P HARMACEUTICAL B IOTECHNOLOGY PHR 403 Chapter 2: Fermentation Technology (L1-L2) Course Instructor : Md. Samiul Alam Rajib Senior Lecturer Department of Pharmacy BRAC University

C HAPTER OVERVIEW Fermentation and Industrial Microbiology- Overview Principles of Fermentation Types of Fermentation Microorganisms and their metabolites useful for fermentation Basics of Bioprocessing and Bioreactors Preparation of different fermented Biopharmaceuticals: Alcohol Penicillin Streptomycin Riboflavin (Vitamin B 2 ) Fermentation and Industrial Microbiology- Overview Principles of Fermentation Types of Fermentation Microorganisms and their metabolites useful for fermentation Basics of Bioprocessing and Bioreactors Preparation of different fermented Biopharmaceuticals: Alcohol Penicillin Streptomycin Riboflavin (Vitamin B 2 )

D EFINITION OF FERMENTATION “Fermentation is the breakdown of larger molecules”- Conventional “any process for the production of useful products through mass culture of microorgnisms”- Microbiological “numerous oxidation-reduction reactions in which organic compounds, used as source of carbon and energy, acts as acceptor or donors of hydrogen ions. The organic compounds used as substrate give rise to various product as of fermentation which accumulate in the growth medium”- Riviere, 1977 “Fermentation is the breakdown of larger molecules”- Conventional “any process for the production of useful products through mass culture of microorgnisms”- Microbiological “numerous oxidation-reduction reactions in which organic compounds, used as source of carbon and energy, acts as acceptor or donors of hydrogen ions. The organic compounds used as substrate give rise to various product as of fermentation which accumulate in the growth medium”- Riviere, 1977

F ERMENTATION AND I NDUSTRIAL M ICROBIOLOGY First generation processes Fermentation based processes using ‘native’ organisms Wine- and beer-making Baking Second generation processes Use of native or engineered enzymes/genes to enhance first generation processes Textile industry Starch industry Third generation processes Direct use of recombinant and engineered systems Plant, insect and animal cell culture First generation processes Fermentation based processes using ‘native’ organisms Wine- and beer-making Baking Second generation processes Use of native or engineered enzymes/genes to enhance first generation processes Textile industry Starch industry Third generation processes Direct use of recombinant and engineered systems Plant, insect and animal cell culture

F ERMENTATION T ECHNOLOGY In the context of Microbial biotechnology the term “fermentation” refers to the growth of large quantities of cells under anaerobic or aerobic conditions within a vessel called a fermenter or bioreactor Similar vessels are used in processes involving cell-free and immobilized enzyme transformations In the context of Microbial biotechnology the term “fermentation” refers to the growth of large quantities of cells under anaerobic or aerobic conditions within a vessel called a fermenter or bioreactor Similar vessels are used in processes involving cell-free and immobilized enzyme transformations

W HY DO WE USE F ERMENTATION ? A HISTORICAL PERSPECTIVE Video Documentary

A PPLICATION OF F ERMENTATION IN PRODUCTION OF B IOPHARMACEUTICALS Fermentation techniques are used in production of different biopharmaceuticals, such as: Microbial cell (Biomass) Yeast Microbial enzymes Glucose isomerase Microbial metabolites Penicillin Food products Cheese, yoghurt, vinegar Vitamins B 12, riboflavin Transformation reactions Steroid biotransformation Fermentation techniques are used in production of different biopharmaceuticals, such as: Microbial cell (Biomass) Yeast Microbial enzymes Glucose isomerase Microbial metabolites Penicillin Food products Cheese, yoghurt, vinegar Vitamins B 12, riboflavin Transformation reactions Steroid biotransformation

P RINCIPLES OF F ERMENTATION R ESPIRATION AND F ERMENTATION Process of converting glucose to a useable form of energy Respiration uses Oxygen and is very efficient Fermentation does not need Oxygen but is very inefficient Process of converting glucose to a useable form of energy Respiration uses Oxygen and is very efficient Fermentation does not need Oxygen but is very inefficient

P RINCIPLES OF F ERMENTATION ATP Adenosine Tri-phosphate A compound that “powers” the cell All cell parts need ATP to work properly Without ATP, the cell dies. Adenosine Tri-phosphate A compound that “powers” the cell All cell parts need ATP to work properly Without ATP, the cell dies.

P RINCIPLES OF F ERMENTATION ATP So where can we get ATP? Glycolysis, Cell respiration or fermentation So where can we get ATP? Glycolysis, Cell respiration or fermentation

P RINCIPLES OF F ERMENTATION L INK WITH G LYCOLYSIS Cells cannot break down large compounds Glucose must be changed so the cell can use it. Through a series of chemical reactions, glucose is changed to pyruvic acid During this process, 6 molecules of ATP are produced (8 are produced, but 2 are used up) Cells cannot break down large compounds Glucose must be changed so the cell can use it. Through a series of chemical reactions, glucose is changed to pyruvic acid During this process, 6 molecules of ATP are produced (8 are produced, but 2 are used up)

P RINCIPLES OF F ERMENTATION P YRUVIC ACID Energy compound produced at the end of glycolysis

Glucose Pyruvic Acid 8 ATP’s produced Glycolysis 2 ATP’s used Principles of Fermentation

P RINCIPLES OF F ERMENTATION R ESPIRATION ( AN AEROBIC PROCESS ) Process of converting pyruvic acid to ATP in the presence of oxygen One pyruvic acid molecule can produce 30 molecules of ATP By-product of respiration is CO 2 + H 2 0 Takes places inside each cell, in the mitochondria (organelle)

C 6 H 12 O 6 + 6O 2 = 6CO 2 +6 H 2 O T HIS REACTION PRODUCES 30 ATP S ! Respiration

Glucose Pyruvic Acid 8 ATP’s produced Glycolysis Respiration CO 2 and H 2 O 30 ATP’s produced 2 ATP’s used

W HAT IF THERE IS NO, OR NOT ENOUGH OXYGEN ? Fermentation

F ERMENTATION ( AN ANAEROBIC PROCESS ) Process of producing some ATP without oxygen present Much less efficient Only 2 ATP’s are produced from each glucose molecule All cells are capable of fermentation Three types Lactic Acid fermentation Alcoholic fermentation Gas producing fermentation. Process of producing some ATP without oxygen present Much less efficient Only 2 ATP’s are produced from each glucose molecule All cells are capable of fermentation Three types Lactic Acid fermentation Alcoholic fermentation Gas producing fermentation.

