enteric bacteria as example 15 Enzymes
gene regulation 3, 7 See also specific enzymes
development 13 degradation of insoluble substrates,
and physiology 16/ mass transfer 345
Depolymerization of cell wall, kinetic immobilized cells as catalysts—See
analysis of enzyme reaction 35 also Immobilized cells
Design—See Reactor design preparation and reaction
Diauxic growth performance 377-392
dissolved oxygen as indicator 171/ comparison of kinetics, with
example of cybernetic approach .... 164 soluble enzymes 40/, 42/, 43
Dictoystelium discoideum kinetics 38-48, 343, 345
feeding behavior in mixed cultures 215 production, strategies for
predator for Escherichia coli 254 optimization 189-191
Didinum nasutum, feeding behavior production of heparinase by
in mixed cultures 214,215 Flavobacterium heparinum 191-192/
D N A synthesis in bacteria 140 strategies for optimization 191-192/
Protocooperation in mixed S
cultures 213-214 Saccharomyces cerevisiae
Proton pumps—See Ion pumps cell division cycle 151, 152/
Pseudomonas and Hyphomicrobiwn, fed-batch fermentation 181-184
interactions in mixed cultures 214 computer control 181-184
Pseudomonas ovalis colonies, dis- strategy for fermenter
solved oxygen contours 395-401 operation 181-184
mathematical model of growth
R kinetics 150-154
Reactor design Saccharomyces italicus
See also Reactor design fundamentals mutation 189,191/
constraints 335, 336 optimization of maltase
effect of flow 339-342 production 189, 191/
effect of interfacial phe- Scale-up in reactor design 370, 374
nomena 338, 340/ Schizosaccharomyces pombe
effect of interparticle transfer cell cycle 145/
rates 339-342 flow cytometer measure-
kinetics and transport phe- ments 144, 146, 147/
nomena 335-35
effect of mixing 342 kinetics 142,144-150
rate controlling steps 336-338 trajectory tracking kinetics 149/, 150
stagnant reactor 339 Serratia marcescens
systems approach 355-358 mutation for production of
Reactor design fundamentals 335-376 isoleucine 87/
heat transfer 368, 369 production of isoleucine 84-86/
hydrodynamics 358-359 Sewage treatment 250
mass transfer 359-365 See also Thiobacillus A 2 , T.
process synthesis at reactor level 369 neapolitanus, Spirillum G 7
reactor control 369-373/ Single-cell kinetics, steady-state
scale-up 370 models 138
Reactor equipment performance Single-cell metabolic models 135-158
bubble columns 345, 346 Single-cell protein
mechanically stirred tanks 347-351 continuous culture 185-187
systems with stationary internals 346 optimizing production 185-187
Reaction mechanism source, Hansenula polymorpha 187, 188/
modified Michaelis-Menten 464-467/ Size distribution of starch
for process whose rates determination by size exclusion
saturate in substrate chromatography 445-449
concentration 464-467/ during enzymatic hydrolysis ... 443-461
Reaction performance, immobilized Size exclusion chromatography,
cells as catalysts—See Immo- procedure 445-449
bilized cells Size of starch
Reaction rate laws effect of recrystallization 457, 458/
development, for processes whose effect of temperature 448
rates saturate in substrate Solid state fermentation
concentration 466, 468-477 of grains 423
thermal sensitivity 470-477 of lignocellulose 421-442
Reaction rates, intraparticle 342-345 choice of microorganisms 426
Reactors mechanism of fungal growth 426-437
effect of start-up conditions on physiological aspects 437,438
growth of Schizosac- pretreatment of lignocelluloses 424-426
charomyces pombe 146, 147/ requirements 437, 438
microbial, population balance Spirillum G7 as example 232, 234/, 235/
equations 135, 136 Thiobacillus neapolitanus as
Regulatory mechanisms, microbial 71-92 example 232, 234/, 235/
Repressor mechanisms of gene Specialists and generalists
regulation 9/ growth rate 230,231