Industrial Biotechnology for biotechnology students

Industrial Biotechnology for biotechnology students

1. Industrial Biotechnology

2. Industrial biotechnology Industrial biotechnology can be defined as “The exploitation of enzymes, microorganism and plants to produce energy, industrial chemicals and consumer goods”. It often referred as “WHITE BIOTECHNOLOGY”. It is the collection of the scientific techniques & technologies to improve both the efficiency and environmental footprint of modern industrial production. Microbial technology constitutes the core of industrial biotechnology. Microbial technology refers to the use of microbes to obtain a product or service of economic value. It is also called fermentation.

3. Fermentation

4. Introduction Fermentation is a metabolic process where microorganisms like bacteria or yeast break down carbohydrates, like sugars, into simpler substances like alcohol or acids, often in the absence of oxygen. This process is widely used in food production (e.g., yogurt, bread, alcoholic beverages) and other industrial applications.

5. Principle of Fermentation The main principle of fermentation is to derive energy from carbohydrates in the absence of oxygen. Glucose is first partially oxidized to pyruvate by glycolysis. Then pyruvate is converted to alcohol or acid along with regeneration of NAD+ which can take part in glycolysis to produce more ATP. Fermentation yields only about 5% of the energy obtained by aerobic respiration.

6. Basic principles of Fermentation Technology Selection, growth, maintenance and further manipulation of microorganisms used for the industrial production purpose: Media preparation (formulation) for industrial fermentation Inoculum development Bioreactor/fermentation vessel design Control of the fermentation conditions: Temperature, pH, aeration, agitation Product recovery: biomass/ metabolite recovery Disposal of effluent/waste produced from the process

7. 1. Selection, growth, maintenance and further manipulation of microorganisms used for the industrial production purpose: Selection: selecting the right microorganism that metabolizes a certain substrate and gives the desired product. From where? Either environmental samples or from microbial culture collection centers. Growing the selected Microorganism: culturing of the MO in synthetic culture medium Maintenance of the MO: maintaining the identified microorganism for continuous use in the industry, including culture preservation. Further manipulation/ Strain improvement: Genetic modification on the microorganism to enhance certain property of the microbe; may be rate of metabolism, avoidance of undesired biproduct or to give it a an ability to metabolize a new substrate

8. 2. Media preparation (formulation) for industrial fermentation All micro-organisms require water, sources of energy, carbon, nitrogen, mineral elements, vitamins and oxygen for their growth. On a small scale - device a medium containing pure compounds satisfy the growth but may be unsuitable for use in a large-scale process or it will be unaffordable economically. Therefore, for industrial scale fermentation, a medium of bulk quantity containing all the necessary nutritional requirements of the microbe is mandatory.

9. Media preparation (formulation) for industrial fermentation Types of media Synthetic media Semi-synthetic media Complex media

10. Synthetic media Synthetic media are media composed of pure ingredients in carefully measured concentrations dissolved in double distilled water, thus the exact chemical composition of the medium is known. Typically, it contains a simple sugar as the carbon and energy source, an inorganic nitrogen source and various mineral salts and, if necessary, growth factors (purified amino acids, vitamins, purines and pyrimidines). Useful in research and laboratory situations where experiment requires high accuracy.

11. Examples Simmons citrate agar: Used to determine an organism's ability to utilize citrate as a sole carbon source. Mineral glucose medium: A simple medium with a defined carbon source (glucose) and mineral salts. Czapek Dox Medium: A standard medium for growing fungi, particularly Aspergillus species. Dubos's medium with tween 80: Used for growing specific bacteria, where the exact components and their concentrations are known.

12. Semi synthetic media Largely chemically defined but one or more poorly specified components of variable but a controlled composition. eg.Yeast extract, beef extract. This type of media are useful in research and laboratory situations –particular organism growth. Plant, animal, fish and microbial extracts have been routinely used in the past to supply vitamins and essential growth factors for specific organisms. Examples include Potato Dextrose Agar (PDA), Corn Meal Agar (CMA), and nutrient agar.

13. Complex media/Crude media Complex media in microbiology refers to growth media with undefined or unknown chemical compositions, often derived from natural sources like animal or plant extracts. Examples include nutrient broth, tryptic soy broth, MacConkey agar, and chocolate agar. These media are used because they can support the growth of a wide range of microorganisms, including those with complex nutritional requirements.

