Bioprocess Principles Chapter1
Bioprocess Principles Chapter1
Fermentation products wine, bread etc., Invention of microbes responsible for these conversion science of microbiology Penicillin fermentation sterility concepts, pilot plant concepts Single cell production by ICI air lift fermenters, continuous cultivation, process control
Biotechnology Products
Microbial biomass Enzymes Primary and secondary metabolites Recombinant products Biotransformation
Operations in Fermentation
Utility
Fermentation bay
Downstream Processing
STORES
Utilities
Steam 4 BAR & 1.5 BAR Air 1.5 BAR Chilled water Cooling water Deionised water Softwater MCC
Fermentation bay
Seed Fermenter Main fermenter Acid, Alkali & Antifoam tanks Nutrient feeding Harvest tank
Microbiology Laboratory
Media preparation Autoclaving Inoculation chamber Microscopic observation Quality control Culture stock maintenance
Plates 15 days Stab/slant 6 months Glycerol stock 6 months Lyophilisation 15 years Sand cultures 15 years Liquid nitrogen storage 15 years
Culture Validation
Bioreactor
How a bioreactor differs from a chemical reactor ? Both are agitated tanks Bioreactor should be capable of being operated aseptically for number of days Adequate aeration and agitation should be provided to meet the metabolic requirements which will vary from time to time
Material of Construction
Lab fermenters are made up of glass Pilot and industrial scale fermenters are made up of Stainless steel Steel having chromium more than 4% are called us stainless steel. The corrosion resistance of stainless steel depend on the existence of a thin hydrous oxide film on the surface of the metal
The composition of this film varies with different steel alloys and manufacturing process This oxide film is stabilized by chromium If the film is damaged it will repair itself when exposed to air or oxidizing agent. Minimum amount of chromium needed to resist corrosion will depend on the corroding agent in environment (acid, alkali, salt etc) Increasing chromium enhances the corrosion resistance.
10 13% chromium will develop effective oxide film Also addition of nickel to this high percent chromium steel enhances the resistance to corrosion and give strength. Addition of molybdenum improves the resistance of stainless steel to solution of salts or halogens such as chlorides. Corrosion resistance can also be increased using tungstan silicone etc.
Cr Cr Cr Cr
Ni Mo : 17 12 2 Ni Mo : 18 14 3 Ni Mo : 17 13 4 Ni : 18.5 10
Normally the main tank is made up of 316 grades and jackets are made up of 304 to reduce the cost.
Height to Diameter ratio : Height of the fermenter is one of the critical parameters in the fermenter design. If the height of the fermenter is more the bubble residence time is more and better oxygen efficiency. H/D ratio of the fermenter 2 to 3 is preferred.
For Design Purpose the effective working volume of the reactor is considered to be 80% of Total volume of the reactor. Fermenter will be running 330 days per annum
80%
Mixing
Mixing is a physical operation which reduces non uniformities in fluid by eliminating gradients of concentration, temperature and other properties
Mixing in the Bioreactor involves
Blending the soluble medium components eg. Sugars Dispersing air in the form of small bubbles Suspension of solid particles such as cells Promoting heat transfer and dispersion of immiscible liquids such as antifoam
Circular flow
Baffle
Normally four Baffles are installed in the Bioreactor Purpose of Baffle is to avoid the vortex formation and increasing the aeration efficiency Baffles are normally 1/10th of the dia of the vessel. Increasing the size of the baffle increase the agitation effect marginally. Decreasing that will have substantial drop in efficiency. Baffles should be installed such that gap exist between the vessel and baffle which minimizes dead space and avoid microbial growth
Propeller
Turbine
Pitched Blade
Types of agitator
Multiple impellers
H/D :23 Imp dia P : 0.3 0.5 D Baffle : 0.1 D W/P : 1.5 2.0 P/Z : 0.5-1.0
Problem:
In a lab scale bioreactor production of gluconic acid in optimized medium is 180 g/l. The time duration of cultivation is 36 hours and downtime for cleaning, sterilization, medium preparation etc. is 12 hours. You have been asked to design a fermentor for the production of 2000 Tonnes per annum. State your assumptions and geometrical ratios for your design?
