Accepted Manuscript

Title: A process for complete biodegradation of shrimp waste

by a novel marine isolate Paenibacillus sp. AD with
simultaneous production of chitinase and chitin
oligosaccharides

Authors: Aditya Kumar, Deepak Kumar, Nancy George,

Prince Sharma, Naveen Gupta

PII: S0141-8130(17)34341-6
DOI: https://doi.org/10.1016/j.ijbiomac.2017.12.024
Reference: BIOMAC 8685

To appear in: International Journal of Biological Macromolecules

Received date: 7-11-2017

Revised date: 22-11-2017
Accepted date: 4-12-2017

Please cite this article as: Aditya Kumar, Deepak Kumar, Nancy George, Prince
Sharma, Naveen Gupta, A process for complete biodegradation of shrimp waste
by a novel marine isolate Paenibacillus sp.AD with simultaneous production
of chitinase and chitin oligosaccharides, International Journal of Biological
Macromolecules https://doi.org/10.1016/j.ijbiomac.2017.12.024

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A process for complete biodegradation of shrimp waste by a novel marine isolate

Paenibacillus sp. AD with simultaneous production of chitinase and chitin

oligosaccharides

Aditya Kumar, Deepak Kumar, Nancy George, Prince Sharma, Naveen Gupta*

Department of Microbiology, Panjab University Chandigarh 160014

To whom Correspondence should be addressed

Dr. Naveen Gupta

Department of Microbiology, BMS Block, South Campus

Panjab University, Chandigarh, India

E-mail: [email protected]

Mob.: + 91-9872692680, 9041062680

Fax.: +91- 172-2541770

Abstract

Disposal of chitinaceous waste is a major problem of seafood industry. Most of the known
chitinolytic organisms have been studied with respect to pure chitin as substrate. Use of these
organisms for degradation of seafood waste has not been explored much. In present study a
marine bacterium capable of proficiently degrading shrimp waste with co-production of value
added products like chitinase and chitin oligosaccharides was isolated from seafood waste
dumping sites. On 16s rRNA and biochemical analysis bacterium was found to be a novel
species of genus Paenibacillus.Under optimized condition complete shrimp waste
degradation (99%) was achieved along with chitinase yield of 20.01IUml-1. SEM and FTIR
showed the structural changes and breakage of bonds typical to that of chitin, which indicated
that this process can be used for the degradation of other chitinaceous material also. Thin
layer chromatography revealed the presence of chitin oligosaccharides of various degree of
polymerization in the hydrolysate. Complete degradation of shrimp waste by Paenibacillus
sp. AD makes it a potential candidate for the bioremediation of seafood waste at large scale.
Concomitant production of chitinase and chitin oligosaccharides further makes the process
economical and commercially viable.

Keywords: Paenibacillus sp. AD, Chitinase, Shrimp waste degradation, Optimization, Chitin

oligosaccharides, Thin layer chromatography.

1. Introduction

The world seafood industry plays an important role in feeding of a significant part of the

world population. Estimated total sea food demand is going to be 183 million tons by 2030

[1]. With the growing demand for Indian seafood products across the world, the business of

seafood in India is increasing rapidly.

Seafood processing activities generate potentially large quantities of wastes like shrimp

shells, crab shell, prawn waste and fish scales from inedible fish parts and endoskeleton shell

parts from the crustacean peeling process. Generally, 50 to 70 per cent of seafood raw

material goes as waste. Management of this huge waste is a problem for sustainability of

seafood processing industry [2].The major part of the waste material generated by sea food

industry is the chitinaceous waste [3]. It has been found that waste generated on the

processing of shrimps and crabs contains up to 50% of chitin by dry weight [4, 5]. Chitin, a

highly insoluble biopolymer, is one of the most abundant organic compounds in nature (after

cellulose). Chitin is composed of linear chains of ß-1, 4-linked N-acetyl D-glucosamine

residues that are highly cross-linked by hydrogen bonds.

The conventional method for seafood waste disposal includes ocean dumping, incineration

and land filling which lead to environmental pollution and bio-fouling. The insolubility of

chitin and its inertness to most chemical agents has increased the search for alternative

disposal methods such as biological processing. Moreover, after bioremediation of sea food

waste, its byproducts can be used for various purposes e.g. preparation of pharmaceutically

important chitin oligosaccharides, N-Acetyl –D –glucosamine [6], production of single cell

protein [7] ethanol [8] etc. In addition to this chitinase produced during this processing can be

used for the various biotechnological applications e.g. for controlling pathogenic fungi,

isolation of protoplasts from fungi, yeast and synthesis of pharmaceutically important

compounds etc. [9]. Hence bio-processing of seafood waste is not only a step towards
environmental protection but there is also a potential for making huge money via its by-

product/s utilization.

A number of microorganisms including bacteria [10, 11, 12] as well as fungi [7, 13, 14] have

been reported to produce chitinase but their commercial applications are restricted due to low

enzyme yield, high production cost and less stability of the enzyme. Moreover most of these

organisms have been reported to utilize pure chitin as a carbon source [15, 16]. Some

organisms have been reported to produce chitinase [17, 18] and chitin oligosaccharides [19,

20, 10] utilizing seafood waste. But no systematic study has been done to optimize conditions

for the effective degradation of sea food waste and develop a bioremediation strategy using

chitinolytic organisms.

In this perspective the present study was undertaken to isolate a potent chitinolytic bacterium

which could efficiently degrade seafood waste. Significant variables influencing shrimp

waste degradation and chitinase production were identified using the Plackett–Burman (PB)

design. The levels of the significant variables were further optimized using Central

Composite Design (CCD) to develop a process for the optimal degradation of shrimp waste

and concomitant production of industrially important extracellular chitinase and bioactive

chitin oligosaccharides. Further the hydrolytic efficacy of chitinase was evaluated by

analysing degraded shrimp waste and hydrolysate by SEM, FTIR and TLC.

2 Material and methods

2.1 Substrate and Chemicals

Shrimp flakes, nutrient broth, yeast extract, beef extract, agar agar NH 4NO3, gelatin, urea,

starch, D-glucose, fructose, galactose, sucrose, Ehrlich’s Reagent A.R. (p-Di-methyl

aminobenzaldehyde, A.R.), were purchased from HIMEDIA (India), Potassium tetraborate

was procured from Sigma and other reagents required for the enzyme assay i.e. NaH2PO4,
 Na2HPO4, K2HPO4, KH2PO4, MgSO4 FeSo4.7H2O, MnCl2, ZnSO4, HCl and glacial acetic

acid were bought from Fisher Scientific UK. Chitin oligosaccharide standards were

purchased from Qingdao BZ Oligo Biotech Co., Ltd. China

2.2 Colloidal chitin preparation

Colloidal chitin was prepared according to the modified method as described by [21]. Chitin

flakes were crushed and 5g of chitin powder was added to 60 ml of concentrated HCl and

incubated for 2 to 3 h at 28- 30°C with constant stirring. Then 200ml of ice-cold distilled

water was added and kept for overnight incubation at 4°C. Precipitates obtained were

washed repeatedly with distilled water until its pH became 7.

