AN INTRODUCTION TO FERMENTATION PROCESS

What is fermentation Conversion of sugar to alcohol using yeast. Chemical conversion of carbohydrates into alcohols or acids. The process is often used to produce wine and beer Also employed in preservation to create lactic acid in sour foods such as pickled cucumbers, kimchi and yogurt. The science of fermentation is known as zymology. With the goal of producing a specific chemical product.

The chemistry of fermentation Aerobic & anaerobic cellular respiration Glycolysis Alcoholic fermentation Lactic acid fermentation

Aerobic Cellular Respiration Aerobic means ‘with air”. This type of respiration needs oxygen for it to occur so it is called aerobic respiration Glucose + oxygen  Carbon dioxide + Water + Energy The chemical equation is: 𝑪 𝟔 𝑯 𝟏𝟐 𝑶 𝟔 + 𝟔𝑶 𝟐 → 𝟔𝑪𝑶 𝟐 +𝟔 𝑯 𝟐 𝑶+𝟐𝟗𝟎𝟎 𝐤𝐣 3 stages – glycolysis – citric acid cycle – electron transport chain

Stages of Aerobic Celllular Respiration In glycolysis, a net of 2 molecules of ATP, or chemical energy, are produced. The citric acid cycle produces another 2 molecules of ATP The electron transport chain produces 28 molecules of ATP Oxygen is used in aerobic cellular respiration as the final electron acceptor in the electron transport chain, which is part of why it’s able to create so much ATP

Anaerobic Cellular Respiration In anaerobic cellular respiration, the only step of this process that occurs is glycolysis

What is fermentation It is a process by which the living cell is able to obtain energy through the breakdown of glucose and other simple sugar molecules without requiring oxygen Fermentation results in the production of energy in the form of two ATP molecules, and produces less energy than aerobic process of cellular respiration Louis Pasteur in the 19th century used the term fermentation in a narrow sense to describe the changes brought about by yeast and other microorganism growing in the absence of air (anaerobically) He also recognized that ethyl alcohol and carbon dioxide are not the only product of fermentation Fermentation is also used more broadly to refer to the bulk growth of microorganisms on a growth medium often with the goal of producing a specific chemical product

Lactic Acid Fermentation In lactic acid fermentation, the pyruvic acid from glycolysis is reduced to lactic acid by NADH, which oxidized to NAD+. This commonly occurs in muscle cells. Lactic acid fermentation allows glycolysis to continue by ensuring that NADH is returned to its oxidized state (NAD+)

Products of Fermentation The end products of fermentation differ depending on the organism Lactic acid and lactate, carbon dioxide, and water – produced from many bacteria, fungi, protists and animals cells (notably muscle cells in the body) Ethyl alcohol, carbon dioxide and water – produced from yeast and most plant cells

Products of Fermentation Fermentation products include: Food products : Milk - yogurt, kefir, fresh and ripened cheeses Fruits - wine, vinegar Vegetable – pickles, soy sauce Meat – fermented sausage, salami Industrial chemical: Solvent – acetone, butanol, ethanol, enzymes, amino acids Specialty chemicals – vitamins, pharmaceuticals

Products of fermentation

The range of fermentation process There are five major groups of commercially important fermentations: Those that produce microbial cells (or biomass) as the product. Those that produce microbial enzymes Those that produce microbial metabolites. Those that produce recombinant products. Those that modify a compound which is added to the fermentation - the transformation

The component parts of a fermentation process The formulation of media to be used in culturing the process organism during the development of the inoculums and in the production fermenter. The sterilization of the medium, fermenters and ancillary equipment. The production of an active, pure culture in sufficient quantity to inoculate the production vessel. The growth of the organism in the production fermenter under optimum conditions for product formation. The extraction of the product and its purification. The disposal of effluents produced by the process.

Interrelationships between the six component parts

Industrial penicillin manufacturing Case Study Industrial penicillin manufacturing

What is penicillin? Group of antibiotics produced by the Penicillium fungi Effective against actively growing Gram positive bacteria Some penicillin like amoxicillin are also effective against Gram negative bacteria, except Pseudomonas aeruginosa. It is a group of closely related compounds, not a single compound Examples: Amoxicillin, ampicillin, phenooxymethlpenicillin Around 50 drugs that are penicillin

Discovery and Production of Penicillin

How to produce penicillin By fermentation Fermenters Purpose of fermenter – provide contained, controlled and homogeneous environment in which the fermentation can proceed in a manner that is both safe and practical and which optimizes the particular objective of the fermentation. Other primary factors include cost, reliability and safety. For reactor being designed for specific purpose, there are a number of important parameters that will greatly affect performance.

Fermenter consideration Reactor size: optimum rates of production Reactor configuration: mechanical agitation or will a bubble column Mode of operation: will it be fed or continuously fed? Conditions inside the reactor: how will conditions (pH, temperature, …) be controlled? Economic requirements: Easy to operate aseptically Reasonably flexible regarding process requirements Low power consumption Stable under fluctuating conditions Cheap, robust, simple and well understood for scale- up

Specific conditions for penicillin production Most penicillin form filamentous broths. This means they can be difficult to mix due to their high viscosity. Also increasing viscosity of the broth can hinder oxygen transfer Solution: bubble column (air lift reactors) – would distribute the oxygen equally and also to agitate the medium Penicillin is an aerobic organism; oxygen supply is critical Optimum pH for penicillin growth is 6.5: maintain pH efficiently Strain stability problems (mutations): careful strain maintenance is required Biomass doubling is about 6h: provisions must be made

Media consideration Provide all the elements required for the synthesis of cell materials and the formation of desired product Provide favorable environment for the culture in question Be cost effective

Media Formulation

Primary and Secondary Metabolism cc

Production method Primary or secondary production? Secondary metabolites are only produced in times of stress when resources are low and the organism must produce these compounds to kill off its competitors to allow t to survive It is these conditions that we wish to duplicate in order to achieve the maximum amount of product from our fermentation

Overall process Seed culture Addition of solvent Medium Heat sterilization Fermentation Biomass removal Centrifugation extraction Extraction Fluid bed drying Final product (Penicillin G) Addition of solvent

Overall process

Quiz 6

Question 1 Identify at least 10 types of food, beverages and/or snack that were produced (at any level) with the aid of microorganisms

Question 2 Provide an overview of the production of one of the product you answered in Question 1