Fermentation Technology Lec-2 2025 PDF

Fermentation Technology 1. Microbial cultures: a. Isolation of microorganisms: From soils and muds of river and lakes.  Shotgun technique samples of free living microorganisms are isolated from plant materials, soil, sewage, water and waste streams then screened for the desirable traits.  Enrichme...

Fermentation Technology 1. Microbial cultures: a. Isolation of microorganisms: From soils and muds of river and lakes.  Shotgun technique samples of free living microorganisms are isolated from plant materials, soil, sewage, water and waste streams then screened for the desirable traits.  Enrichment culture technique samples of environmental samples are enriched with the target substrate to encourage the growth of those organisms with the desirable traits b. Culture Collections: Microbial culture collections provide a rich source of microorganisms that are of potential interest. American Typing Culture Collection (ATCC) National Collection of Type Cultures (NCTC). The culture used in biotechnology should ideally exhibit the followings: 1- Be grown in pure culture, 2- Genetically stable, 3- Amenable to genetic manipulation, 4- Grow rapidly on non-expensive raw materials, 5- Limited or no need for vitamins and additional growth factors, 6- Efficient production of the target product, 7- Safe and non-pathogenic, 8- Ready harvesting from the fermentation medium, 9- Grow well under workable conditions (pH, Temperature, et.) 10- Production of limited by-product 2- Culture maintenance: 1. Storage on agar slopes at 4oC and subcultured every 3 months 2. Storage of spore in water. Disadvantage: spore germination 3- Lyophilization (freeze-drying): Cultures in a small tube or ampoule is rapidly frozen, followed by drying under vacuum, then sealed under vacuum. It is the best method for culture maintenance. 4- Storage in glycerol at –70oC in deep freezer 5- Storage under liquid nitrogen ( at -150 C). 6- Soil cultures (more suitable for fungi). Moist sterile soil is inoculated with the microbial cultures and then incubated, dry at room temperature and stored. Bioreactors It is a device in which the organisms are cultivated and motivated to form the desired products. It is a vessel in which cell cultivation is done under sterile conditions and proper environmental condition.  Bioreactors are the containment vehicles of any biotechnology-based production process, be it for brewing, organic or amino acids, antibiotics, enzymes, vaccines or for bioremediation. It must be designed to give the correct environment for optimizing the growth and metabolic activity of the biocatalyst. Bioreactors range from simple stirred or non-stirred open containers to complex aseptic integrated systems involving varying levels of advanced computer control. Standards of materials used in bioreactor (fermenter) design 1. All materials and solutions entering the bioreactor must be corrosion resistant to prevent trace metal contamination of the process. 2. The materials must be non-toxic. 3. The materials of the bioreactor must withstand repeated sterilization with high-pressure steam. 4. The bioreactor stirrer system, entry ports and end plates must be easily machinable and sufficiently rigid not to be deformed or broken under mechanical stress. 5. Transparent bioreactor materials should be used if possible (visual inspection). Guidelines for the optimization of the bioreactor system 1. The bioreactor should be designed to exclude entrance of contaminating organisms. 2. The culture volume should remain constant (no leakage or evaporation). 3. The dissolved oxygen level must be maintained, and culture agitation for aerobic organisms. 4. Environmental parameters such as temperature, pH, etc., must be controlled and the culture volume must be well mixed.  Types of bioreactors: According to sterility ❑Non-aseptic systems: where it is not absolutely essential to operate with entirely pure cultures Examples: Brewing ‫تخمير‬, effluent disposal systems ‫محطات تحلية مياه الصرف الصناعي‬ ❑Aseptic systems: sometimes sterility is a Non-aseptic system prerequisite for successful product Brewing cheese formation. This type of process involves considerable challenges on the part of engineering construction and operation. Examples: antibiotics, vitamins, polysaccharides and recombinant proteins. Aseptic system Types of bioreactors: According to oxygen requirements Aerobic system ❑Aerobic systems: where oxygen and agitator shafts are used for aerobic processes. Aerobic bioreactors could be either stirred tank or non-stirred vessels ❑Anaerobic Systems: anaerobic bioreactors or digesters have long been used to treat sewage matter. In the absence of free oxygen, certain microbial consortia are able to convert biodegradable organic material to methane, carbon dioxide and new microbial biomass. Anaerobic system Types of bioreactors: According to operation method (fermentation method) 1. Batch