2.3 Isolation and screening of chitinase producing bacteria

Chitinase producing bacteria were isolated on minimal medium reported by HSU and

Lockwood, 1975 [22]. Soil samples from coastal areas of Chennai, India and sea food waste

dumping sites of Kerala, India were added in minimal medium containing shrimp waste (1%)

for enrichment, after 5 days appropriate dilutions were plated on minimal medium plates

containing colloidal chitin (0.5%). The plates were incubated at 37º C for 72 h. The bacteria

showing different colony morphology and zone of hydrolysis were shortlisted.

2.4 Selection of the isolate on the basis of chitinase yield and degradation of shrimp waste

Shortlisted bacterial isolates were screened for the shrimp waste degradation and presence of

extracellular chitinase. 20 ml broth of minimal medium containing 1% shrimp waste was

inoculated with 1% inoculum of overnight grown cells and incubated at 37°C for 96 h. Then

the cultures were centrifuged at 10000rpm for 15 min at 4 °C and supernatant was used as an

extracellular enzyme. The pellet was washed thoroughly and dried at 80°C to determine the

weight of residual shrimp waste. The strain/s exhibiting maximum shrimp waste degradation

and high extracellular chitinase activity were selected for further studies.
2.5 Chitinase Assay procedure

Chitinase activity was determined by using colloidal chitin (1%) as substrate at 45°C for 5

min. in 0.1 M phosphate buffer (pH 7) and then centrifuged at 5,000rpm for 10 min. The

supernatant was further used for analysis of reducing amino sugars by the Reesieg method

[23]. One unit (IU) of chitinase activity was defined as the amount of enzyme required to

release one micromole of N-acetyl D-glucosamine (GlcNAc) per minute by one ml of

enzyme under the standard assay conditions.

2.6 Identification of selected strain

The cellular morphology of the bacterial isolate was examined by Gram staining.

Physiological and biochemical characterization was done according to Bergey’s Manual of

Determinative Bacteriology [24]. Further identification of bacterium was done by 16S rRNA

sequencing wherein the genomic DNA was isolated and 16S rRNA fragment was amplified.

The product was sequenced using universal primers and sequence analysis was carried out.

Phylogenetic tree was constructed with 1000 bootstrap replicates using MEGA 7 software

[25].

2.7 Optimization of chitinase production and shrimp waste degradation in submerged

fermentation (SmF)

2.7.1 Optimization by one variable at a time method (OVAT)

The effect of various physiochemical factors on shrimp waste degradation and chitinase

production was analyzed by varying one factor at a time keeping the other factors constant.

Different parameters were optimized which includes incubation time, different media, pH,

temperature, carbon source, nitrogen source and metal ions. All the experiments were carried

out in triplicates.
2.7.2 Selection of significant factors by Plackett-burman (PB)

On the basis of results of one variable at a time studies and data available in literature, 11

different parameters viz.: A: NaCl, B: Casein, C: Yeast Extract, D: Inoculum Size, E:

Glucose, F: KCl, G: MgSO4, H: MgCl2, J:Ammonium Sulfate, K: Fructose, L: Di-Sodium

hydrogen phosphate were selected and design was employed to choose significant parameters

among them.

Each factor was investigated at two levels high (+) and low (-). A design of total 12

experiments was generated by using the software Design Expert 8.0.7.1 (Table 2). The effect

of individual factors on enzyme activity was calculated according to the following equation:

∑ Pi+ – ∑ Pi— ………….. Eq. 1

Ei= N

Where Ei is the effect of parameter i under study, Pi+ and Pi- are responses of trials at which

the parameter was at its high and low level respectively, and N is the total number of trials.

From the Pareto chart, the factors showing maximum positive effect were selected.

2.7.3 Central composite design (CCD)

The variables which showed positive effect (ammonium sulfate, sodium chloride, magnesium

sulfate and potassium chloride) on shrimp waste degradation as well as chitinase production

were optimized using CCD. The minimum and maximum range of the selected variables i.e.

ammonium sulfate (0.2-0.5g), sodium chloride (0.2-0.5g), magnesium sulfate (0.05-0.1g) and

potassium chloride (0.0002-0.0005g) were used in 30 combinations (Table S1). The RSM

model was validated further for predicted versus actual responses.

TABLE S1
2.8 Structural analysis of shrimp waste and hydrolysate after treatment with

Paenibacillus sp. AD

2.8.1 Scanning electron microscopy (SEM)

SEM was used to determine the effect of Paenibacillus sp. AD on shrimp flakes degradation.

Samples of shrimp flakes untreated and treated with Paenibacillus sp. AD were fixed with

10% glutaraldehyde and dehydrated with a gradient of ethanol and hexamethyl disilazane

(HMDS). The samples were then gold coated and observed under SEM.

2.8.2 Fourier-transformed infrared spectra analysis (FTIR)

The Paenibacillus sp. AD treated and untreated shrimp flakes samples were embedded in

KBr disk with Perkin Elmer-hydraulic press and subjected for FTIR with a Perkin Elmer-

spectrum 400 FT-IR/FT-FIR spectrometer at room temperature.

2.8.3 Analysis of hydrolytic products by Thin Layer Chromatography (TLC)

Hydrolyzed products were detected by TLC using silica gel plates 60 F 254. All samples

were applied in equal quantities (2 µl) and were resolved by using N- Butanol: Ethanol:

water (2 : 1 : 1) as mobile phase and spots were detected by spraying with diphenylamine

aniline phosphoric acid reagent mixed in ethanol followed by heating at 100°C for 5 min.

[26]. A mixture consisting of monomer, dimer, trimer and tetramer of N-acetyl D-

glucosamine was used as a standard.

3. Results and discussion

3.1 Isolation screening and selection of chitinase producing bacteria

Soil samples were collected from coastal areas of Chennai and sea food waste dumping areas

of Kerala, India. Samples were enriched with shrimp waste (1%) and chitinolytic bacteria

were isolated on the basis of zone of clearance on colloidal chitin agar plates. Among these
strains Isolate AD was selected for further studies owing to its ability to effectively degrade

shrimp waste and produce high yield of extracellular chitinase.