14. Nutrients that complex media should contain Carbon sources: for growth and energy Nitrogen sources: protein synthesis Minerals: for Enzyme functions Growth regulators: preformed cellular components (vitamins, aa, fatty acids, sterols) for growth Water: water is the major component of industrial fermentation media and it is necessary for many of its ancillary services such as heating, cooling and rinsing. Therefore clean water of consistent composition is required from permanent sources.

15. Criteria for media selection Produce maximum yield of product Produce the maximum concentration of biomass Will permit maximum rate of product formation Will be of consistent quality Will be available throughout the year. Cause minimal problems during medium sterilization. Minimum yield of undesirable product

16. 3. Inoculum Development The inoculum is the amount of fermented medium containing microorganisms in their active growth state. It serves as starter culture ( Ersho ) for the large scale fermentation process. The inoculum should satisfy the following criteria It must be in active and healthy state, helps to minimize the lag phase for the large scale fermentation. It must be sufficiently large volume to provide optimum number of Mos for the large fermenter The microbes must be in the suitable morphological form Must be free of contamination Must retain its product-forming capabilities

17. Why are microorganisms used in Fermenters? They are small and so can be grown economically in fermenters on either solid or liquid media They have a fast growth rate and so can produce large yields in short periods of time They can be genetically modified with relative ease to synthesize non-natural products of interest May be able to tolerate unusual growth conditions (e.g. extremophiles can grow in high temperatures or acidic conditions)

18. Growth curve of Microorganism

19. 4. Bioreactor/fermentation vessel design A fermentation process requires a fermenter for the successful production of the desired product. Fermenters used in biotechnological productions are known as Bioreactors. Bioreactor is the large vessel containing large quantity of nutrient media maintaining favorable conditions for the producer microorganisms. For a specific fermentation process, the bioreactor type must be chosen in terms of construction material or design.

20. Size for different fermenters The sizes of the fermenter are divided into the following groups. 1. The microbial cell fermenter (mm cube) 2. Shake flask fermenter (100-1000ml) 3. Laboratory fermenter (1-50 L) 4. Pilot scale fermenter(50 - 5000 dm 3 ) 5. Industrial scale fermenter (25-500 m 3 )

21. Different Operating Modes: Batch culture A closed system which contain initial, limited amount of nutrients After a certain time the growth rate of the culture decreases until growth ceases, mainly due to the depletion of nutrients Primary metabolites are produced during exponential phase & secondary metabolites are produced during stationary phase Batch fermentation is used to produce biomass, primary metabolites & secondary metabolites

22. Continuous Culture Exponential growth is prolonged by addition of fresh medium. Added medium displaces an equal amount of culture from the vessel. steady state-- If the medium is fed continuously to a culture in a vessel at a suitable rate, the formation of a new biomass by the culture is balanced by the loss of the cells from the vessel at this rate, the biomass concentration will be maximum and constant

23. Fed–batch culture Batch cultures which are fed sequentially with medium, without removal of culture fluid Variable volume fed batch culture- --- Same initial medium or a solution of the limiting substrate at the same concentration as that in the initial medium is added , resulting in an increase in volume Fixed volume fed batch culture----- A very concentrated solution of the limiting substrate is added at a low rate, resulting in an insignificant increase in volume

24. Diagram of a Standard Fermenter

25. Part of Bioreactor Vessel: The main container where the biological process takes place. Control Systems: These maintain optimal conditions like temperature, pH, and dissolved oxygen levels. Agitation/Mixing: Ensures uniform distribution of nutrients and cells, preventing settling. Aeration/Gas Supply: Provides oxygen for aerobic processes. Monitoring and Sensors: Used to track key parameters and ensure the process is running smoothly. Sterilization: Bioreactors are designed to be sterilized to prevent contamination.

26. Types of Bioreactor Stirred Tank Bioreactors: The most common type, using impellers for mixing. Bubble Column Bioreactors: Mixing is achieved by introducing gas into the liquid. Airlift Bioreactors: Similar to bubble columns, but with a distinct riser and downcomer for enhanced mixing. Packed Bed Bioreactors: Cells are immobilized on a solid support within the reactor. Fluidized Bed Bioreactors: Particles carrying cells are kept in suspension by fluid flow.

