Fermentation 1 Power Point Presentation

Dr. Nermin Hassan Ibrahim Ass. Prof. of Medical Microbiology and Immunology

What is fermentation?y Pasteurs definition: life without air, anaerobe redox

reactions in organismsy New definition: a form of metabolism in which the end

products could be further oxidized For example: a yeast cell obtains 2 molecules of ATP per molecule of glucose when it ferments it to ethanol2

What is fermentation techniques (1)?yTechniques for large-scale production of microbial products. It must both provide an optimum environment for the microbial synthesis of the desired product and be economically feasible on a large scale. yThey can be divided into surface (emersion) and submersion techniques. yThe latter may be run in batch, fed batch, continuous reactors yIn the surface techniques, the microorganisms are cultivated on the surface of a liquid or solid substrate. These techniques are very complicated and rarely used in industry3

What is fermentation techniques (2)?yIn the submersion processes, the microorganisms grow in a liquid medium. Except in traditional beer and wine fermentation, the medium is held in fermenters and stirred to obtain a homogeneous distribution of cells and medium. yMost processes are aerobic, and for these the medium must be vigorously aerated. yAll important industrial processes (production of biomass and protein, antibiotics, enzymes and sewage treatment) are carried out by submersion processes.4

Some important fermentation productsProduct Ethanol Glycerol Lactic acid Acetone and butanol E-amylase Organism Saccharomyces cerevisiae Saccharomyces cerevisiae Lactobacillus bulgaricus Clostridium acetobutylicum Bacillus subtilis Use Industrial solvents, beverages Production of explosives Food and pharmaceutical Solvents Starch hydrolysis5

Some important fermentation products

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Some important fermentation products

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Some important fermentation products

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Wine-making fermenter

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General Aspects of Fermentation Processes

y All cells break down complex organic compounds into

simpler molecules. Cells use some of the energy that is released in this process to make ATP.y What is cellular respiration?

Cellular respiration- is a complex process in which cells make ATP by breaking down organic compounds.

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What is glycolysis?y Glycolysis- is the first biochemical pathway, which yields small amounts of ATP.

Glycolysis consist of two pathways: 1. Glycolysis Oxygen present Aerobic respiration ATP 2. Glycolysis Oxygen Absent Fermentation (anaerobic). y C6H1206 2 CH3CH2OH + 2 CO2 + 2 ATP y C6H1206 + 6O2 6CO2 + 6H2O + 16-18 ATP

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y What is anaerobic respiration?

Anaerobic respiration- makes ATP without the use of oxygen, instead uses sugar.y What is aerobic respiration?

Aerobic respiration- is the process of making ATP with the use of oxygen and produces much larger amount of ATP than anaerobic respiration.y So Glycolysis can be a pathway in which one six-carbon molecule of glucose is oxidized to produce two threecarbon molecules of Pyruvic acid. y Aerobic respiration has two major stages: Krebs cycle, and the electron transport chain.

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Glycolysis Substrate-level phosphorylation

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y Fermentation is an energy yielding process where

organisms converts organic molecules (such as sugar) into energy, carbon dioxide or/and ethanol depending on the respiration pathway.

y Fermentation has been practiced for years and has

resulted in synthesis of different kinds of food such as bread, wine and beer.

y There is two types of fermentation: 1) Homolactic fermentation. 2) Ethanol Fermentation.

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y Homolactic fermentation:

Homolactic Fermentation breaks down the pyruvate into Lactate.

It occurs in the muscles of animals when they need energy faster than

the blood can supply oxygen. It also occurs in some kinds of bacteria (such as lactobacilli) and

some fungi. It is this type of bacteria that converts lactose into lactic acid in

yogurt, giving it its sour taste. These lactic acid bacteria can be classed as homofermentative, where

the end product is mostly lactate, or heterofermentative, where some lactate is further metabolized and results in carbon dioxide, acetate or other metabolic products17

y Ethanol Fermentation : Ethanol fermentation (performed by yeast and some types of

bacteria) breaks the pyruvate down into ethanol and carbon dioxide. It is important in bread-making, brewing, and wine-making.

Usually only one of the products is desired; in bread-making, the alcohol is baked out, and, in alcohol production, the carbon dioxide is released into the atmosphere or used for carbonating the beverage. When the ferment has a high concentration of pectin, minute

quantities of methanol can be produced.18

y Often fungi or streptomycetes (bacteria)

y Grow rapidly & produce product in relatively short period.

y Organisms can be manipulated in large-scale cheap culture to

produce one or more products in high concentration.

y Organisms are genetically stable with infrequent mutation19

y Able to grow in inexpensive liquid culture media (waste

from other industries): eg

Corn steep liquor from corn milling (rich in N & growth factors)

Whey from cheese production (rich in lactose & minerals)

y Should not be pathogenic (humans, animals, plants)

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Fermentation mediumy Medium

nutritional, hormonal, and fulfilling

requirement of cellsy In most cases, the medium is independent of the

bioreactor design and process parametersy Even small modifications in the medium could

change cell line stability, product quality, yield, operational parameters, and downstream processing.21

ySummarize in 2

sentences

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:y Lag Phase y Exponential Phase y Stationary Phase y Death Phase

(Growth curve is from Shuler p. 161)

Lag Phasey This is the first phase in the fermentation process y The cells have just been injected into a new environment

and they need time to adjust accordingly y Cell growth is minimal in this phase.

Exponential Phasey The second phase in the fermentation process y The cells have adjusted to their environment and rapid

growth takes place y Cell growth rate is highest in this phase

Exponential Phase (Continued)y At some point the cell growth rate will level off and

become constant y The most likely cause of this leveling off is substrate limited inhibitionSubstrate limited inhibition means that the microbes do not have enough nutrients in the medium to continue multiplying.

Stationary phasey This is the third phase in the fermentation process y The cell growth rate has leveled off and become

constant y The number of cells multiplying equals the number of cells dying

Death phasey The fourth phase in the fermentation process y The number of cells dying is greater than the number of

cells multiplyingThe 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

Metabolitesy Metabolites are metabolism.

the

intermediates

and

products

of

y The term metabolite is usually restricted to small molecules. y A primary metabolite is directly involved in normal growth, development, and reproduction. y A secondary metabolite is not directly involved in those processes, but usually has an important ecological function. Examples include antibiotics and pigments.

Primary metabolite : formed during growth phase Secondary metabolite: formed near the end of growth phase frequently during near or in stationary phase29

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Secondary metabolitesy produced from substrates provided by primary metabolism y not essential for growth and reproduction. y formation is extremely dependent on growth conditions. y often produced as a group of closely related products

e.g. a particular streptomycete strain is known that can produce > 30 related but different anthracycline antibioticsy possible to induce dramatic overproduction of secondary

metabolites (different from primary metabolites which usually cannot be significantly overproduced).31

y Most secondary metabolites are complex organic

molecules requiring a large number of specific enzymatic reactions for synthesis e.g. tetracycline synthesis (72 separate enzymatic steps). e.g. erythromycin synthesis ( 25 steps)y No one organism produces all of these secondary

metabolites & there are many steps between the amino acid & the antibiotic32

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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

Media for Industrial FermentationsThe media is the feed solutiony It must contain the essential nutrients needed for the

microbe to grow

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

The Development of Inocula for Industrial FermentationsThe inoculum is the starter culture that is injected into the fermentery 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- 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

Design of a FermenterFactors to consider when designing a fermentery

y y y y

Aseptic and regulatory capability, long-term reliability Adequate aeration and agitation Low power consumption Temperature and pH controls Sampling facilities

Instrumentation and ControlThe 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

Aeration and AgitationMost 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

Pharmacy collegeDr. Nermin Hassan Ibrahim

Pharmaceutics/ Microbiology Department

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