3.2 Identification of Isolate AD

The organism was identified on the basis of morphology, biochemicals and 16S rRNA

analysis (Accession no. MG183682). Analysis demonstrated that Isolate AD was novel,

showing only 95.79% sequence similarity with type strain Paenibacillus chitinolyticus NBRC

15660 (Fig.1). This novel isolate could be distinguished on the basis of biochemical analysis

also; as it was different from Paenibacillus chitinolyticus NBRC 15660 with respect to

catalase, lactose reduction, and casein hydrolysis [27] (Table 1). It was evident from the

analysis that Isolate AD can be classified as representative of a novel species of genus

Paenibacillus for which the name Paenibacillus sp. AD was designated. The strain was

deposited in Microbial Type Culture Collection (MTCC), Chandigarh India (MTCC no.

12619).

FIGURE 1

TABLE 1

3.3 Optimization of biodegradation of shrimp waste and chitinase production

3.3.1. Optimization of physicochemical factors by one variable at a time (OVAT)

method

Classical OVAT method was used for optimization of shrimp waste degradation and enzyme

production. 3 fold and 4 fold increase in shrimp waste degradation and chitinase yield

respectively, was achieved under the following optimized conditions, incubation time 96h,

minimal medium [22] supplemented with 1 % shrimp waste, pH 7, temperature 37°C and

0.5% ammonium sulfate (Table S2). The maximum increase in degradation and enzyme yield
was achieved in minimal medium with the addition of ammonium sulfate. Ammonium sulfate

has been reported to increase chitinase yield in case of Serratia marcescens and Aspergillus

sp. Sl-13 also [28, 29].

TABLE S2

3.3.2. Optimization by statistical methods

3.3.2.1 Screening of important components using Plackett-Burman (PB) Design

The effect of 11 different parameters was estimated for shrimp waste degradation and

chitinase production. Shrimp waste degradation ranging from 10% to 69% and enzyme yield

ranging from 3.84 IUml-1 to 5.28 IUml-1 was observed (Table 2). Influence of various

parameters on shrimp waste degradation and chitinase production has been represented in

pareto chart (Fig. 2). Out of 12 factors, ammonium sulfate, sodium chloride, magnesium

sulfate, disodium hydrogen orthophosphate, potassium chloride and inoculum size showed a

positive effect in case of shrimp waste degradation (Fig. 2A) while in case of chitinase yield

ammonium sulfate, sodium chloride, potassium chloride, magnesium sulfate and disodium

hydrogen orthophosphate showed the positive effect (Fig. 2B). Out of all these positive

factors ammonium sulfate, sodium chloride, magnesium sulfate and potassium chloride were

common factors which had a high positive influence on shrimp waste degradation as well as

chitinase production.

Ammonium sulfate has been reported to enhance the chitinase production in case of Serratia

marcescens XJ-01 and Serratia marcescens JPP [30, 28].

However, Jholopara et al, 2013 has reported that organic nitrogen sources like peptone served

as better nitrogen sources for chitinase production than ammonium sulfate in case of Bacillus

spp.[31]. Similarly, NaCl has been reported to have a positive effect on chitinase production

from Aeromonas hydrophila SBK1 and Lysinibacillus fusiformis B-CM18 [19, 32]. This
might be due to the requirement of Na+ ions for the amino acid transport and also to maintain

the intracellular solute concentration in marine bacteria [33].

Chitinase production is affected by the presence trace elements like minerals in the

production medium. Mg2+ ions are known to play a crucial role in cell growth enzyme

production and stability. Enhancement of chitinase yield by Paenibacillus sp. AD is in

agreement with Jholapara et al. 2013 and Kumar et al. 2017 [31, 34] which have reported the

positive influence of MgSO4 and KCl on chitinase production by Bacillus spp. and Humicola

grisea respectively. Another report which states that MgSO4 concentration can increase

chitinase production is in case of Bacillus pumilus [35]. There is no report available where

different parameters have been analyzed and optimized for shrimp waste degradation.

Therefore, in order to obtain the maximum shrimp waste degradation and enzyme production

optimal concentration of these parameters was investigated with Response Surface

Methodology (RSM).

TABLE 2

FIGURE 2

3.3.2.2 Central composite design (CCD) in Response surface methodology (RSM)

A CCD was formulated and maximum shrimp waste degradation (99%) and chitinase yield

(20.01IUml-1) were obtained when 0.35% ammonium sulfate, 0.35% sodium chloride,

0.075% magnesium sulfate and 0.00035% potassium chloride were added to the medium.

Response for shrimp waste degradation and enzyme yield are represented in (Table 3). By

applying multiple regression analysis on the experimental data, predictive quadratic

polynomial equations (Eq.2, Eq.3) were constructed to describe the correlation between

responses and the parameters.

TABLE 3
Model equations

R1= +98.50 +4.87* A +6.12* B +0.29* C -0.71* D +1.69*A*B +0.56*A*C

+2.94* A* D -0.69* B* C -1.81*B*D +2.06 *C* D -3.80 * A2 -2.68* B2 -1.80* C2 -6.68* D2 Eq. 2

R2 = +19.43 +0.77*A +0.80* B -0.065* C -0.13* D +0.38 * A* B -0.031 *A * C -0.048*A*D

+5.625E-003 *B*C -0.44*B* D +0.026 *C* D -2.26 * A2 -1.49*B2 -1.95* C2 -2.44*D2 ..........Eq. 3

Where, R1 is the response 1 (Shrimp waste degradation, %age) and R2 is response 2

(Chitinase yield, IUml-1). A, B, C and D were the coded values of ammonium sulfate,

sodium chloride, magnesium sulfate and potassium chloride respectively.

The analysis of variance for the response surface quadratic model has been summarized in

(Table 4, 5). The p values < 0.0001 indicated that the linear, interactive, and squared terms all

had quite significant influence on both the responses. The p values for the lack of fit were

0.0725 and 0.0731 for shrimp waste degradation and chitinase yield respectively, indicating

that this quadratic model adequately fit into the data. The determination coefficients R1 2 0.9960

and R22 0.9774 indicated that the predicted and experimental values had perfect coherence with

each other.

The values of adjusted R12 and R22 (Table 4, 5) suggested that the variation in the responses

was attributed to the independent variables and only 3.92 % and 3.9% of the total variations

could not be explained by the models. Therefore this model could be used for the prediction

of shrimp waste degradation and chitinase yield from Paenibacillus sp. AD.

