Microorganisms in Fermentation PDF

# Microorganisms in Fermentation - Depending on the ATP generating metabolic pathways, organisms can be classified into 2 groups: - **Autotrophs**: In autotrophs, generation of ATP is done by electron transport chain, which follows oxidation of organic material prepared during photosynthesis....

# Microorganisms in Fermentation - Depending on the ATP generating metabolic pathways, organisms can be classified into 2 groups: - **Autotrophs**: In autotrophs, generation of ATP is done by electron transport chain, which follows oxidation of organic material prepared during photosynthesis. - **Heterotrophs**: ATPs are generated by oxidation of organic compounds in the growth substrates, which maybe done either in presence or absence of oxygen. - In aerobic organisms (which oxidize food material in the presence of oxygen), the process is called cellular respiration and in anaerobes, it's called anaerobic respiration or fermentation. - The process of fermentation was used only to produce alcohol from molasses. But nowadays the development of genetic engineering has made it possible to use the process of fermentation to produce many compounds including vaccines, antibiotics & enzymes, and for biological conversion of many substances. ## Microorganisms Used in Fermentation - Includes bacteria, fungi, algae, & actinomycetes. Commonly used species include: - Bacteria: Example - Bacillus subtilis - Algae: Example - Spirulina maxima - Fungi: Aspergillus oryzae - Actinomycetes: Streptomyces griseus ## Culting the Microorganism - After isolation of microorganism, they are grown on culture medium provided that the medium contains all the necessary nutrients & the environmental conditions are favourable. - Different types of culture medium are used for different purposes. Some of the common types are: ### Batch Culture - It's the simplest method where microorganism are grown on a limited amount of medium until essential nutrients are exhausted or toxin byproducts inhibit the growth. ### Continuous Culture - Continuous culture systems are designed to maintain microbial populations in a steady state bu continuously supplying fresh nutrients and removing an equal volume of culture. - This approach prevents nutrient depletion and the accumulation of toxic byproducts, allowing cells to remain in the exponential growth phase. - By carefully contolling the rate of nutrient addition and culture removal, there is the production of new biomass resulting in a stable condition. ### Fed-Batch Culture - A fed-batch culture is a fermentation process where fresh nutrients are continuously or intermittently added to the bioreactor without removal of original culture from the fermenter. - Products obtained from microorganism are: - Primary Metabolites - Secondary Metabolites - Enzymes - Microbial Biomass ## Biologicals obtained from Fermentation ### Amino Acid: L-Lysine - Lysine is commercially produced by using 2 species of bacteria in a 2 stage process: - **Stage 1**: Involves formation of diaminopimelic acid (DAP) by E.coli - **Stage 2**: Involves release of decarboxylase enzyme from organism Enterobacter aerogenes <start_of_image> diagram showing the process of producing amino acid: L-lysine: ```mermaid graph LR subgraph E.coli A[Grown on \n medium containing \n Glycerol, corn steep \n liquor & (NH4)2HPO4] B[E.coli grown on medium under \n controlled conditions of \n pH temp & aeration] end subgraph Enterobacter aerogenes C[DAP decarboxylase \n produced by this \n organism is added] D[Optimum quantity \n of DAP is produced] end A-->B B-->D D-->[3 Days incubation]-->C C-->E[DAP is converted to L-lysine] E-->F[L-lysine is later extracted] ``` ## Vitamins from Fermentation ### Riboflavin (Vit B2) - Riboflavin is essential for the growth and reproduction of both humans and animals. It is produced commercially by microbial fermentation. - Various microorganisms can be used to produce riboflavin. - It is a byproduct of acetone butanol fermentation, which utilizes Cl. butylicum and Cl.acetobutylicum, it is commercially produced