1. Biotechnology practical course Second Year Clinical

2. Microbial Fermentations What Is Fermentation? What Is Fermentation? Fermentation has always been an important part of our lives: foods can be spoiled by microbial fermentations, foods can be made by microbial fermentations, and muscle cells use fermentation to provide us with quick responses. Fermentation could be called the staff of life because it gives us the basic food, bread. But how fermentation actually works was not understood until the work of Louis Pasteur in the latter part of the nineteenth century and the research which followed. Fermentation has always been an important part of our lives: foods can be spoiled by microbial fermentations, foods can be made by microbial fermentations, and muscle cells use fermentation to provide us with quick responses. Fermentation could be called the staff of life because it gives us the basic food, bread. But how fermentation actually works was not understood until the work of Louis Pasteur in the latter part of the nineteenth century and the research which followed. Louis Pasteur Louis Pasteur

3. Microbial fermentations Microbial fermentations Microbial fermentations have other benefits. For one, they don't use toxic reagents or require the addition of intermediate reagents. Microbiologists are now looking for naturally occurring microbes that produce desired chemicals. In addition, they are now capable of engineering microbes to enhance production of these chemicals. In recent years, microbial fermentations have been revolutionized by the application of genetically-engineered organisms. Many fermentations use bacteria but a growing number involve culturing mammalian cells. Some examples of products currently produced by fermentation are listed in Tables 1. Microbial fermentations have other benefits. For one, they don't use toxic reagents or require the addition of intermediate reagents. Microbiologists are now looking for naturally occurring microbes that produce desired chemicals. In addition, they are now capable of engineering microbes to enhance production of these chemicals. In recent years, microbial fermentations have been revolutionized by the application of genetically-engineered organisms. Many fermentations use bacteria but a growing number involve culturing mammalian cells. Some examples of products currently produced by fermentation are listed in Tables 1. Microbial fermentations Tables 1 Microbial fermentations Tables 1

4. Tables 1 Tables 1 ORGANISMAPPLICATIONPRODUCT Bacillus subtilis (bacterium) AntiobioticBacitracin Streptomyces venezuelae (bacterium) Antiobiotic Chloramphenico l Aspergillus niger (fungus) Food flavoring Citric acid Streptomyces fradiae (bacterium) AntibioticNeomycin

5. How Does Fermentation Work in Biotechnology? In the pharmaceutical and biotechnology industries, fermentation is any large-scale cultivation of microbes or other single cells, occurring with or without air. In the teaching lab or at the research bench, fermentation is often demonstrated in a test tube, flask, or bottle-in volumes from a few milliliters to two liters. At the production and manufacturing level, large vessels called fermenters or bioreactors are used. A bioreactor may hold several liters to several thousand liters. Bioreactors are equipped with aeration devices as well as nutrients, stirrers, and pH and temperature controls. In the pharmaceutical and biotechnology industries, fermentation is any large-scale cultivation of microbes or other single cells, occurring with or without air. In the teaching lab or at the research bench, fermentation is often demonstrated in a test tube, flask, or bottle-in volumes from a few milliliters to two liters. At the production and manufacturing level, large vessels called fermenters or bioreactors are used. A bioreactor may hold several liters to several thousand liters. Bioreactors are equipped with aeration devices as well as nutrients, stirrers, and pH and temperature controls. fermenters or bioreactors fermenters or bioreactors

6. During production, technicians monitor temperature, pH, and growth in the bioreactors to ensure that conditions are optimum for cell growth and product. Bioreactors are used to make products such as insulin and human growth hormone from genetically engineered microorganisms as well as products from naturally-occurring cells, such as the food additive xanthan.

7. Microbial Growth Kinetics Microbial Growth Kinetics describe how the microbe grows in the fermenter. This information is important to determine optimal batch times. The growth of microbes in a fermenter can be broken down into four stages: Microbial Growth Kinetics describe how the microbe grows in the fermenter. This information is important to determine optimal batch times. The growth of microbes in a fermenter can be broken down into four stages: Lag Phase Lag Phase Exponential Phase Exponential Phase Stationary Phase Stationary Phase Death Phase Death Phase (Growth curve is from Shuler p. 161)

8. Microbial Growth Kinetics Lag Phase Lag Phase This is the first phase in the fermentation process This is the first phase in the fermentation process The cells have just been injected into a new environment and they need time to adjust accordingly The cells have just been injected into a new environment and they need time to adjust accordingly Cell growth is minimal in this phase. Cell growth is minimal in this phase.