The spherical curves in fig. 3 and fig. 4 indicated that the interaction between ammonium

sulfate, sodium chloride, magnesium sulfate and potassium chloride were significant and

maximum responses were located inside the design boundary, which validated the tested

ranges of parameters. According to fig. 4 and fig. 5, the relative significance of the impact of

parameters on shrimp waste degradation as well as enzyme yield was in the following order:
ammonium sulfate> sodium chloride> magnesium sulfate> potassium chloride which was in

accordance with the results.

3.3.2.3 Validation of the experimental model

The validation of the statistical model and regression equation was conducted by cultivating

Paenibacillus sp. AD under predicted optimal conditions in triplicate. The observed

experimental values were 99% and 20.56 IUml -1 for shrimp waste degradation and chitinase

production respectively. The close relationship between the predicted values (98.5% and

19.43 IUml-1) and experimental response values from the validation experiment revealed the

validity and acceptability of the statistical model for the optimization. After optimization,

shrimp waste degradation and chitinase yield were enhanced by 8.25 and 45.6 fold

respectively than the un-optimized conditions.

No detailed optimization study has been reported for shrimp waste degradation. In some

preliminary reports only 20 - 25% degradation has been reported in 5 days with Bionectria

CBNR BKRR spp. [5] and Fusarium solani sps. [17]. Effective degradation of shrimp waste

has been reported with Bacillus licheniformis SSCL10 and Bacillus subtilis SSCL 14,

however time taken to achieve this degradation was very long 12- 15 days [18] whereas in

present study complete degradation of the waste was achieved in only 4 days.

While in most of the available reports on chitinase production by bacteria very low enzyme

yield has been achieved even after optimization. In case of Bacillus pumilus [35]

Chitiolyticbacter meiyuanensis SYBC-H1[36] and Humicola grisea [34] only 0.9IU, 5.17IU

and 0.172IU of chitinase yield respectively has been reported after statistical optimization by

RSM. Similarly, optimization of chitinase production in case of Humicola grisea led to only

1.43 fold enhancement [34] Chitinase yield comparable to that of present study has been

reported from Aeromonas hydrophila SBK1 [19].

TABLE 4

TABLE 5

FIGURE 3

FIGURE 4

3.4 Structural analysis of shrimp waste and hydrolysate after treatment with

Paenibacillus sp. AD

Visible morphological changes were observed in shrimp flakes on treatment with

Paenibacillus sp. AD. To further analyze the structural changes scanning electron

microscopy (SEM) and Fourier-transformed infrared spectra (FTIR) analysis was done.

Hydrolysate obtained after degradation of shrimp waste was also analyzed for by-product/s.

3.4.1 Scanning electron microscopy (SEM)

Shrimp flakes before the treatment showed almost a smooth surface (Fig. 5A). However, after

fermentation shrimp flakes became cracked and a number of pores was formed, which clearly

indicate the degradation of chitin by the enzyme during the treatment (Fig. 5B). Similar

changes have been shown on the treatment of shrimp waste with chitinase from Aeromonas

hydrolytica SBK1 [19] and Paenicillium sp. LYG0704 [37]. Results indicate that chitinase

from Paenibacillus sp. AD is very effective in chitin hydrolyses and can also be used for the

degradation of other chitinaceous materials, as principally in all the sources same structure of

chitin is present.

FIGURE 5
3.4.2 FTIR analysis

Various bonds present in chitin and their corresponding spectral bands have been summarized

in table S3. The FTIR spectra of untreated shrimp flakes, shrimp flakes treated with

Paenibacillus sp. AD and its hydrolysate obtained after degradation are shown in fig. 6.

Untreated shrimp waste (Fig.6A) showed peaks at 1552 cm -1, 1621 cm-1, 3257 cm-1 and 1375

cm-1 which were typical to that of chitin [38, 39].

While in treated shrimp flakes (Fig. 6B) peaks at 1259.96 cm -1 and 1203.97 cm-1 which

corresponds to acetyl and amide groups disappeared due to breakage of these bonds. Peak

size at 1417 cm-1 which corresponds to –OCH3 group in case of untreated sample was

increased along with a band shift to 1432cm -1 due to aromatic skeletal vibrations within

plane deformation. There was change in the intensities of other peaks also. All these changes

indicate degradation of chitinaceous material.

While in case of FTIR spectrum of hydrolysate (Fig.6C) there was a substantial increase in

the peak size around 3257-3363 cm -1 after enzymatic treatment. It was attributed to breaking

of bonds in chitin chain and formation of –OH groups. All these changes indicate the

formation of chitin hydrolysis products like chitin oligosaccharides.

TABLE S3

FIGURE 6

3.4.3 Analysis of hydrolytic products by TLC

The hydrolysate obtained after degradation of shrimp waste were subsequently analyzed by

TLC using standard chitin oligomers as reference viz. GlcNAc, (GlcNAc) 2, (GlcNAc)3,

(GlcNAc)4 (Sigma-Aldrich, USA). TLC analysis revealed that a mixture of chitin

oligosaccharides viz. N-acetyl glucosamine (GlcNAc); chitobiose, (GlcNAc) 2; chitotriose,

(GlcNAc)3, chitotetroses (GlcNAc)4 and oligosaccharides of higher degree of polymerization

were produced (Fig 7). Results matched with that of FTIR analysis. As oligosaccharides of

various degree of polymerization are produced in the hydrolysate they can be used as

potential prebiotic agents and for other therapeutic purposes. [19] has showed the formation

of only N-acetyl glucosamine (GlcNAc) and chitobiose (GlcNAc) 2 after degradation of

shrimp waste by Aeromonas hydrophila SBK11.

FIGURE 7

4. Conclusion

The present study reports the isolation of a novel species of genus Paenibacillus and its

application for the bioremediation of sea food waste along with the production of chitinase

and chitin oligosaccharides. Optimization resulted in significant increase in degradation

(8.25 fold) and chitinase production (45.6 fold). Under optimal conditions 99%

degradation of shrimp waste and notable levels of extracellular chitinase and chitin

oligosaccharides of various degree of polymerization were produced by Paenibacillus sp.

AD. As per our knowledge this is the 1st report of complete biodegradation of shrimp

waste by a bacterium. The process can be utilized in the efficient management of chitinous

sea food waste. Moreover it is also useful for the production of chitinase which has

various biotechnological applications and bioactive chitin oligosaccharides which can be

used as pre-biotic and therapeutic agents.

Supplementary material

Supplementary data associated with this article can be found in online resource file.
Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

Financial support from UGC -SAP New Delhi, and UGC JRF fellowship to Mr Aditya

Kumar is highly acknowledged.