Air Filter
Rupture disk
Drain Valve
Sampling valve
Ports
Small reactor -12 mm ports Pilot scale 19 and 25 mm ports Big reactors - any size depending on the probes.
INSTRUMENTATION
Measurement type
Inline measurement : The sensor is an integrated part of the fermentation equipment and measure the process variable real time continuously. Online measurement: Continuous/discontinuous measurement but the sensor is not an integrated part of the bioreactor but connected to the system Offline sensor: Discontinuous measurement and the sensor is not an integrated part of the system. Samples have to be connected and measured manually
When evaluating sensors to use in measurement and control it is important to consider the following:
Response time Gain and sensitivity Accuracy Ease and speed of calibration Reliability Material of construction Sterilization
Bioreactor variables
Physical variables
Chemical variables
pH, Dissolved oxygen, Dissolved carbon dioxide, ion concentration, substrate concentration
Biological variables
Temperature Measurement
Mercury in glass thermometers, thermocouples, bimetallic thermometers, thermistors, Metal resistance thermometers etc.,
Metal resistance thermometers are most commonly used. Mercury in glass thermometers are used for precise calibration of other measurement systems
The known electrical voltage is applied across the bridge and change in the voltage occurs when the resistance changes according to the temperature
Pressure Measurement
Pressure is one of the critical measurements mainly for the safety of the bioreactor. Always bioreactors are operated under positive pressure i.e pressure above atmospheric pressure to prevent contamination and better oxygen transfer. Pressure will buidup in the bioreactor during sterilization
Devices
C tube bourdon gauge - manual If a wire is subject to strain its electrical resistance changes.
Flow Measurement
Gas flow measurement area rotameter Mass flow meter Liquid flow weighing the mass or metering pump
Rotameter
Advantages: A rotameter requires no external power or fuel, it uses only the inherent properties of the fluid, along with gravity, to measure flow rate. A rotameter is also a relatively simple device that can be mass manufactured out of cheap materials, allowing for widespread use in places such as thirdworld countries. Disavantages: Due to its use of gravity, a rotameter must always be vertically oriented and right way up, with the fluid flowing upward. Due to its reliance on the ability of the fluid or gas to displace the float, graduations on a given rotameter will only be accurate for a given substance. The main property of importance is the density of the fluid; however, viscosity may also be significant. Floats are ideally designed to be insensitive to viscosity; however, this is seldom verifiable from manufacturers specs. Either separate rotameters for different densities and viscosities may be used, or multiple scales on the same rotameter can be used. Rotameters normally require the use of glass (or other transparent material), otherwise the user cannot see the float. This limits their use in many industries to benign fluids, such as water. Rotameters are not easily adapted for reading by machine; although magnetic floats that drive a follower outside the tube are available
Speed
Weight
Foam
Biomass Measurement
pH Probe
The pH probe measures pH as the activity of hydrogen ions surrounding a thin-walled glass bulb at its tip. The probe produces a small voltage (about 0.06 volt per pH unit) that is measured and displayed as pH units by the meter
Oxygen sensors have a thin organic membrane covering a layer of electrolyte and two metal electrodes. Oxygen diffuses through the membrane at a rate proportional to its partial pressurethe greater the oxygen partial pressure, the more oxygen diffuses through the membrane. Oxygen meters measure the current as oxygen is reduced at the cathode and more oxygen diffuses through the membrane. Since the diffusion current is directly proportional to the concentration of dissolved oxygen, the calibrated meter simply converts measured current into concentration units.