by direct fermentation using Ascomycetes species, Eremothecium ashbyii, and Ashbya gossypii. ## Diagram Showing how riboflavin is produced ```mermaid graph LR subgraph Ashbya gossypii A[Ashbya gossypii \n (A filamentous \n hemiascomycete)] B[Grow on \n Culture media] end subgraph Culture Conditions C[Glucose+ low level corn \n oil = improves yield] D[Medium contains semi- \n purified sugar like glucose, \n additional crude organic \n soyabean meal, purine or its \n derivatives & cobalt (2- \n 10ppm)] E[Temp.26-28°C \n pH at 6-7.5] end A-->B B-->D D-->E E-->[4-6 days fermentation]-->F[Culture is submerged & \n aerated. High level of \n aeration is avoided since it \n inhibits mycelium production \n & yield is reduced] B-->[Fermentation through]-->C C-->[Stimulates production]-->F ``` ## How riboflavin is Produced - **Organism**: Ashbya gossypii: This filamentous hemiascomycete. - **Culture Medium**: The fungus is grown on a culture medium. The addition of low-level corn oil along with glucose is noted to improve riboflavin yield. - **Controlled Growth Conditions**: - **Temperature**: The culture is maintained at 26-28°C. - **pH**: The pH is controlled within a range of 6 to 7.5. - **Fermentation Period**: The fermentation lasts for 4-6 days. - **Aeration**: A high level of aeration is avoided. - **Fermentation Output**: Under these conditions, Ashbya gossypii stimulates riboflavin production, which is later harvested for commercial use. ## Diagram showing the cycle of riboflavin production ```mermaid graph LR A(Growth is rapid;\n vitamin production is very little.\n Glucose is rapidly used)-->B[pH becomes\n acidic due to\n pyruvic acid\n release.] B --> C(Pyruvate Concentration\n is reduced) C --> D(At the end, growth is\n completely stopped\n due to exhaustion of\n glucose.) C --> E(Sporulation Occurs) E --> F(Maximum production of Riboflavin,\n remains as cell bound Flavin\n dinucleotide (FAD) & in the form of\n smaller amounts of Flavin \n mononucleotide (FMN).) F --> G(Ammonia accumulates due to\n increased deaminase activity) F --> H(Autolysis Occurs) H --> I(Free Riboflavin is released\n along with FAD & FMN.) ``` ## Phases of Riboflavin Production - **Phase 1: Rapid Growth Phase** - **Growth Characteristics**: During this initial phase, growth is rapid, and glucose is consumed quickly. - **Vitamin Production**: Minimal riboflavin (vitamin B2) production occurs at this stage. - **pH Shift**: the pH becomes acidic due to the release of pyruvic acid as a byproduct of glucose metabolism. - **End of Phase**: Growth eventually halts once glucose is exhausted, signaling the transition to the next phase. - **Phase 2: Sporulation and Riboflavin Production** - **Sporulation**: The cells undergo sporulation, a process of forming spores. - **Pyruvate and Ammonia**: The concentration of pyruvate decreases, while ammonia accumulates. - **Maximum Riboflavin Production**: At this point, riboflavin is produced in significant amounts. It is mainly stored as cell-bound flavin dinucleotide (FAD) and, to a lesser extent, as flavin mononucleotide (FMN). - **Phase 3: Autolysis and Riboflavin Release Autolysis**: The calls undergo autolysis (self-digestion), breaking down their cellular structures. - **Release of Riboflavin**: During autolysis, free riboflavin, along with FAD and FMN, is released into the medium, making it accessible for extraction. ## Additional Information - The word fermentation is derived from the Latin word "fevere", which means to boil. - The conventional definition of fermentation is breakdown of larger molecules into smaller & simple molecules using microorganisms. - In biotechnology fermentation means any process by which microorganisms are grown in large quantities to produce any type of useful materials. - Biochemically, fermentation involves several oxidation-reduction reactions in which the organic compounds act as donors or acceptors of hydrogen ions. - The organic compounds used as substrates give rise to various products of fermentation. - ATP is the energy-rich molecule in almost all types of organisms.