References

1. K. Jayathilakan, K. Sultana, K. Radhakrishna, A.S. Bawa, Utilization of byproducts

and waste materials from meat, poultry and fish processing industries: a review. J.

Food Sci. Technol. 49(2012) 278–293.

2. W. Russ, R.M. Pittroff, Utilizing waste products from the food production and

processing industries. Crit. Rev. Food Sci. Nutr. 44 (2004) 57–62.

3. H. Tsujibo, H. Orikoshi, K. Shiotani, Characterization of chitinase C from marine

bacterium Alteromonas sp. strain O-7, and its corresponding gene and domain

structure. Appl. Environ. Microbiol. 64 (1998) 472–478.

4. H. Meruvu, D.S.R. Reddy, Chitinous Seafood Waste for Production Of A Fungicidal

Enzyme. Int. J. Inn. Res. Dev. 1 (2012) 108-111.

5. B. Krishnaveni, R. Ragunathan, Chitinase production form sea food waste by plant

pathogen Bionectria CBNR BKRR sps and its application in Bioremediation studies.

Int. Res. J. Med. Sci. 2 (2014a) 15-19.

6. M.S. Brzezinska, U. Jankiewicz, M. Walczak, Biodegradation of chitinous substances

and chitinase production by the soil actinomycete Streptomyces rimosus. Int.

Biodeterior. Biodegradation. 84(2013)104–110.

7. N.S. Patil, J.P. Jadhav, Single Cell Protein Production Using Penicillium

ochrochloron Chitinase and Its Evaluation in Fish Meal Formulations. J. Microb.

Biochem. Technol. S4: 005(2014). doi:10.4172/1948-5948.S4-005.

8. K. Inokuma, M. Takano, K. Hoshino, Direct ethanol production from N-

acetylglucosamine and chitin substrates by Mucor species. Biochem. Eng. J.72 (2013)

24– 32.

9. R. Hamid, M.A. Khan, M. Ahmad, M.M. Ahmad, M.Z. Abdin, J. Musarrat, S. Javed,

Chitinases: An update. J. Pharm. Biol. Sci. 5(2013) 21-9.

10. A. Mathur, A. Rawat, G. Bhatt, Isolation of Bacillus producing chitinase from soil:

Production and purification of chito-oligosaccharides from chitin extracted from fresh

water crustaceans and antimicrobial activity of chitinase. Recent. Res. Sci. Technol.

3(2011) 01-06.

11. S.K. Karunya, D. Reetha, P. Saranraj, Optimization and purification of chitinase

produced by Bacillus subtilis and its antifungal activity against plant pathogens. Int. J.

pharm. biol. sci. arch. 2(2011) 1680-1685.

12. N.R. Mubarik, I. Mahagiani, A. Anindyaputri, Chitinolytic bacteria isolated from chili

rhizosphere: chitinase characterization and its application as bio-control for whitefly

(Bemisia tabaci Genn.). Am. J. Agri. Bio. Sci. 5(2010) 430-435.

13. N.N. Nawani, B.P. Kapadnis, A.D. Das, A.S. Rao, S.K. Mahajan, Purification and

characterization of a thermophilic and acidophilic chitinase from Microbispora sp. V2

J. Appl. Microbiol. 93(2002) 965-975.

14. S. Meena, R.K. Gothwal, J. Saxena, S. Nehra, M.K. Mohan, P. Ghosh, Effect of metal

ions and chemical compounds on chitinase produced by a newly isolated

thermotolerant Paenibacillus sp. BISR-047 and its shelf-life. Int J. Curr. Microbiol.

Appl. Sci. 4(2015) 872-881.

15. V. Thiagarajan, R. Revathia, K. Aparanjinib, Extra cellular chitinase production by

Streptomyces sp. PTK19 in submerged fermentation and its lytic activity on PTK2

cell wall. Int. J. Curr.Sci. 1(2011) 30-44.

16. M. Zarei, S. Aminzadeh, H. Zolgharnein, Serratia marcescens B4A chitinase product

optimization using Taguchi approach. Iran. J. Biotechnol. 8(2010) 252-262.

17. B. Krishnaveni, R. Ragunathan, Chitinase production from marine wastes by

Aspergillus terreus and its application in degradation studies. Int. J. Curr. Microbiol.

Appl. Sci. 3(2014b) 76-82.

18. S. Abirami, K. Yogalsakshmi, A.S.R. Pushpa, M. Kananan, Screening and

identification of chitin degrading bacteria from shrimp shell waste dumping soil

environment and its media optimization for chitinase enzyme production. World J.

Pharm. Pharm. Sci. 5 (2016) 743-757.

19. S.K. Halder, C. Maity, A. Jana, A. Das, T. Paul, P.K.D.M. Mohapatra, B.K. Pati, K.C.

Mondal, (2013) Proficient biodegradation of shrimp shell waste by Aeromonas

hydrophilla SBK-1 for the concomitant production of antifungal chitinase and

antioxidant chitosaccharides. Int. Biodeterior. Biodegradation. 79(2013) 88-97.

20. M. Jolanda, A.V. Munster, A.Y.P. Sanders, A. Geralt, A.T. Kate, A.L. Dijkhuizen,

J.E.C. Marc, A.B.V.D. Maarel, Kinetic characterization of Aspergillus niger chitinase

CfcI using a HPAEC-PAD method for native chitin oligosaccharides. Carbohydr.

Res. 407(2015) 73-78.

21. M.A. Faramarzi, M. Fazeli, Y. Tabatabaei, S. Adrangi, K. Jami, A.l. Ahmadi, N.

Tasharrofi, F.A. Mohseni, Optimization of Cultural Conditions for Production of

Chitinase by a Soil Isolate of Massiliatimonae.Biotechnol. 8(2009) 93-99.

22. S.C. Hsu, J.L. Lockwood, Powdered chitin agar as a selective medium for

enumeration of actinomycetes in water and soil. Appl. Environ. Microbiol. 29(1975)

422-426.

23. J.L. Reissig, J.L. Strominger, L.F. Leloir, A modified colorimetric method for

estimation of N-acetylamino sugar. J. Biol. Chem. 217(1955) 959-699.

24. J.G. Holt, N.R. Krieg, P.H.A. Sneath, J.T. Staley, S.T. Williams, Wilkins, Bergey’s

Manual of Determinative Bacteriology 9th Eds Waverly Baltimore MD, 1994.

25. S. Kumar, G. Stecher, K. Tamura, MEGA7: Molecular Evolutionary Genetics

Analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33(2016) 1870-1874.