The reactions are as follows: At cathode: O2 + 4H+ + 4 e- 2H2O At anode: 2H2O O2 + 4H+ + 4 e-
ASSIGNMENT
Temperature measurement Pressure measurement and foam measurement pH PROBE DO Probe O2 Analyser CO2 analyser Mass spectrometer gas analysis Online biomass analysis
Downstream Processing
Cell Disruption
Concentration
Product Polishing
Fermentation Broth
Extracellula r
Harvesting
Intracellula r
Supernatunt
Cells
Cell Disruption
Methods
Homogeniser
Operating pressure Number of passes Valve design Operating temperature Type of cells and conditions under which grown
Disruption does not depend on cell concentration
Commercial high speed bead mills have been originally constructed for wet comminution of fine solids in thick viscous suspensions.
Operational parameters
Sonication
In principle, the high-frequency is generated electronically and the mechanical energy is transmitted to the sample via a metal probe placed into the cell-containing sample resulting in breaking open the cells. Ultrasound frequency of 20-50kHz is applied to the sample. Disadvantages
Heat generated by the ultrasound process must be dissipated. High noise levels (most systems require hearing protection and sonic enclosures) Yield variability
Enzymatic lysis Enzymes can hydrolyze the walls of microbial cells and when sufficient wall has been removed the internal osmotic pressure can burst the periplasmic membrane.
The cell wall of yeast and bacteria are different. Hence in general the lytic systems are specific for the type of cell.
Yeast cell walls have two main layers an outer layer of protein mannan complex and inner glucan layer. Essentially for cell breakage a specific wall lytic protease for outer layer and a lytic glucanase to degrade the inner layer is essential. For gram positive bacteria single enzyme is sufficient to break the peptidoglycan layer but for gram negative for breaking the outer layer detergents have to be used and then enzyme treatment.
Chemical Lysis
Solubilization detergents
Lipid dissolution organic solvents Alkali treatment
Osmotic shock
Every cell membrane maintains substantial osmotic gradient : however a drastic reduction in extracellular concentrations of solutes from 0.15 M to 0.001 M will tend to burst the cells that donot have cell walls such as animal cells
Most animal cells are quickly lysed by a rapid transition to distilled water.
Bacterial and plant cells are protected against osmotic lysis by cell walls.
Chelating agents
EDTA the most common chelating agent, binds the divalent cations Mg2+ and Ca2+ present in the outer membrane of gram negative bacteria.
The divalent cations stabilize the structure of the outer membrane by binding lipopolyscachharide molecules to each other as well as to outer membrane proteins.
When EDTA removes the divalent cations from the outer membrane a large portion of the LPS molecules are also removed.
Mostly periplasmic proteins are removed by this way. Also higher mol.wt proteins may not permeabilize through this eg -galactosidase.
Solvents
Organic solvents dissolves the cell wall and so destabilizes. Eg. Toluene, for e.coli and yeast, DMSO for plant cells, ethyl acetate for yeast etc.
Detergents
Nonionic detergents are able to break the plasma membrane by lipid solubilisation and are commonly used to lyse animal cells. Triton x-100 (polyoxyethylene[9-10]p-t-octylphenol) is one of the common non ionic detergent used
Concentration
at saturation, it is of sufficiently high molarity that it causes the precipitation of most proteins it does not have a large heat of solution, allowing heat generated to be easily dissipated its saturated solution (4.04 M at 20 C) has a density (1.235g/cc) that does not interfere with the sedimentation of most precipitated proteins by centrifugation its concentrated solutions are generally bacteriostatic in solution it protects most proteins from denaturation.
When two polymers or polymer and salt are mixed in particular concentrations two immiscible phases are formed and when we do this with fermentation broth or cell lysates partioning of protein occurs. In the plot between polymer-polymer or polymer-salt concentrations, the curve separating the single phase and two phases of that system is called as Binodal curve. In ATPE, two immiscible phases are formed when polymers such as polyethylene glycol (PEG) are mixed with other polymers, such as dextran, ficoll or salts (ammonium sulphate, sodium sulphate etc) in particular concentrations.