Maximizing the efficiency of fermentation process
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The document discusses strategies to maximize the efficiency of the fermentation process by maintaining optimal growth conditions, including monitoring parameters such as oxygen concentration, pH, temperature, and mixing. It emphasizes the importance of managing high-density cell cultures, enhancing plasmid stability, minimizing acetate production, and improving protein secretion to achieve higher yields in fermentation. Various methods and considerations for ensuring the successful production of recombinant proteins in microbial systems are outlined.
Maximizing the efficiency of fermentation process
- 1. B Y A B H I S H E K G I R I MAXIMIZING THE EFFICIENCY OF FERMENTATION PROCESS M.sc-II SEM- III P-I
- 2. WHY TO MAXIMISE? • In any type of fermentation process that is used to grow cells, it is necessary to monitor & control culture parameters, such as I. Dissolved oxygen concentration. II. pH, Temperature & III. Degree of mixing. • Changes in any of these parameters can have a dramatic effect on the yield of cells & the stability of the protein product.
- 3. OPTIMAL GROWTH (OXYGEN) • The maximal oxygen demand in a fermentation, Qmax = Xμmax/Yo2 • Where, X = cell mass μmax = maximal specific growth rate. Yo2 = growth yield based on oxygen consumed. • Oxygen supplied in the form of sterilized air. • However, introducing air produces bubbles, & thus the rate of transfer of oxygen to the cells is insufficient. • Thus, fermenter design should monitor these changes in the culture.
- 4. OPTIMAL GROWTH (pH) • Most microorganisms grow optimally between 5.5 & 8.5. • However, cellular metabolites are released into the growth medium. • Therefore, the pH must be monitored & either acid or base must be added as needed to maintain the pH. • After adding fermentation broth should be mixed throughout so that the pH is same in entire reaction vessel.
- 5. OPTIMAL GROWTH (TEMPERATURE) • Microorganisms grown at a temperature below the optimum grow slowly & have a reduced rate of cellular production. • Whereas if grown at a temperature above the optimum growth there may be premature induction of the expression of the target protein. • If it is under control of temperature-sensitive repressor, or induction of a heat shock response, will produce proteases that lower the yield of the protein product.
- 6. OPTIMAL GROWTH (MIXING) • Adequate mixing of a microbial culture is essential to assure an adequate supply of nutrients to the cells & prevention of the accumulation of any toxic metabolic by- product in local, poorly mixed regions. • Effective mixing is easily attained with small-scale cultures, but it is one of the major problem with the scale of fermentation is increased.
- 7. OPTIMAL GROWTH (AGITATION) • Agitation of the fermentation broth affects other factors, such as 1. The rate of transfer of oxygen from the gas bubbles to the liquid medium & then from the medium to the cells, 2. Efficient heat transfer, 3. Accurate measurement of specific metabolites in the culture fluid, & 4. Efficient dispersion of added solutions such as acids, bases, & antifoaming agents. • On these grounds, it might be concluded that the more mixing there is, the better the growth.
- 8. • However, excessive agitation of a fermentation broth can cause hydomechanical stress (shear) • Thus damages larger microbial or mammalian cells, & a temperature increase, which may also decrease cell viability. • Thus a balance must be struck between the need to provide through mixing & the need to avoid damage to the cells.
- 9. ADDITIONAL CONSIDERATION FOR GEM • For scaled-up fermentations that has nothing to do with the technical aspects of the process but depends instead on whether a GeM is being used. • Although most recombinant microorganisms are not hazardous, it is nevertheless important to ensure they are not inadvertently released into the environment. • Therefore, fail-safe system are used to prevent accidental spills of the live recombinant organisms & to contain them if they occur. • Furthermore, all organisms must render them nonviable before they are discharged from the production facility
- 10. HOW TO MAXIMIZE? 1. 1.HIGH-DENSITY CELL CULTURES
- 11. HIGH-DENSITY CELL CULTURES • A major objective of fermentation is to maximise the volumetric production. • In practice, cell concentration of more than 50 gm. cells/liter of culture have been obtained with fed-batch cultures of recombinant E-coli. • Some nutrients like carbon & nitrogen can inhibit cell growth if they are present at too high concentration. • In addition, fermentations that use complex media containing peptone or yeast extract, can vary from one media to another & are not always reproducible.