26. N.N. Nawani, D. Prakash, B.P. Kapadnis, Extraction, purification and

characterization of an antioxidant from marine waste using protease and chitinase

cocktail. World J Microbiol. Biotechnol. 26(2010) 1509–1517.

27. C. Jui-Hsing, L. Jo-Hsin, L. Mei-Chun, C. Poh-Shing, A.B. Arun, Y. Chiu-Chung, C.

Wen-Ming, Paenibacillus contaminans sp. nov., isolated from a contaminated

laboratory plate. Int. J. Syst. Evol. Microbiol. 59 (2009) 125–129.

28. K. Wang, P.S Yan, L.X Cao, Optimization of nutrients for chitinase production by

Serratia marcescens JPP1 against aflatoxin using statistical experimental design. J.

Chem. Pharm. Res. 5(2013) 447-449.

29. N. Rattanakit, S. Yano, A. Plikomol, M. Wakayama, T. Tachiki, Purification of

Aspergillus sp. S1-13 chitinases and their role in saccharification of chitin in mash of

solid-state culture with shellfish waste. J. Biosci. Bioeng. 103(2007) 535-541.

30. J.L. Xia, J. Xiong, R.Y. Zhang, Production of chitinase and its optimization from a

novel isolate S. marcescens XJ-01. Indian J. Microbiol. 51(2011) 301-306.

31. R.J. Jholapara, R.S. Mehta, C.S. Sawant, Optimization of cultural conditions for

chitinase production from chitinolytic bacterium isolated from soil sample. Int. J.

Pharma. Bio. Sci. 4(2013) 464-471.

32. R.K. Singh, D.P. Kumar, M.K. Solanki, P. Singh, A.K. Srivastva1, S. Kumar, P.L.

Kashyap, A.K. Saxena, P.K. Singhal, D.K. Arora, Optimization of media components

for chitinase production by chickpea rhizosphere associated Lysinibacillus fusiformis B-

CM18. J. Basic Microbiol. 52(2012) 1–10.

33. O. Simon, Stanley, Y. Richard, Morita, Salinity Effect on the Maximal Growth

Temperature of Some Bacteria Isolated from Marine Environments.. J. Bacteriol. 95

(1968) 169-173.

34. M. Kumara, A. Brara, V. Vivekanandb, N. Pareeka, Production of chitinase from

thermophilic Humicola grisea and its application in production of bioactive

chitooligosaccharides. Int. J. Biol. Macromol. 104(2017) 1641–1647.

35. N. Tasharrofi, S. Adrangie, M. Fazelia, H. Rastegarc, M.R. Khoshayandd, M.A.

Faramarzia, Optimization of chitinase production by Bacillus pumilus using plackett-

burman design and response surface methodology. Iran. J. Pharm. Res. 10(2011) 759-

768.

36. Z. Hao, Y. Cail, X. Liao, X. Zhang, X. Fang, D. Zhang, Optimization of nutrition

factors on chitinase production from a newly isolated Chitiolyticbacter meiyuanensis

SYBC-H1. Braz. J. Microbiol. 2(2012) 177-186.

37. Y.G. Lee, K.C. Chung, S.G. Wi, J.C. Lee, H.J. Bae, Purification and properties of a

chitinase from Paenicillium sp. LYG 0704. Protein Expr. Purif. 65 (2009) 244-250.

38. M.L. Duarte, M.C. Ferreira, M.R. Marvao, J. Rocha, Determination of the degree of

acetylation of chitin materials by 13C CP/MAS NMR spectroscopy. Int. J. Biol.

Macromol. 28(2001) 359-363.

 39. R. Ravindra, K.R. Krovvidi, A.A. Khan, Solubility parameter of chitin and chitosan.

Carbohydr. Polym. 36(1998)121-127.

40.

41. Figures

Paenibacillus vulneris CCUG 53270(T) (HE649498)

100

83 Paenibacillus yunnanensis YN2(T) (KJ914577)

39 AM900778 s PA215 (AM900778)

23
Paenibacillus xylanisolvens X11-1(T) (AB495094)

Paenibacillus soli DCY03(T) (DQ309072)

AB680850 s NBRC 15377 (AB680850)

72
Paenibacillus ehimensis KCTC 3748(T) (AY116665)

Paenibacillus sp.AD

66 P. chitinolyticus NBRC 15660(T) (BBJT01000029)

85
Paenibacillus gansuensis B518(T) (AY839866)

42. 0.020

43. Fig. 1

44.

Design-Expert® Software
(Chitinase Activity)^1 Pareto Pareto Chart
A: Nacl 57.69 J Chart 19.67
J
B: Casein C
C: Yeast
extract D:
Inoculum size E:
48.08
Glucose
F: KCl H Bonf erroni Limit 14.7818
G: MgSO4 14.75
H: MgCl2
t- V a lu e o f | E f f e c

J: Ammonium Sulphate 38.46

t - V a lu e o f | E f f e c t |

K: Fructose
L: Na2HPO4 AC
Positive Effects
Negative 28.85 9.83
Effects
AG FG
B
t|

19.23
L F Bonf erroni Limit 14.7818 t-Value Limit 4.30265
4.92 L
H
9.62 K E

Dt -Value Limit 4. 30265

0.00 0.00

1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11

Rank Rank
 A B

Fig.2

A B C

105 105 105

S h r im p w a s te D e g r a d a

100
S h r im p w a s te D e g ra d a

S h r im p w a s t e D e g r a d a

100

95 100
95

90 90

95
85 85

80 80
90
ti o n

ti o n
tio n

0.50 0.50 0.10 0.50 0.00 0.50

0.10
0.09 85
0.43 0.43 0.09 0.43 0.000.43
0.08
0.35 0.35 0.08 0.35 0.00 0.35
0.27 0.27 0.07 0.27
0.07 D: Potassium Chloride 0.00 0.27 A: Ammonium Sulphate
B: Sodium A: Ammonium Sulphate C: Magnisium Sulphate 0.06
0.06 A: Ammonium 80
Chloride 0.20 0.20 0.05 0.20
Sulphate 0.00 0.20

75

D E F

105
105
105
S h r im p w a s te D e g r a d a

S h r im p w a s te D e g r a d a
S h rim p w a s t e D e g ra d a t

100

100
95 100

90

85 95
95

80

75
90
90
ti o n

tio n
io n

0.00 0.50 0.00 0.10 0.10

0.10 0.50 85
0.10 0.00 0.09
85 0.000.43
0.09 0.43 0.00 0.09
0.09 0.08 0.08
0.08 0.08
0.07
0.35 0.00 0.35 D: Potassium Chloride 0.07
0.07 D: Potassium Chloride 0.00 0.27 B: Sodium Chloride 0.00 0.06 0.07 C: Magnisium Sulphate
C: Magnisium Sulphate 0.06
0.06
0.27
B: Sodium Chloride 80 0.06
80 0.00 0.20 0.00 0.05
0.05 0.20