Culture The microorganisms are inoculated into a fixed volume of medium. As growth takes place nutrients are consumed and products of growth (biomass, metabolites) accumulate. Disadvantages: After a while, cell multiplication ceases due to exhaustion or limitation of nutrient(s)and accumulation of toxic excreted waste products. Types of bioreactors: According to operation method (fermentation method) 2. Fed-Batch Culture The gradual addition of concentrated components of the nutrient ,such as carbohydrates, so increasing the volume of the culture. Advantages: Prolonging the life of a batch culture and thus, increasing the yield by various substrate feed methods. Types of bioreactors: According to operation method (fermentation method) 3. Continuous Culture Depends on fresh medium entering a batch system at the exponential phase of growth with a corresponding withdrawal of medium plus cells. Advantages: Gives nearly balanced growth with little fluctuation of nutrients, metabolites or cell numbers or biomass. Constant volume in the bioreactor. Biotechnology process Upstream and Downstream processing  Upstream processing (USP) involves all factors and processes leading to and including, fermentation and consists of: 1- Producer organism -Selecting the suitable producer -Industrial strain improvement to enhance productivity - Maintenance of strain purity - Preparation of suitable inoculum -Development of selected strains to increase the economic efficiency of the process 2-Fermentation media 3- Fermentation conditions  Downstream processing (DSP) includes all processing following fermentations 1-primary recovery 2-product purification 3-finishing processes  Improvement of product quality and quantity by manipulation of microorganisms Upstream manipulations such as: a. Screening program to select the active strain b. Improvement of selected strain by mutation c. Improvement of the existing process and products by recombinant DNA technology (Insulin production) d. The use of cell fusion and cell hybridization for generation of cell hybrides with high productivity (Nitrogen fixation and monoclonal antibody MCA) Maximization of the yield through manipulation of the fermentation conditions: 1- Manipulation of the culture conditions : A- Control of oxygen level in the cultural growth of saccharomyces yield different product: Hexose ------------aerobic------------ baker’s yeast Hexose ------------anaerobic---------- alcohol B- Citric acid production by Aspergillus niger spores could be produced by surface culture or submerged culture. Submerged culture is preferred due to: 1-needs less space and labor 2- more economic with high yield 3- ease of sterilization, where low energy is required C- Removal of metal cations (Fe+2 & Mg+2) from the media for citric acid fermentation usually improve the yield. 2- Instruction of mathematical modeling prediction of the best conditions for maximizing yield, cost reduction and scaling up the process 3- Improvement of the capacity of the biocatalyst (microorganisms. or enzyme) e.g. by immobilization for maximizing the yield and the production 4- Downstream processing Using proper methods for separation and isolation, recovery and purification of the product to maximize the yield and reduce lost during extraction and purification (ion exchange and chromatography) Role of genetic engineering in down stream process (DSP): DSP can be improved by : 1- Modification of the organism to suppress by-product formation, 2- Recombinant protein products may be designed to be excreted outside the cell, 3- Microbial cell wall may be modified to increase its permeability to the product 4- Recombinant protein products may be engineered with a high affinity for certain separation matrix.  2- Fermentation media  Microbial fermentation is designed for production of : 1- Biomass 2- Specific metabolite: (Primary metabolite, secondary metabolites) 3- Enzymes 4- Fermentation processes that can transform some compounds Choice of media: 1- Locally available 2- Sources for C & N 3- Might support precursor for the product 4- Ease of handling 5- Maximum yield of the product 6- Sterilization requirements 7- Formulation, mixing and viscosity characteristics Examples: whey, corn steep liquor, molasses, skimmed milk.  