Biotechnology Lecture 1 Fall 2024 PDF

Microbiology & Immunology department Biotechnology PM 906 Lecture 1 Dr. Amal Emad Ali Professor of Microbiology & Immunology  Lecture Overview Bioprocess/Fermentation technology Components of fermentation process Bioreactor: sti...

Microbiology & Immunology department Biotechnology PM 906 Lecture 1 Dr. Amal Emad Ali Professor of Microbiology & Immunology  Lecture Overview Bioprocess/Fermentation technology Components of fermentation process Bioreactor: stirred tank bioreactor Modes of Fermentation: Batch, Fed-Batch, Continuous Scaling Up Stages of Bioprocess/ Fermentation Upstream Processing Downstream Processing  Bioprocess/ Fermentation technology A bioprocess is a specific process that uses complete living cells or their components (e.g., bacteria, enzymes, plant or mammalian cell) to obtain desired products. Constitute a significant part in most old & new biotechnology Applications: Production of biopharmaceuticals, food industry, biofuels, wastewater treatment, chemicals industry.  Fermentation processes Fermentation is an industrial process utilizing living cells for the production of commercially valuable products either aerobically or anaerobically. In fermentation living cells are allowed to grow under defined conditions in a fermenter (bioreactor). The defined conditions include the use of the proper substrate (medium) and the proper environmental parameters (e.g. temperature, agitation, pH and aeration). All must be optimized to achieve the highest yield and quality at the lowest cost possible.  Most fermentations are performed as submerged fermentation in closed aseptic conditions in a bioreactor (example: antibiotics, amino acids, enzymes). Some fermentations are carried in an open bioreactor under non aseptic conditions (example: some food and alcoholic beverage, wastewater treatment).  Components of fermentation process Biological system: Microbial culture, animal or plant biocatlyst Substrate Product Raw materials Specific conditions  Cultures: (microbial, animal and plant) Biocatalyst Microbial cultures Microbial cultures are either obtained from culture collections e.g. American type culture collection (ATCC) or usually isolated (from soil, air ….etc.) by enrichment technique (maintain conditions that favor isolation of the required microorganism). In industry, microorganisms act like chemical factories. Those ones intended to be used in industry should be: 1-Should be pure culture i.e. not contaminated with other species or low producing strains. 2-Produce a large amount of the required product. 3-Easily cultivated and maintained. 4-Be genetically stable (low rate of mutation). 5-Grow rapidly on inexpensive and readily available media. 6-Produce the desired product under workable conditions (pH, O2 temperature,….etc.).  Characteristics of raw materials used in fermentation process 1. Produce maximum yield of the product per gram substrate used. 2. Cheap and available locally throughout the year. 3. Causes minimal problems during the fermentation, product separation and waste disposal. 4. Of definite composition ( chemically defined) and easily processed Easily transported and sterilized. Chemically defined are usually used in laboratory research, but in industrial fermentation the use of pure defined chemicals would be too expensive, accordingly complex less defined substrates are used to reduce the production costs.  Bioreactor There are many types of bioreactors Continuous stirred tank bioreactor is the most widely used type for production of biopharmaceuticals  Motor Inlets Air filter power Compressor unit Monitors Outlets Air exhaust sampler Agitator shaft Baffle Cooling or heating coils Impeller Diagram showing design of …… Air sparger at …… (bioreactor)the Stirred tank the end of air  In aerobic fermentations it is important to achieve the optimal oxygen concentration. It is supplied from compressed filtered air to the bioreactor and forced through a sparger to facilitate the dispersion of oxygen to liquid medium. The medium as well as air bubbles are mixed by impellers arranged on an agitator shaft attached to bottom or top sealed motor. Careful design is required to achieve optimal oxygen concentration throughout the fermentation medium since oxygen transfer in large volume fermenters is difficult. Large fermenters are also supplied with internal baffles that help in mixing. Aeration and mixing are relatively expensive due to the high- energy costs needed. Fermenters may be supplied with pumps to supply acid or alkali in order to adjust the pH during the fermentation course. Also pumps to supply nutrients or antifoams may be also required.  Small fermenter vessel up to 20 liters are usually made of glass, however , large volume vessel are usually made of stainless steel. It is necessary that fermenter and substrate solutions be sterilized (mostly inside the fermenter) before the addition of the inoculum (microbial strain). Small fermenters are sterilized in autoclave, while large fermenters are sterilized by steam generated from distant boiler. There are three modes in which submerged fermentation is carried out: (i) Batch mode, (ii) Fed-batch mode and (iii) Continuous mode. Fermentation mode A-Batch B-Fed-batch C-Continuous fermentation fermentation fermentation  A- Batch Fermentation The fermenter containing the sterilized culture, inoculated with the microorganism and then incubation is allowed to proceed under optimal conditions for the required period of time. Nothing is added to the fermenter (closed system) during the entire fermentation process except 🔘Air 🔘Antifoam agent 🔘Acid or alkali to control the ph.  