75
75

Fig.3
 A B C

21 22
21
20

19
20
C h it in a s e A c t iv

C h it in a s e A c t iv
18 20

C h itin a s e A c t iv
19
17

16
18
15 18

14
it y

it y
it y
16
17

0.50 0.50 0.10 0.10 0.50 0.00 0.50

0.43 0.43 0.09 0.09 0.43 14 0.000 .43

0.08 0.08 0.000 .35
0.35 0.35 16 0.35
0.07 D: Potassium Chloride 0.000.27 A: Ammonium Sulphate
0.27 0.27
C: Magnisium Sulphate 0.070.06 0.27 A: Ammonium Sulphate
B: Sodium Chloride A: Ammonium Sulphate
0.20 0.20 0.06
0.05 0.20 0.00 0.2 0
12
15

14

D E F

22 22

22
20
20
C h itin a s e A c t iv ity

20

C h it in a s e A c t iv
18
18
C h itin a s e A c t

16
16
18
14
14
12
iv it y

it y
12
16

0.00 0.50 0.00 0.10

0.10 0.50 0.10
0.10 0.09 14 0.000.43 0.00 0.
0.09 0.43 0.09
0.08 0.08 0.00 0.35 0.00 0.08 09
0.35 0.07 0.08
0.07 D: Potassium Chloride 0.00 0. 27 B: Sodium Chloride 0. 07
0.00
C: Magnisium Sulphate 0.07 0.06 0.06 0.27
B: Sodium D: Potassium 0.06
0.06 C: Magnisium Sulphate
12 0.00 0.20 0.00 0.05
0.05 0.20
Chloride Chloride

Fig. 4

A B

Fig. 5
 A

B
RC SAIF PU, Chandigarh

95.1

2132,94 855,94 776 ,94

932 ,93
90

85

1452 ,83
1406,83
80

%T
75 1116,78

70

1632,70
1585 ,69

65

3363 ,64

60.0
4000.0 360 0 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
c m-1

Fig. 6
 . Fig. 7

Fig. 1: Unrooted maximum likelyhood phylogenetic tree of the Paenibacillus sp. AD and

species of related genera (GenBank accession numbers are shown in parentheses).

Fig.2 Pareto chart showing effect of different factors on (A) Shrimp waste degradation (B)

Chitinase yield by Paenibacillus Sp. AD

Fig.3 Three dimensional response surface plots for the effect of A) Sodium Chloride and

Ammonium Sulfate, B) Magnesium Sulfate and Ammonium Sulfate, C) Potassium

Chloride and Ammonium Sulfate, D) Magnesium Sulfate and Sodium Chloride, E)

 Potassium Chloride and Sodium Chloride and F) Potassium Chloride and Sodium Chloride

for the shrimp waste degradation from Paenibacillus sp. AD

Fig. 4: Three dimensional response surface plots for the effect of A) Sodium Chloride and

Ammonium Sulfate, B) Magnesium Sulfate and Ammonium Sulphate, C) Potassium

Chloride and Ammonium Sulfate, D) Magnesium Sulfate and Sodium Chloride , E)

Potassium Chloride and Sodium Chloride and F) Potassium Chloride and Sodium Chloride

for the yield of chitinase from Paenibacillus sp. AD

Fig. 5 Scanning electron micrographs of (A) Untreated shrimp flakes (B) Paenibacillus sp. AD

treated shrimp flakes

Fig. 6 FTIR spectra of (A) Shrimp flakes (B) Paenibacillus sp. AD treated shrimp flakes (C)

Supernatant obtained after treatment

Fig. 7 TLC analysis of chitin oligosaccharides from shrimp waste

TABLE

Table 1 Comparison of biochemical characteristics of Paenibacillus sp. AD and

Paenibacillus chitinolyticus NBRC15660

Characteristics Paenibacillus sp. AD Paenibacillus chitinolyticus NBRC15660

Catalase -ve +ve

Oxidase +ve +ve

Nitrate reduction +ve +ve

Acid production from

 Glucose +ve +ve

Lactose -ve +ve

Xylose -ve -ve

Hydrolysis of Casein +ve Weak response

Table 2: Plackett-burman design for screening of significant factors affecting shrimp

waste degradation and chitinase production by Paenibacillus sp. AD

Factor Factor Factor Factor Factor Factor Factor Factor Factor Factor Factor Response Response

1 2 3 4 5 6 7 8 9 10 11 1 2

G: J: Shrimp

A: B: C:Yeast D:Inoculum E: F: MgSO H: Ammonium K: L: waste Chitinase

Run Nacl Casein extract size Glucose KCl 4 MgCl2 sulphate Fructose Na2HPO4 degradation Activity

% % Uml-1

(W/V) % (W/V) % (W/V) % %(W/V) % (W/V) (W/V) % (W/V) % (W/V) % (W/V) % (W/V) %

1 0.01 0.1 0.1 0.1 0.1 0 0.05 0 0 0 0.1 29 3.84

2 0.1 0.1 0.01 1 0.1 0 0.01 0 0 0.1 0 43 4.16

3 0.1 0.1 0.1 0.1 0 0 0.05 0 0.1 0.1 0 47 4.64

4 0.01 0.01 0.01 0.1 0 0 0.01 0 0 0 0 30 3.84

5 0.01 0.1 0.1 1 0 0 0.01 0 0 0.1 0.1 10 2.64

6 0.01 0.01 0.1 0.1 0.1 0 0.01 0 0.1 0.1 0 30 3.84

7 0.1 0.01 0.1 1 0 0 0.05 0 0 0 0 30 3.84

8 0.01 0.01 0.01 1 0 0 0.05 0 0.1 0.1 0.1 69 5.28

9 0.1 0.01 0.1 1 0.1 0 0.01 0 0.1 0 0.1 46 4.64

10 0.1 0.1 0.01 0.1 0 0 0.01 0 0.1 0 0.1 68 4.64

11 0.1 0.01 0.01 0.1 0.1 0 0.05 0 0 0.1 0.1 48 4

12 0.01 0.1 0.01 1 0.1 0 0.05 0 0.1 0 0 49 4

Table 3: Central composite design matrix with experimental and predicted values of

shrimp waste degradation and chitinase production from Paenibacillus sp. AD

Factor Shrimp waste degradation Chitinase activity

% (W/V) (%) (IUml-1)