L ACTIC A CID F ERMENTATION “Feeling the burn!” When a cell runs out of oxygen, it makes ATP by repeating the glycolysis process. Fermentation replaces a special compound lost during glycolysis Fermentation does not produce ATP, but merely allows the cell to go through the glycolysis process repeatedly. “Feeling the burn!” When a cell runs out of oxygen, it makes ATP by repeating the glycolysis process. Fermentation replaces a special compound lost during glycolysis Fermentation does not produce ATP, but merely allows the cell to go through the glycolysis process repeatedly.

L ACTIC A CID FERMENTATION Fermentation uses sugar much faster than respiration This is why exercise is better if your cells are functioning in this mode (anaerobic). Lactic acid and carbon dioxide are the by products of fermentation. If you produce them, your body must get rid of them quickly, or they can cause damage to your cells. Fermentation uses sugar much faster than respiration This is why exercise is better if your cells are functioning in this mode (anaerobic). Lactic acid and carbon dioxide are the by products of fermentation. If you produce them, your body must get rid of them quickly, or they can cause damage to your cells.

A LCOHOLIC F ERMENTATION Similar to lactic acid fermentation, but the by- product is ethyl alcohol, instead of lactic acid, and carbon dioxide The carbon dioxide produces the air spaces in bread, and the ethyl is the toxin that impairs brain function when consumed. Similar to lactic acid fermentation, but the by- product is ethyl alcohol, instead of lactic acid, and carbon dioxide The carbon dioxide produces the air spaces in bread, and the ethyl is the toxin that impairs brain function when consumed.

Glucose Pyruvic Acid 8 ATP’s produced Glycolysis Respiration CO 2 and H 2 O 30 ATP’s produced 2 ATP’s used Fermentation Ethyl alcohol or lactic acid and CO 2 2 ATP’s produced Is oxygen present? YesNo

S OMETHING TO THINK ABOUT What is the advantage of aerobic respiration vs anaerobic? Why wouldn’t all cells just do aerobic? In what situations would anaerobic be better? What is the advantage of aerobic respiration vs anaerobic? Why wouldn’t all cells just do aerobic? In what situations would anaerobic be better?

M ICROBIAL G ROWTH K INETICS 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 Exponential Phase Stationary Phase Death Phase (Growth curve is from Shuler p. 161) 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 Exponential Phase Stationary Phase Death Phase (Growth curve is from Shuler p. 161)

M ICROBIAL G ROWTH K INETICS Lag Phase 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 Cell growth is minimal in this phase. Lag Phase 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 Cell growth is minimal in this phase.

M ICROBIAL G ROWTH K INETICS Exponential Phase The second phase in the fermentation process The cells have adjusted to their environment and rapid growth takes place Cell growth rate is highest in this phase Exponential Phase The second phase in the fermentation process The cells have adjusted to their environment and rapid growth takes place Cell growth rate is highest in this phase

M ICROBIAL G ROWTH K INETICS Exponential Phase (Continued) 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 Substrate limited inhibition means that the microbes do not have enough nutrients in the medium to continue multiplying. Exponential Phase (Continued) 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 Substrate limited inhibition means that the microbes do not have enough nutrients in the medium to continue multiplying.

M ICROBIAL G ROWTH K INETICS Stationary phase This is the third phase in the fermentation process The cell growth rate has leveled off and become constant The number of cells multiplying equals the number of cells dying Stationary phase This is the third phase in the fermentation process The cell growth rate has leveled off and become constant The number of cells multiplying equals the number of cells dying

M ICROBIAL G ROWTH K INETICS Death phase The fourth phase in the fermentation process 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 Death phase The fourth phase in the fermentation process 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

M ICROBIAL P RODUCTS  Primary metabolite:  Produced during Exponential phase  Basically intermediate metabolites  Further needed in growth or energy yielding catabolism.  Vitamins, amino acids, nucleotides, protein, carbohydrate, lipids, acetone, ethanal, butanol, organic acids etc.  Secondary metabolite:  Produced during idiophase  Toxins, alkaloids and antibiotics.  Produced by a limited number of microorganisms.  Enzymes:  Both extracellular (maximum) and intracellular enzymes are produced.  Intracellular enzyme production possible, but with difficulties.  Extra cellular- Amylases, Cellulase, invertase, β-galactosidases (Lactase), esterase, lipase, proteases.  Primary metabolite:  Produced during Exponential phase  Basically intermediate metabolites  Further needed in growth or energy yielding catabolism.  Vitamins, amino acids, nucleotides, protein, carbohydrate, lipids, acetone, ethanal, butanol, organic acids etc.  Secondary metabolite:  Produced during idiophase  Toxins, alkaloids and antibiotics.  Produced by a limited number of microorganisms.  Enzymes:  Both extracellular (maximum) and intracellular enzymes are produced.  Intracellular enzyme production possible, but with difficulties.  Extra cellular- Amylases, Cellulase, invertase, β-galactosidases (Lactase), esterase, lipase, proteases.