Production of metabolites Trophophase Idiophase Stationary phase Log b Death phase Number of cells Log phase Lag phase Time Microbial growth curve  Production of useful metabolites 1- During logarithmic phase (Trophase)----- production of primary metabolites 2- During stationary phase (Idiophase)----- production of secondary metabolites  Products of Primary Metabolism Essential for m.o and is concerned with the release of energy, Synthesis of important macromolecules such as: proteins, nucleic acids , organic acids, vitamins associated with microbial growth their maximum production occurs in the log phase of growth  Products of secondary metabolism Has no apparent function in the organism. produced in response to a restriction in nutrients. restricted to some species of plants and microorganisms have unusual chemical structures Microbial secondary metabolites include antibiotics, pigments, toxins, enzyme inhibitors, immunomodulating agents, receptor antagonists and agonists, pesticides, Some products of microbial secondary metabolism Product Organism Use Antibiotics Penicillin Penicillium Clinical use Streptomycin Streptomyces Clinical use Cephalosporin Cephalosporium Clinical use Anti-tumor Agents Actinomycin Streptomyces Clinical use Bleomycin Streptomyces Clinical use Toxins Aflatoxins Aspergillus Food toxin Alkaloids Ergot alkaloids Claviceps Pharmaceutical industry Primary metabolites Fungal Fermentation Citric acid Gluconic acid  Citric acid  Culture -- mutant of Aspergillus niger  Medium- molasses, skimmed milk and ammonium nitrate  K ferricyanide is added to remove heavy metals and phosphate from molasses  Conditions: 1- aerobic 2- Temperature: 25-30 oC 3- pH : 3.5 High pH is usually result in gluconic acid formation Methanol is added to reduce the toxic effect of heavy metals  Recovery: 1- Mycelia is removed by filtration 2- The filtrate is heated with CaCO3  neutralization; boil; filter; cool, centrifugation -- The crystal (Ca citrate) is dissolved in hot water, decolorize and the acid is liberated by H2SO4. Yield Improvement is achieved by: 1-Use mutant of Aspergillus niger deficient glucose oxidase and consequently unable to produce gluconic acid from glucose. 2-The medium is formulated with minimum level of iron (an activator of aconitase), and sugar concentration must be at least 140 g/L. 3- Submerged culture is preferred as it gives high yield Uses: - food industry in production of soft drink -Pharmaceutical and cosmetic industries -Preservative and in detergent industry  Gluconic acid Uses: 1- Pharmaceutical, metal and leather industry 2- Calcium and Ferrous gluconate is used as a Ca & Fe source for the body Culture : by Aspergillus niger in submerged fermentation Fermentation period: 36-48 h further incubation -- utilization of gluconic acid when all glucose is utilized, further incubation results in utilization of gluconic acid. Condition: aerobic at pH 5.5 using Ca carbonate To prevent ppt of Ca gluconate, 0.1% boron is added Recovery Mycelia was removed and the acid is recovered as Ca gluconate  Primary metabolites Bacterial fermentation Lactic acid Vitamins Amino acids Dr. Rasha Hashem  Lactic acid Lactic acid is commonly used in food industry  Calcium lactate is used in the treatment of calcium deficiency,  Iron lactate is used in treatment of anemia. Culture --- Lactobacillus sp. Medium--- whey or molasses and suitable N source Conditions  1- anaerobic or microaerophilic fermentation 2- Temperature - 45-60 oC 3- pH - 5.0-6.0 controlled by addition of CaCO3 4- Time  4-6 days in batch fermentation Dr. Rasha Hashem in acid-resistant fermenter  Recovery: For recovery the fermentation mesh is boiled to coagulate the microbial proteins which is trapped on a filter and dried for use as animal feed supplement. The filtrate, which contains the soluble calcium lactate, is concentrated then subjected to further purification. N.B. (I) Homofermenters - lactic acid only Heterofermenters  lactic acid + other acids + alcohol (2) Fermentation process is not liable for contamination ? due to: 1- Insufficient oxygen 2- High temperature 3- Acidic pH Dr. Rasha Hashem Vitamins Dr. Rasha Hashem  Vitamin B12 (cyanocobalamine) Culture ---- Propionobacterium Medium--- Soya bean meal, yeast extract, corn steep liquor, meat extract, Cobalt Conditions  Two stages process (1)- anaerobic 2 days-- formation of cobinamide; then aerobic  3-4 days  formation of cobalamine (2)- continuous addition of carbohydrate increase the yield (3)- addition of radioactive cobalt for production of radioactive B12 Dr. Rasha Hashem Uses B12 is used for treatment of pernicious anemia Recovery: KCN is added Cobalamine is then converted to cyanocobalamin with KCN N.B. 1- Yield of vitamin is proportional to cell mass 2- the vitamin could also produced by Bacillus megaterium Dr. Rasha Hashem  Vitamin B2 (Riboflavin) Culture : Ashbya gossypii (fungal fermentation) The organism is classified as cotton pathogen and is not suitable for production of riboflavin in Egypt Medium: corn steep liquor, peptone, soybean oil The vitamin is extracellular and mycelium bounded Bounded vitamin is released by heat treatment at 120 C for 1 h, Mycelium is separated and culture filtrate is used as an animal feed Dr. Rasha Hashem  Amino acids  L-Glutamic acid Culture : Corynebacterium glutamicum sucrose and molasses is used as a C source Precursor: alpha-ketoglutaric acid pH is controlled by ammonia High PO4 ---- cause growth and production inhibition Dr. Rasha Hashem Lysin Culture : Corynebacterium glutamicum cane molasses is used as a C source pH is controlled by ammonia L- tryptophan