MIC 303 INDUSTRIAL AND ENVIRONMENTAL MICROBIOLOGY

1. MIC 303INDUSTRIAL AND ENVIRONMENTAL MICROBIOLOGY FERMENTATION TECHNOLOGY Mdm Aslizah Mohd Aris

2. STRUCTURE OF A BATCH BIOREACTOR

3. Fermenter • Are structures designed to optimize the growth conditions of the specific organisms that we want • Control oxygen, pH, medium, temperature and nutrients antifoaming • Stirred tank reactor • Air lift reaction

4. STRUCTURE OF A BATCH BIOREACTOR • Basic function of a fermenter: • to provide a controlled environment for the growth of microorganism or a defined mixture of microorganism to obtain desired product. • Two basic types of fermenter are used: • A glass vessel with round or flat bottom and a top flanged carrying plate. • A glass cylinder with stainless steel top and bottom plates. • The thickness of construction materials increase with scale. • It is important to select materials that can withstand repeated steam sterilization.

5. Factors to be considered: • Vessel should be capable of being operated aseptically- for a no of days/ long operation. • Adequate aeration and agitation- mixing should not cause damage to the organism. • Power consumption- should be low as possible. • Temperature controlled system. • pH controlled system. • Sampling facilities should be provided. • Evaporation losses from the fermenter should not be excessive. • Vessel should be designed to require the minimal use of labour in operation, harvesting, cleaning and maintenance. • The vessel should be suitable for a range of process.

6. The vessel should be constructed to ensure smooth internal surfaces, using welds instead of flange joints whenever possible. • The vessel should be of similar geometry to both smaller and larger vessels in the pilot plant or plant to facilitate scale up. • The cheapest materials which enable satisfactory results to be achieved should be used • Should be adequate service provisions for individual plants.

7. INDUSTRIAL FERMENTORS Side view

8. Top view

10. Fermentor sizes for Industrial Processes

11. Advantages of bioreactor over large flask containers Larger cultures volume can be grown. Ability to control substrate environment. Sterilization and cleaning are designed in place. Improved aeration and mixing characteristics result in improved cell growth and high final cell densities. A high degree of automation is possible. Process reproducibility is improved. It offers aseptic sampling and harvest systems for in-process sampling.

12. TYPICAL FERMENTATION PROCESS

13. STARTER CULTURES

14. STARTER CULTURE • Starter culture: Inoculum. • Isolation of Industrially Important Microorganisms • 1st Stage: screening of microorganism that are potential in industrial application • Purpose of isolation: to obtain either pure or mixed cultures that are able to carry out the desired reaction (organic acid transformation and biodegradable plastic) or produce the desired products (enzyme, antibiotic, amino acid etc) • Two main isolation methods: • Enrichment Liquid Culture • Solidified Media

15. Criteria of Inoculum Culture must be in a healthy active state. Must be available in sufficient large volume to provide optimum inoculum size (normally 10% from total volume). Suitable morphological form. Free from contamination. Retain it product-forming capabilities

16. Important Criteria of Inoculum for Fermentation Processes • Nutritional characteristics of the organism. • Very cheap medium • Pre-determined medium (ex: the use of methanol as an energy sources) • Optimum temperature of the organism • The use of an organism of optimum temperature above 40ºC reduces considerably the cooling cost of a large scale fermentation) • The reaction of organism with the equipment use and the suitability of the organism to the type of process to be used • If an airlift fermentor to be used, culture of microorganism with low viscosity should be used.

17. The stability of organism and its amendability to genetic manipulation. • The productivity of the organism. • Yield based (Yp/s) of the substrate consume (g product/substrate) and overall productivity (g/L product / h) • The ease of product recovery. • Contamination during fermentation should be taken into account • Extracellular/Intracellular- determine the type of product • Quality of the product • Ex: Glucoamylase produce by different microorganism produce different type of product

18. INOCULUM PREPARATION

19. Stage of Inoculum Preparation Depend on the scale of production fermenter.

20. MEDIA FORMULATION

21. Media for Industrial Fermentations • The media is the feed solution. • It must contain the essential nutrients needed for the microbe to grow. • Factors to be considered: • Quality consistence and availability. • Ensure there are no problems with media prep or other aspects of production process. • Ex: Sugar cane, molasses

22. MEDIA FORMULATION • Basic requirement of microorganism: • Water • Source of energy – carbon, nitrogen, mineral element, vitamin • Oxygen (if aerobes) • The constituents of medium must satisfy: • Cell biomass requirement. • Metabolite production. • Energy supply – biosynthesis , cell maintenance.

23. Criteria of Fermentation Medium Produce maximum yield of product or biomass per gram of substrate used. Produce maximum concentration of product / biomass. Permit max rate of product formation. Minimize yield of undesired products. Consistent quality and be readily available throughout the year. Minimal problems during media making and sterilization. Minimal problems in production process such as aeration, agitation, purification and waste treatment. Cheap substrate/ simpler procedure.

24. Composition of Fermentation Medium Carbon source Nitrogen source Mineral Element Chelators Growth Factors Buffers Precursors Inhibitors/ Inducers

25. Carbon source • Factors influencing the choice of carbon source: • The rate which carbon source is metabolized influence: • Formation of biomass. • Formation of primary metabolite. • Formation of secondary metabolite. • The main product of a fermentation process also determine the choice of carbon source. • The purity of carbon source – avoid undesirable product. Ex: metallic ion must be removed from carbohydrate source used in some citric acid process.

26. The method of media preparation particularly sterilization may affect the suitability of carbohydrates for individual fermentation process. • Ex: Sterilize sugar separately from nitrogen source + mineral- to avoid reaction of ammonium ions and amino acid to form black nitrogen (blackening agent) containing compounds which will partially inhibit the growth of microorganisms. • Government legislation. • Ex: European Economy Community (EEC): the use of beet sugar and molasses is encouraged and the minimum price controlled.

27. Examples of carbon source: • Carbohydrates: • Starch (maize, potato, cassava) • Barley grains • Sucrose (Sugar cane and sugar beet) • Molasses (Beet or cane molasses) • Lactose and crude lactose (Milk whey powder) • Corn steep liquor (by product after starch extraction from maize) • Oils and Fats • Oils: carriers for antifoams in antibiotic processes, contain fatty acids, oleic, linoleic and linolenic acid. • Ex: vegetables oils (olive, maize, cotton seed, tinseed, soya bean etc) • Hydrocarbon and derivatives. • Using n-alkanes for production of organic acid, amino acid, vitamin, protein etc.

28. Nitrogen source • Divided into: • Organic: amino acid, protein, urea • Inorganic: ammonia gas, ammonium salts or nitrates • Factors influencing the choice of nitrogen source: • Metabolic regulation. Ex: control mechanism of nitrate reductase enzyme involved in conversion of nitrate to ammonium ion, repressed by ammonia) • Type of product. Ex: Antibiotic production is influenced by the type of nitrogen source, may be inhibited by ammonium, nitrate or certain amino acid. • Pre-determined aspects of process. Ex: Limiting substrate.

29. Mineral Element • Microorganism require certain mineral element for: • Growth • Metabolism • Macro element: Magnesium, phosphorus, potassium, calcium and chlorine. • Essential component of media • Added as distinct component. • Trace element: Copper, iron, molybdenum and zinc. • Essential but usually present as impurities in other major ingredients.

30. Chelators Insoluble metal phosphate may be eliminated by incorporating low concentration of chelating agents such as EDTA (ethylene diamine tetra acetic acid), citric acid, polyphosphates. Form complexes with metal ions – may be gradually utilized by microorganisms

31. Growth Factors • