Biotechnology Biotechnology practical coursepractical course

Second Year ClinicalSecond Year Clinical

Microbial FermentationsMicrobial Fermentations

What Is FermentationWhat Is Fermentation??Fermentation has always been an important part Fermentation has always been an important part

of our lives: foods can be spoiled by microbial of our lives: foods can be spoiled by microbial fermentations, foods can be made by microbial fermentations, foods can be made by microbial fermentations, and muscle cells use fermentation fermentations, and muscle cells use fermentation to provide us with quick responses. Fermentation to provide us with quick responses. Fermentation could be called the staff of life because it gives us could be called the staff of life because it gives us the basic food, bread. But how fermentation the basic food, bread. But how fermentation actually works was not understood until the work actually works was not understood until the work ofof  Louis PasteurLouis Pasteur  in the latter part of the in the latter part of the nineteenth century and the research which nineteenth century and the research which followedfollowed..

Microbial fermentationsMicrobial fermentations Microbial fermentationsMicrobial fermentations  have other benefits. have other benefits.

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

Tables 1Tables 1

PRODUCTPRODUCT APPLICATIONAPPLICATION ORGANISMORGANISM

BacitracinBacitracin AntiobioticAntiobiotic Bacillus Bacillus subtilis subtilis (bacterium)(bacterium)

ChlorampheChloramphenicolnicol

AntiobioticAntiobiotic Streptomyces Streptomyces venezuelae venezuelae (bacterium)(bacterium)

Citric acidCitric acid Food flavoringFood flavoring Aspergillus Aspergillus niger (fungus)niger (fungus)

NeomycinNeomycin AntibioticAntibiotic Streptomyces Streptomyces fradiae fradiae (bacterium)(bacterium)

How Does Fermentation How Does Fermentation Work in BiotechnologyWork in Biotechnology??

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

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

Microbial Growth Microbial Growth KineticsKinetics

• Microbial Growth Microbial Growth Kinetics describe how the Kinetics describe how the microbe grows in the microbe grows in the fermenter. This fermenter. This information is important information is important to determine optimal to determine optimal batch times. The growth batch times. The growth of microbes in a of microbes in a fermenter can be broken fermenter can be broken down into four stages:down into four stages: Lag Phase Lag Phase Exponential PhaseExponential Phase Stationary PhaseStationary Phase Death PhaseDeath Phase

(Growth curve is from Shuler p. 161)(Growth curve is from Shuler p. 161)

Microbial Growth Microbial Growth KineticsKinetics

• Lag PhaseLag Phase This is the first phase in the This is the first phase in the

fermentation processfermentation process The cells have just been injected The cells have just been injected

into a new environment and they into a new environment and they need time to adjust accordinglyneed time to adjust accordingly

Cell growth is minimal in this Cell growth is minimal in this phase. phase.

Microbial Growth Microbial Growth KineticsKinetics

• Exponential PhaseExponential Phase The second phase in the The second phase in the

fermentation processfermentation process The cells have adjusted to their The cells have adjusted to their

environment and rapid growth environment and rapid growth takes placetakes place

Cell growth rate is highest in this Cell growth rate is highest in this phasephase

Microbial Growth Microbial Growth KineticsKinetics

• Exponential Phase (Continued)Exponential Phase (Continued) At some point the cell growth rate At some point the cell growth rate

will level off and become constantwill level off and become constant The most likely cause of this leveling The most likely cause of this leveling

off is substrate limited inhibition off is substrate limited inhibition •Substrate limited inhibition means Substrate limited inhibition means that the microbes do not have that the microbes do not have enough nutrients in the medium to enough nutrients in the medium to continue multiplying. continue multiplying.

Microbial Growth Microbial Growth KineticsKinetics

• Stationary phaseStationary phase This is the third phase in the This is the third phase in the

fermentation processfermentation process The cell growth rate has leveled The cell growth rate has leveled

off and become constantoff and become constant The number of cells multiplying The number of cells multiplying

equals the number of cells dyingequals the number of cells dying

Microbial Growth Microbial Growth KineticsKinetics

• Death phaseDeath phase The fourth phase in the fermentation The fourth phase in the fermentation

processprocess The number of cells dying is greater The number of cells dying is greater

than the number of cells multiplyingthan the number of cells multiplying•The cause of the death phase is The cause of the death phase is usually that the cells have consumed usually that the cells have consumed most of the nutrients in the medium most of the nutrients in the medium and there is not enough left for and there is not enough left for sustainabilitysustainability

Media for Industrial Media for Industrial FermentationsFermentations

• The media is the feed solutionThe media is the feed solution It must contain the essential nutrients It must contain the essential nutrients

needed for the microbe to growneeded for the microbe to grow• Factors of consideration when choosing Factors of consideration when choosing

mediamedia-Quality consistence and availability-Quality consistence and availability-Ensure there are no problems with -Ensure there are no problems with Media Prep or other aspects of Media Prep or other aspects of production process Ex. Cane production process Ex. Cane molasses, beet molasses, cereal grainsmolasses, beet molasses, cereal grains

SterilizationSterilization

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

sterilization of the feed solution sterilization of the feed solution

The Development of Inocula The Development of Inocula for Industrial for Industrial FermentationsFermentations

• The inoculum is the starter culture that is The inoculum is the starter culture that is injected into the fermenterinjected into the fermenter It must be of sufficient size for optimal It must be of sufficient size for optimal

growth kineticsgrowth kinetics• Since the production fermenter in industrial Since the production fermenter in industrial

fermentations is so large, the inoculum volume fermentations is so large, the inoculum volume has to be quite largehas to be quite large

- A seed fermenter is usually required to - A seed fermenter is usually required to produce the inoculum volumeproduce the inoculum volume

-The seed fermenter-The seed fermenter’’s purpose is not to s purpose is not to produce product but to prepare inoculumproduce product but to prepare inoculum

Design of a FermenterDesign of a Fermenter• Factors to consider when Factors to consider when

designing a fermenterdesigning a fermenter Aseptic and regulatory Aseptic and regulatory

capability, long-term capability, long-term reliabilityreliability

Adequate aeration and Adequate aeration and agitationagitation

Low power consumptionLow power consumption Temperature and pH Temperature and pH

controlscontrols Sampling facilities Sampling facilities

(14 L fermenter shown is a (14 L fermenter shown is a copyright of New copyright of New Brunswick Scientific)Brunswick Scientific)

Instrumentation and Instrumentation and ControlControl

•The success of a fermentation The success of a fermentation process is highly dependent on process is highly dependent on environmental factorsenvironmental factorsThe fermenter needs to be able The fermenter needs to be able

to control such factors as to control such factors as temperature, pH, and temperature, pH, and dissolved oxygen levelsdissolved oxygen levels

Aeration and AgitationAeration and Agitation

• Most industrial fermentations are Most industrial fermentations are aerobic processes meaning that the aerobic processes meaning that the production microbe requires oxygen production microbe requires oxygen to growto grow The oxygen demand is met by The oxygen demand is met by

sparging air through the sparging air through the fermentation vessel and using an fermentation vessel and using an agitator increase the amount of agitator increase the amount of dissolved oxygendissolved oxygen

Industrial and Commercial Industrial and Commercial ApplicationsApplications

Food IndustryFood Industry~ Beer~ Beer~ Bread~ Bread~ Cheese~ Cheese~ Wine~ Wine

Pharmaceutical IndustryPharmaceutical Industry~ Insulin~ Insulin~ Vaccine ~ Vaccine

AdjuvantsAdjuvants

EnergyEnergy~ Fuel Ethanol~ Fuel Ethanol

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