A:Ammonium B:Sodium C:Magnisium D:Potassium

Run Actual Predicted Residual Actual Predicted Residual
sulphate chloride sulphate chloride
1 0.5 0.2 0.1 0.0005 89.00 88.25 0.75 11.51 11.07 0.44

2 0.5 0.5 0.05 0.0002 98.00 97.71 0.29 14.52 13.96 0.56

3 0.5 0.2 0.05 0.0002 78.00 77.08 0.92 10.71 10.73 -0.019

4 0.35 0.35 0.025 0.00035 90.00 90.71 -0.71 12.18 11.75 0.43

5 0.5 0.5 0.1 0.0002 95.00 93.92 1.08 14.22 13.73 0.49

6 0.5 0.5 0.1 0.0005 98.00 98.88 -0.88 12.52 12.54 -0.019

7 0.35 0.35 0.075 0.00035 98.00 98.50 -0.50 19.82 19.43 0.39

8 0.35 0.35 0.075 0.00065 71.00 70.38 0.62 9.95 9.40 0.55

9 0.5 0.5 0.05 0.0005 95.00 94.42 0.58 12.29 12.67 -0.38

10 0.35 0.35 0.125 0.00035 92.00 91.88 0.12 11.63 11.50 0.13

11 0.2 0.2 0.1 0.0002 75.00 74.42 0.58 10.11 9.66 0.45

12 0.35 0.35 0.075 0.00035 99.00 98.50 0.50 19.34 19.43 -0.087

13 0.2 0.5 0.05 0.0005 77.00 76.54 0.46 10.00 10.42 -0.42

14 0.2 0.5 0.1 0.0002 86.00 85.54 0.46 10.53 11.40 -0.87

15 0.35 0.35 0.075 0.00035 98.00 98.50 -0.50 18.90 19.43 -0.53

16 0.2 0.2 0.05 0.0005 70.00 69.92 0.083 10.05 10.47 -0.42

17 0.65 0.35 0.075 0.00035 93.00 93.04 -0.042 11.07 11.91 -0.84

18 0.2 0.5 0.1 0.0005 79.00 78.75 0.25 10.50 10.41 0.090

19 0.35 0.35 0.075 0.00035 99.00 98.50 0.50 20.01 19.43 0.58

20 0.35 0.05 0.075 0.00035 76.00 75.54 0.46 12.26 11.87 0.39

21 0.2 0.2 0.1 0.0005 74.00 74.88 -0.88 9.25 10.45 -1.20

22 0.2 0.2 0.05 0.0002 78.00 77.71 0.29 9.18 9.79 -0.61

23 0.35 0.35 0.075 0.00035 98.00 98.50 -0.50 19.50 19.43 0.073

24 0.05 0.35 0.075 0.00035 73.00 73.54 -0.54 10.24 8.84 1.40
25 0.5 0.2 0.05 0.0005 80.00 81.04 -1.04 11.46 11.22 0.24

26 0.35 0.35 0.075 0.00035 99.00 98.50 0.50 18.99 19.43 -0.44

27 0.5 0.2 0.1 0.0002 75.00 76.04 -1.04 10.26 10.48 -0.22

28 0.35 0.35 0.075 0.00005 72.00 73.21 -1.21 9.92 9.91 0.013

29 0.2 0.5 0.05 0.0002 92.00 91.58 0.42 11.14 11.51 -0.37

30 0.35 0.65 0.075 0.00035 99.00 100.04 -1.04 15.24 15.07 0.17

Table 4: Analysis of variance (ANOVA) for response surface model developed for

optimum shrimp waste degradation

Sum of Mean F p-value

Source Squares Df Square Value Prob > F

Model 3308.22 14 236.3 267.51 < 0.0001 Significant

A-Ammonium

sulfate 570.37 1 570.37 645.71 < 0.0001

B-Sodium chloride 900.37 1 900.37 1019.29 < 0.0001

C-Magnesium

sulfate 2.04 1 2.04 2.31 0.1492

D-Potassium

chloride 12.04 1 12.04 13.63 0.0022

AB 45.56 1 45.56 51.58 < 0.0001

AC 5.06 1 5.06 5.73 0.0302

AD 138.06 1 138.06 156.3 < 0.0001

BC 7.56 1 7.56 8.56 0.0104

BD 52.56 1 52.56 59.5 < 0.0001

CD 68.06 1 68.06 77.05 < 0.0001

 A2 396.5 1 396.5 448.87 < 0.0001

B2 196.57 1 196.57 222.54 < 0.0001

C2 89.07 1 89.07 100.84 < 0.0001

D2 1222.86 1 1222.86 1384.37 < 0.0001

Residual 13.25 15 0.88

Lack of Fit 11.75 10 1.18 3.92 0.0725 not significant

Pure Error 1.5 5 0.3

Cor Total 3321.47 29

Model fitting C.V= 1.09 R-Sq=99.60% R-Sq (Pred)= 97.90% R-Sq(Adj)=99.23%

Table 5: Analysis of variance (ANOVA) for response surface model developed for optimum

chitinase yield

Sum of Mean F p-value

Source Squares Df Square Value Prob > F

Model 370.6 14 26.47 46.24 < 0.0001 Significant

A-Ammonium

sulfate 14.09 1 14.09 24.62 0.0002

B-Sodium

chloride 15.28 1 15.28 26.69 0.0001

C-Magnesium

sulfate 0.1 1 0.1 0.17 0.6817

D-Potassium

chloride 0.38 1 0.38 0.67 0.4265

AB 2.27 1 2.27 3.97 0.0648

AC 0.015 1 0.015 0.026 0.8735

 AD 0.037 1 0.037 0.065 0.8026

BC 5.06E-04 1 5.06E-04 8.84E-04 0.9767

BD 3.16 1 3.16 5.52 0.0329

CD 0.011 1 0.011 0.018 0.894

A2 140.47 1 140.47 245.38 < 0.0001

B2 60.83 1 60.83 106.27 < 0.0001

C2 104.35 1 104.35 182.29 < 0.0001

D2 163.7 1 163.7 285.96 < 0.0001

Residual 8.59 15 0.57

not

Lack of Fit 7.61 10 0.76 3.9 0.0731 significant

Pure Error 0.98 5 0.2

Cor Total 379.19 29

Model fitting C.V= 5.86 R-Sq=97.74% R-Sq (Pred)= 88.07% R-Sq(Adj)=95.62%