9. Microbial Growth Kinetics Exponential Phase Exponential Phase The second phase in the fermentation process The second phase in the fermentation process The cells have adjusted to their environment and rapid growth takes place The cells have adjusted to their environment and rapid growth takes place Cell growth rate is highest in this phase Cell growth rate is highest in this phase

10. Microbial Growth Kinetics Exponential Phase (Continued) Exponential Phase (Continued) At some point the cell growth rate will level off and become constant At some point the cell growth rate will level off and become constant The most likely cause of this leveling off is substrate limited inhibition The most likely cause of this leveling off is substrate limited inhibition Substrate limited inhibition means that the microbes do not have enough nutrients in the medium to continue multiplying. Substrate limited inhibition means that the microbes do not have enough nutrients in the medium to continue multiplying.

11. Microbial Growth Kinetics Stationary phase Stationary phase This is the third phase in the fermentation process This is the third phase in the fermentation process The cell growth rate has leveled off and become constant The cell growth rate has leveled off and become constant The number of cells multiplying equals the number of cells dying The number of cells multiplying equals the number of cells dying

12. Microbial Growth Kinetics Death phase Death phase The fourth phase in the fermentation process The fourth phase in the fermentation process The number of cells dying is greater than the number of cells multiplying The number of cells dying is greater than the number of cells multiplying The cause of the death phase is usually that the cells have consumed most of the nutrients in the medium and there is not enough left for sustainability The cause of the death phase is usually that the cells have consumed most of the nutrients in the medium and there is not enough left for sustainability

13. Media for Industrial Fermentations The media is the feed solution The media is the feed solution It must contain the essential nutrients needed for the microbe to grow It must contain the essential nutrients needed for the microbe to grow Factors of consideration when choosing media Factors of consideration when choosing media -Quality consistence and availability -Ensure there are no problems with Media Prep or other aspects of production process Ex. Cane molasses, beet molasses, cereal grains

14. Sterilization Sterilizing the feed solution is essential because the media cannot contain foreign microbes because this could severely hinder the growth of the production microbe Sterilizing the feed solution is essential because the media cannot contain foreign microbes because this could severely hinder the growth of the production microbe Most popular method is heat sterilization of the feed solution Most popular method is heat sterilization of the feed solution

15. The Development of Inocula for Industrial Fermentations The inoculum is the starter culture that is injected into the fermenter The inoculum is the starter culture that is injected into the fermenter It must be of sufficient size for optimal growth kinetics It must be of sufficient size for optimal growth kinetics Since the production fermenter in industrial fermentations is so large, the inoculum volume has to be quite large Since the production fermenter in industrial fermentations is so large, the inoculum volume has to be quite large - A seed fermenter is usually required to produce the inoculum volume -The seed fermenter ’ s purpose is not to produce product but to prepare inoculum

16. Design of a Fermenter Factors to consider when designing a fermenterFactors to consider when designing a fermenter Aseptic and regulatory capability, long-term reliability Aseptic and regulatory capability, long-term reliability Adequate aeration and agitation Adequate aeration and agitation Low power consumption Low power consumption Temperature and pH controls Temperature and pH controls Sampling facilities Sampling facilities (14 L fermenter shown is a copyright of New Brunswick Scientific)

17. Instrumentation and Control The success of a fermentation process is highly dependent on environmental factors The success of a fermentation process is highly dependent on environmental factors The fermenter needs to be able to control such factors as temperature, pH, and dissolved oxygen levels The fermenter needs to be able to control such factors as temperature, pH, and dissolved oxygen levels

18. Aeration and Agitation Most industrial fermentations are aerobic processes meaning that the production microbe requires oxygen to grow Most industrial fermentations are aerobic processes meaning that the production microbe requires oxygen to grow The oxygen demand is met by sparging air through the fermentation vessel and using an agitator increase the amount of dissolved oxygen The oxygen demand is met by sparging air through the fermentation vessel and using an agitator increase the amount of dissolved oxygen

19. Industrial and Commercial Applications Food Industry Food Industry ~ Beer ~ Bread ~ Cheese ~ Wine Pharmaceutical Industry Pharmaceutical Industry ~ Insulin ~ Vaccine Adjuvants Energy Energy ~ Fuel Ethanol

20. Thanks