- 12. • Acetate, is inhibitory to cell growth, is produced by E.coli both when the cells are grown under oxygen-limited conditions & in the presence of excess glucose. • Using glycerol instead of glucose as the carbon source, lowering the culture temperature or using Gem to shunt acetate into less toxic compounds. • Oxygen may become limited in such cultures. To overcome this problem, the rate of introduction of air, the agitation rate, or both can be increased. • Alternatively, expression of the gene Vitreoscilla hemoglobin can increase uptake of oxygen & improve enzyme production in Bacillus subtilis, increases erythromycin production by Saccharopolyspora erythraea.
- 13. • High density cell cultures are most readily attained in fed- batch cultures. • The addition of nutrients following depletion of some of the original nutrients may be constant, stepwise, or exponential. • Addition of nutrients can be automated. NUTRIENT TIME CONSTANT Specific growth rate NUTRIENT TIME STEPWISE Specific growth rate
- 14. HOW TO MAXIMIZE? 1. 2. INCREASING PLASMID STABILITY
- 15. INCREASING PLASMID STABILITY • Loss of plasmid is the major industrial problem in large-scale growth of recombinant E. coli cells. • Plasmid loss often limits the yield of plasmid-encoded recombinant proteins. • Plasmid instability in bacterial cultures is typically due to unequal distribution of plasmids to daughter cells during growth & cell division. • Once lost, cells grow faster, with the result that cells lacking plasmid eventually dominating the culture. • One approach to avoid this problem is to include antibiotic resistance gene & then add to the culture.
- 16. • In addition to the obvious economic cost of the antibiotic, disposal of spent growth medium is a potential environmental hazard. • One way to deal with this problem is to delete an essential gene from the chromosomal DNA of the host bacterium & at the same time place this gene on the plasmid that is being stabilised. • As a result , only plasmid-carrying cells can grow, making the bacterial strain totally dependent upon maintenance of the plasmid. • With this system, selection that utilizes antibiotics is no longer necessary thereby decreasing both cost & environmental risk.
- 17. HOW TO MAXIMIZE? 1. 3. QUIESCENT E. COLI CELLS
- 18. QUIESCENT E. COLI CELLS • It is difficult to engineer recombinant bacteria to produce large amounts of foreign protein & at the same, time to grow to a high cell density. • It would be advantageous to be able to grow cells to a high density & then shift the allocation of available resources from growth to foreign-protein production. • With this in mind, workers engineered a quiescent cell expression system in which a plasmid-encoded protein is expressed in non-growing but metabolically active cells. • Understanding the commercial potential of this unique system, scientist who developed this approach have applied for patent to protect their intellectual property rights
- 19. QUIESCENT STATE IN E. COLI • Quiescent stage is established by overexpression of Rcd, a regulatory protein, in an hns mutant E. coli. • Hns gene codes for histone like nucleoid-structuring protein. • E.coli gradually cease synthesizing host protein but continue synthesizing plasmid-encoded foreign proteins. • In both batch & fed-batch modes, the quiescent cells produce less biomass & secrete considerably more scFv protein than control E.coli cells engineered to express scFv under the control of the pL promoter.
- 20. Rcd gene is placed under the transcriptional control of the pR promoter while the recombinant protein gene (encoding a single chain antibody variable fragment scFv) was controlled by the pL promoter. The activities of both pR & pL was repressed by a temperature-sentitive cI repressor protein
- 21. HOW TO MAXIMIZE? 1. 4. PROTEIN SECRETION
- 22. PROTEIN SECRETION • High-level cytoplasmic expression in E.coli of different foreign proteins results in the formation of inclusion bodies consisting of insoluble improperly folded proteins. • Even if soluble purifying it from a cytoplasmic extract is major undertaking.
- 23. Investigator observed that expression levels were quite low when they expressed several different foreign proteins under transcriptional control of the strong pm/xyLS promoter/regulator system. Use of translocation signal sequences significantly stimulated the levels of expression of these 3 human proteins. 20-30% of the protein that was produced was found to be insoluble form. A strategy that minimizes the extent of insoluble protein formation needs to be developed.
- 24. HOW TO MAXIMIZE? 1. 5. REDUCING ACETATE
- 25. REDUCING ACETATE • Acetate inhibits both cell growth & protein production & also wastes carbon & energy resources. • Removing acetate from the culture during fermentation can be achieved by several different method including continuous dialysis & the use of macroporous ion-exchange resins. • However, these methods tend to remove nutrients that are necessary for cell growth along with the acetate. • Use of fructose & mannose is used as a carbon source thus lowering levels of acetate & higher yields of protein.
- 26. • Another method is to reduce the uptake of glucose by cells by adding methyl α-glucoside, a glucose analogue. • This effect can also achieved by using an E.coli host cell that contained a mutation in ptsG, a gene encoding enzyme II in the glucose phosphotransferase system. • Ex : In a batch culture both wild-type & ptsG mutant E.coli expressing β-galactosidase activity, Wild-type = 10gm/lit ptsG mutant = 15gm/lit at the same time, the mutant cells synthesized about 25% more β-galactosidase/gm of cells than wild type cells. • it is much easier & quicker to alter host cell by genetic transformation than by mutagenesis & selection, alternative methods were developed.
- 27. • One of these method include introducing a gene encoding the enzyme acetolactate synthase into host cells. • This enzyme catalyses the formation of acetolactate from pyruvate, thereby decreasing the flux through acetyl coA to acetate. • The transformed cells produced 75% less acetate than the nontransformed cells. • Acetoin which is produced in approx. 50- fold less toxic than acetate. The protein yield was also doubled.
- 28. • Another way of converting acetate to acetoin is to redirect it to TCA cycle. • In one study workers overexpressed the gene for the enzyme phosphoenol pyruvate carboxylase, which converts it to oxaloacetate, they obtained 17% increase in the specific growth rate of E.coli cells & 44% decrease in acetate production. • Unfortunately, overexpressing this enzyme also decreases the amount of glucose uptake by the bacterial cells & diminishes the growth rate. • Another group of workers tranformed the host cell with the gene for the enzyme pyruvate carboxylase isolated from gram negative bacteria Rhizobium etli. • Thus acetate levels were decreased & cell yield was increased & synthesis of foreign proteins was also increased
- 29. Addition of pyruvate carboxylase allow s E.coli to use the available carbon more efficiently, directing it away from acetate toward biomass & protein formation. Similarly, this may also be done by converting aspartate to fumarate. To do this, E.coli were transformed with the gene for L- aspartate ammonium lyase (aspartase) under the control of strong tac promoter . Aspartate activity is induced by addition of IPTG at the mid- to late log phase of growth.
- 30. SUMMARY • To maximise the fermentation process optimal growth conditions must be maintained. • In high density cell cultures nutrients like carbon & nitrogen should not exceed the optimal level. • Acetate production must be lowered for maximizing the efficiency. • Plasmid stability must be increased and also insoluble protein secretion must be restricted.
- 31. REFERENCE BOOKS • Bernard R. Glick and Jack J. Pasternack, Molecular Biotechnology – Principles and applications of recombinant DNA, ASM Press, Washington DC. • S. S. Purohit, Biotechnology – Fundamentals and applications, 3rd Edition, Agrobios, India WEB LINKS • http://www.massey.ac.nz/~ychisti/FermentInd.PDF • http://www.rpi.edu/dept/chem-eng/Biotech- Environ/SeniorLab/Fermentation/