27. Basic Function of Fermenter provide a controlled environment for optimum biomass/product yields. power consumption should be minimum. provide easy and dependable temperature control. Facility for sampling should be provided. should have a system for monitoring and regulating pH of the fermentation broth. should require a minimum of labour in maintenance, cleaning, operating and harvesting operations. should have smooth internal surfaces, and joints should be welded wherever possible. Provide adequate mixing and aeration for optimum growth and production of metabolites.

28. Upstream Processing Three main areas: A) Producer microorganism B ) Fermentation media C) Fermentation Process

29. Downstream Processing The processes that follows fermentation: A) Cell harvesting B) Cell disruption C) Product purification from cell extracts or the growth medium

30. 6. Product recovery: biomass/ metabolite recovery Refers to the process conducted after the fermentation process is completed. Many fermentation processes require a purification scheme that help to reduce the fermentation broth to its pure final product. The extraction and purification of fermentation products may be difficult and costly. Recovery costs of microbial products varies from 15% to 70% of the total production cost.

31. Factors for deciding the extraction method The value of the final product. The degree of purity required. The chemical and physical properties of the product. The location of the product in the mixture i.e. whether it is free within the medium or is cell bound. The location and properties of the impurities. The cost-effectiveness of the available alternate purification procedures.

32. 7. Disposal of effluent/waste produced from the fermentation process Every fermentation process generates a residual waste material that leaves behind the desired pure product. Depending on the individual fermentation process, varying types and amounts of waste materials are produced. Typical wastes might include: unconsumed medium components, microbial cells and other suspended solids and waste wash water from cleansing operations. There should be a means or a system to manage these unwanted waste products. The system of managing such wastes is known as; industrial waste treatment/ effluent treatment system.

33. Historically, it was possible to directly dispose wastes into nearby open land or water bodies. This cheap and simple method of waste disposal is very rarely possible and environmentally undesirable. With increasing population density and industrial expansion together with the greater awareness of the damages caused by pollution, the need for treatment and controlled disposal of waste has grown and will continue to grow.

34. So, these days, fermentation industries are legally appreciative to have modern ways of waste disposal system. Sold waste: mostly beneficial, animal feed Liquid waste/effluent: disposed off after treatment. Who is responsible to control the proper way of industrial waste disposal? It varies from country to country. In India, the Ministry of Environment, Forest and Climate Change ( MoEFCC ) and the Central Pollution Control Board (CPCB) are primarily responsible for overseeing the proper disposal of industrial waste. They are supported by State Pollution Control Boards (SPCBs) in each state.

35. Pharmaceutical fermentation Pharmaceutical fermentation is a vital process for producing various medicines and related products. It uses microorganisms to convert organic materials into useful substances, including antibiotics, enzymes, therapeutic proteins, and even some vaccines

36. 1. Antibiotics Penicillin, streptomycin, and erythromycin are examples of antibiotics produced through fermentation. Other antibiotics like mupirocin, fusidic acid, and pleuromutilin are also produced via fermentation.

37. 2. Enzymes: Enzymes like thrombolytics, used to dissolve blood clots, are produced through fermentation. 3. Therapeutic Proteins: Insulin, human growth hormone (HGH), and various recombinant proteins are examples of therapeutic proteins synthesized via fermentation. 4. Vaccines: Some vaccines are also produced using fermentation techniques.

38. 5. Hormones: Fermentation can be used to produce various hormones used in medicine. 6. Immunosuppressants: Drugs like cyclosporin and tacrolimus, used to suppress the immune system, can be produced through fermentation. 7. Materials for Medical Devices: Fermentation is used to produce materials needed for diagnostic kits, drug delivery systems, and other medical devices.

39. 9. Vitamins: Some vitamins, like vitamin B12, can be produced through fermentation. 10. Steroid Hormones: Some steroid hormones, like corticosteroids, can be synthesized using fermentation and chemical modifications, according to the American Chemical Society

40. Benefits It's a cost-effective method for producing complex molecules. It allows for the production of molecules that are difficult or impossible to synthesize chemically. Fermentation is often used to produce active pharmaceutical ingredients (APIs).