Culture : Corynebacterium glutamicum , Esherichia coli Using rDNA technology230 fold increase in tryptophan synthetase enzyme of E. coli Indole----- tryptophan synthetase --------- tryptophan Dr. Rasha Hashem  Extracellular polysaccharides (biopolymers) The glycocalyx (polysaccharides that covers the microbial cell surface). Glycocalyx is also termed exopolysaccharides or biopolymers. If attached firmly to the cell wall, it is called capsule If disorganized and attached loosely, it is described as a slime layer. Or they may be dissolved in the media. Such water-soluble biopolymers are rapidly emerging as source of gums. Dr. Rasha Hashem  Microbial Polymers Microbial polysaccharides: Homo-polysaccharides: dextran and cellulose Heter-polysaccharides: xanthan Production of microbial polymers Polymer Producer organism Applications/uses Dextran Leuconostoc mesenteroids Plasma substitute Pullulan Aureobasidium pullulans Biodegradable film and fibers Xanthan Xanthomonas campestri Stabilizing agent and Dr. Rasha Hashem viscosity controllor Secondary Metabolites Antibiotics Dr. Rasha Hashem  Secondary Metabolites Antibiotics Antibiotics are natural product with small molecular weight that inhibit or kill microorganisms, selectively at low concentrations often products of secondary metabolism antibiotics produced by various bacteria & fungi Bacillus----------- Bacitracin Streptomyces---- Streptomycin Penicillium------ Penicillin Natural antibiotic Semi-synthetic synthetic Dr. Rasha Hashem  Penicillin Penicillin : Acylated 6-amino penicillanic acid (6APA) Biosynthesis of penicillin - condensation of valine and cysteine - 6APA Phenylalanine or phenylacetic acid is added as a precursor- Benzyl- penicillin If the precursor is not added -- mixture of penicillin Dr. Rasha Hashem  Penicillin Culture ---- Penicillium notatum  low yield and pigment production Penicillium chrysogenum  no pigment ; high yield Medium :- lactose (slowly utilizable sugar + corn steep liquor (contain phenylalanine) + CaCO3 and KH2PO4 (buffers) Dr. Rasha Hashem  Phases of fermentation: Phase I (36 h) (growth phase) Glucose is utilized with acid production -> pH 4.0  pH 7.5 due to acid utilization in corn steep liquor and also due to utilization of proteins producing NH3.  At the end of this phase, phenylacetic acid should be added Phase II. (36-75h) (production phase) : Lactose is slowly utilized by the fungus so that pH remains constant at 7.5. During this phase, the fungal growth stopped and penicillin is produced in large amount.  Penicillin is recovered at the end of this phase Phase III : After 75 h, the fungal cells are autolysed with the production of NH3 which rise pH  destruction of penicillin Dr. Rasha Hashem Recovery of penicillin 1- separation of fungal mass by filtration 2- fungal mass is removed and used as animal feed 3- Filtrate is cooled to about 2-3 C, pH adjusted to liberate to free acid 4- Penicillin is extracted by organic solvent, and then extracted back to the aqueous after adjusting pH 5- K+ is added and penicillin is ppt as K salt 6- Penicillin salt is removed by filtration Dr. Rasha Hashem  Modification is achieved by removing their natural acyl group, leaving 6-amino penicillanic acid (6-APA) to which other acyl group can be added to confer new properties.  These semisynthetic penicillins such as methicillin, carbenicillin, oxacillin and ampicillin  Semisynthetic exhibit various improvement including resistance to acids to allow oral administration, degree of resistance to ß-lactamase broaden its spectrum of antibacterial activity. Dr. Rasha Hashem  Streptomycin Culture ------ Streptomyces griseus Medium- : glucose as a C source, soybean - N source Phases of fermentation Phase I (growth phase) --------> rapid growth formation of mycelial mass pH rises due to proteolytic activity and release of ammonia Phase II. : streptomycin accumulates in the medium glucose residue and NH3 are utilized during this phase. Dr. Rasha Hashem  Recovery : 1- Mycelia is separated by filtration and the antibiotic is adsorbed on a charcoal or ion exchange. 2-The antibiotic is ppt with acetone and purified by chromatography  Infection with bacteriophages may cause problems in streptomycin production. To avoid such problem, it is better to isolate and use of phage- resistant mutants for production. Dr. Rasha Hashem  Solid State Fermentation Bacteria and yeasts play a major role in SSF which involve their growth and multiplication on moist solid substrates in the absence of free water. Substrates used : cereal grains, bran, legumes and lignocellulosic materials. Used for: food fermentations producing cheeses & mushroom.. Dr. Rasha Hashem  Good examples for products produced by SSF are: 1-Alpha-amylase: 2-Protease. 3-Pickling of cucumbers: Cucumbers can be fermented by lactic acid bacteria. 4-Cheese ripening: Cheese is ripened by fermentation using certain moulds or by the lactic acid bacteria Dr. Rasha Hashem