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EXAMPLES? Wine Production INTRODUCTION • Fermentation can be defined as the alteration or production of products with the help of microorganisms. • It is process of growing micro organisms in a nutrient media by maintaining physiochemical cond. & thereby converting feed in to desired end product. • Micro organisms serves as a biocatalyst in this reactions • They are designed to produce diff. p’ceutical such as Antibiotics, enzymes, vitamins, amino acids, insulin etc. •Bacteria, Actinomycetes, viruses & fungi can be used •Industrial Biotechnology: the process by which large quantities of cells are grown under aerobic or anaerobic conditions. •The industrial microorganisms are grown under controlled conditions with an aim of optimizing the growth of the organism for production of a target microbial product. •Cell growth, the structure of intracellular enzymes, and product formation depend on the nutritional needs of the cell (salts, oxygen) and on the maintenance of optimum biological conditions (temperature, concentration of reactants, and pH) within narrow limits. FERMENTER • Fermentation is carried out in vessels known as Fermentors • The types of fermentor ranges from simple tank to complex integrated system of automated control. • contains the media to carry out fermentation, and creates environment for fermentation at large scale. The performance of any fermenter depends on the following key factors: • Agitation rate • Oxygen transfer • pH • Temperature • The design and mode of operation of a fermenter mainly depends on the production organism, the optimal operating condition required for target product formation, product value and scale of production. Pure culture: organism, quantity, physiological state Sterilised medium: for microorganism growth Seed fermenter: inoculum to initiate process Production fermenter: Equipment ii) cell separation large model i) drawing the culture medium iii) collection of cell iv) product purification v) effluent treatment. Microbial Growth Kinetics •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 Batch Fermentation Process Microbial Growth Kinetics • 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. Microbial Growth Kinetics • 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 Microbial Growth Kinetics • 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. Microbial Growth Kinetics •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 Types of Fermentation Process • Batch Fermentation • Continuous Fermentation • Fed batch BATCH FERMENTATION • Batch reactors ,simplest type. • Reactor is filled with medium and the fermentation is allowed. • sterilized media components are supplied at the beginning of the fermentation with no additional feed after inoculation. • Once the desired amount of product is present in the fermenter the contents are drained off and the product is extracted. • The reactor is then cleaned, re-filled, re-inoculated and the fermentation process starts again. CONTINUOUS FERMENTATION • Continuous reactors, where fresh media is continuously added and bioreactor fluid is continuously removed. • Sterile medium is added to the fermentation with a balancing withdrawal of broth for product extraction. • As a result, cells continuously receive fresh medium and products and waste products and cells are continuously removed for processing. • The reactor can thus be operated for long periods of time without having to be shut down. • Many times more productive than batch reactors. CONTINUOUS FERMENTATION • does not have to be shut down as regularly • the growth rate of the bacteria in the reactor can be more easily controlled and optimized • cells can also be immobilized in continuous reactors, to prevent their removal. Continuous culture apparatus FED BATCH • Fed batch reactor, most common type of reactor used in industry. • fresh media is continuous or sometimes periodically added. PROCESS OF FERMENTATION • Fermentation process mainly divided into 3 steps 1)Upstream 2)Fermentation 3)Down stream An overview of a typical industrial fermentation process and the movement of materials through a typical fermentation plant are shown in the following figure: Fermentation Plant I. Upstream process • All the operations before starting the fermenter are collectively called upstream process. • such as sterilization of the fermenter, preparation and sterilization of culture medium and the preparation and growth of a suitable inoculums of microbial strain. Upstream Processing Three main areas: A) Producer microorganism • This include processes for obtaining a suitable • Microorganism strain improvement to increase the productivity and yield • Maintenance of strain purity • Preparation of suitable inocullum B ) Fermentation media C) Fermentation Process Inoculums •Incoculum is the substance/ cell culture that is introduced to the medium. The cell then grow in the medium, conducting metabolisms. •Inoculum is prepared for the inoculation before the fermentation starts. •It needs to be optimized for better performance: • Adaptation in the medium • Mutation (DNA recombinant, radiation, chemical addition) 29 Medium composition Fermentation medium consists of: • Macronutrients (C, H, N, S, P, Mg sources water, sugars, lipid, amino acids, salt minerals) • Micronutrients (trace elements/ metals, vitamins) • Additional factors: growth factors, attachment proteins, transport proteins, etc) For aerobic culture, oxygen is sparged 30 Fermenter Downstream Processing • The processes that follows fermentation: • A) Cell harvesting • B) Cell disruption • C) Product purification from cell extracts or the growth medium III. Downstream process • All the operations after the fermentation are known as downstream process. • It includes the purification of fermented products from fermentation broth. • Methods commonly used are distillation, centrifugation, filtration, and solvent extraction. FERMENTER FERMENTATION • The function of the fermenter or bioreactor is to provide a suitable environment in which an organism can efficiently produce a target product—the target product might be • · Cell biomass • · Metabolite • · Bioconversion Product • The sizes of the bioreactor can vary over several orders of magnitudes. • The microbial cell culture (few mm3), shake flask ( 100 -1000 ml), laboratory fermenter ( 1 – 50 L), pilot scale (0.3 – 10 m3) to plant scale ( 2 – 500 m3) are all examples of bioreactors. • Large volume and low value products like alcoholic beverages need simple fermenters and do not need aseptic condition. • High value and low volume products require more elaborate system of operation and aseptic condition. • The Designing of a Bioreactor also has to take into considerations the Unique Aspects of Biological Processes: A) The concentrations of starting materials (substrates) and products in the reaction mixture are frequently low; both the substrates and the products may inhibit the process. • Cell growth, the structure of intracellular enzymes, and product formation depend on the nutritional needs of the cell (salts, oxygen) and on the maintenance of optimum biological conditions (temperature, concentration of reactants, and pH) within narrow limits. B) Certain substances, inhibitors, effectors, precursors, metabolic products influence the rate and the mechanism of the reactions and intracellular regulation. C) Microorganisms can metabolize unconventional or even contaminated raw materials (cellulose, molasses, mineral oil, starch, ores, wastewater, exhaust air, biogenic waste), a process which is frequently carried out in highly viscous media. D) In contrast to isolated enzymes or chemical catalysts, mo’s adapt the structure and activity of their enzymes to the process conditions, whereby selectivity and productivity can change. • Mutations of the microorganisms can occur under sub optimal biological conditions. E ) Microorganisms are frequently sensitive to strong shear stress and to thermal and chemical influences. REQUIREMENTS OF FERMENTERS • There is no universal bioreactor. • The general requirements of the bioreactor are as follows: A) The design and construction of bioreactors must keep sterility from the start point to end of the process. B) Optimal mixing with low, uniform shear. C) Adequate mass transfer, oxygen. D) Clearly defined flow conditions. E) Feeding substrate with prevention of under or overdosing. F) Suspension of solids. G) Gentle heat transfer. H) Compliance with design requirements such as: ability to be sterilized; simple construction; simple measuring, control, regulating techniques; scale-up; flexibility; long term stability; compatibility with up- downstream processes; antifoaming measures. THANK YOU -PHARMA STREET