At the end of the fermentation cycle, the fermenter is shut off and the contents are collected and the product is recovered. Example: production of secondary metabolites which are produced by the depletion of one or more nutrients at the end of the growth phase: antibiotics Production of the 2ry metabolite  B- Fed-Batch Fermentation Nutrients are added at intervals as the fermentation progresses. fed-batch culture is superior to conventional batch culture when controlling concentrations of a nutrient (or nutrients) affects the yield or productivity of the desired metabolite. Example: Production of baker’s yeast  C- Continuous Fermentation Sterile nutrients are added continuously to the fermenter and equivalent amount of product with microorganism are simultaneously harvested out of the fermenter. e.g. Production of Biomass and Primary metabolites which are produced during the logarithmic phase of growth: organic acids (citric acid, gluconic acid, lactic acid) vitamins, amino acids  Scaling up It is the transfer of small scale laboratory fermentation to an industrial large scale. Fermentation processes are usually developed in three stages: i. laboratory scale (flasks, laboratory fermenters). ii. pilot plant scale (usually 50-200 liters). iii. Production scale iv. production scale. laboratory scale pilot plant scale production scale Stages of Bioprocess/ Fermentation  I-Upstream processessing The upstream part of a bioprocess refers to the first step in which microbes/cells are grown, e.g. bacterial or mammalian cell lines, in bioreactors. Upstream processing involves all the steps related to inoculum development, media development, improvement of inoculum by genetic engineering, optimization of growth kinetics so that product development can improve tremendously. The main goal of the upstream process is the transformation of substrates into the desired metabolic products.  Upstream processing includes formulation of the fermentation medium, sterilization of air, fermentation medium and the fermenter, inoculum preparation and inoculation of the medium. The fermentation medium should contain an energy source, a carbon source, a nitrogen source and micronutrients required for the growth of the microorganism along with water and oxygen, if necessary. The fermentation medium which is used for a large scale fermentation, should have the following characteristics: 1. It should be cheap and easily available 2. It should maximize the growth of the microorganism, productivity and the rate of formation of the desired product 3. It should minimize the formation of undesired products Usually, waste products from other industrial processes, such as molasses, lignocelluloses wastes, cheese whey and corn steep liquor, after modifying with the incorporation of additional nutrients, are used as the substrate for many industrial fermentations. Inoculum build up is the preparation of the seed culture in amounts sufficient to be used in the large Fermenter vessel. This involves growing the microorganisms obtained from the pure stock culture in several consecutive fermenter.  Improvement of product quality and quantity by manipulating the process. I- Upstream manipulation 1) Strain development. The productivity of the wild strains are usually too low for economical processes In general such programs include Ø Selection of the biological entities by a screening program to choose the best biocatalyst of the required product. Ø Utilization of mutation techniques to prepare mutants of better characters. Ø Utilization of recombinant DNA (genetic engineering) to improve an existing process or developing a totally new product. Ø Cell fusion for the generation of hybrids with improved productivity. Ø Plant cell and tissue culture. 2) Manipulation of the fermentation conditions to specify the product and maximize the yield e.g. changing the oxygen potential of growth for Saccharomyces yields different product. Saccharomyces + Hexoses aerobic Baker’s yeast Saccharomyces + Hexoses anaerobic alcohol Saccharomyces + Hexoses anaerobic & glycerol bisulphite e.g. citric acid production could be performed by solid- substrate fermentation (low yield)in which the growth of microorganisms in moist, nonsoluble substrate with low water content or by submerged culture in a stirred tank bioreactor (Higher yield). Solid state fermentation 3) Obtainingmathematical models for the fermentation parameters to help in the prediction of the best conditions for maximum yield and cost reduction in scaling up of the process. 4) Improvement of the efficiency of the biocatalyst ( enzyme) by immobilization (discussed later).  II- Downstream processing Downstream processing, all stages that follow the fermentation Application of the proper methods for separation and process, involves suitable techniques and methods for recovery, purification of the product. purification, and characterization of the desired fermentation product. It has the primary aim of efficiently, reproducibly and safely recovering the target product to the required specifications (biological activity, purity) while maximizing recovery yield and minimizing costs. A vast array of methods for downstream processing, such as centrifugation, filtration, and chromatography, may be applied. The choice of the operations used in recovery depends on the nature of the desired product (intracellular or extracellular), physical , chemical nature, its concentration and stability and the required level of purity in the end product. A significant percentage (50–80%) of the total manufacturing costs of biopharmaceutical molecules is incurred during downstream processing  Steps of Downstream process 1. Separation of biomass: Separating the biomass (microbial cells) generally carried out by centrifugation or ultra-centrifugation. If the product is biomass, then it is recovered for processing and spent medium is discarded. If the product is extracellular the biomass will be discarded. Ultrafiltration is an alternative to the centrifugation. 2. Cell disruption: If the desired product is intracellular the cell biomass can be disrupted so that the product should be released. The solid-liquid is separated by centrifugation or filtration and cell debris are discarded. 3. Concentration of broth: The spent medium is concentrated if the product is extracellular. 4. Initial purification of product: According to the physico- chemical nature of the product molecule several methods for recovery of product from the clarified fermented broth were used ( precipitation, solvent extraction, ultracentrifugation, ion-exchange chromatography, adsorption and solvent extraction).  5. Polishing (finishing) of product all impurities must be removed, including any mammalian infecting viruses, bacterial pathogens and their endotoxins. This last step is vital for the safety of the products. Size exclusion chromatography is used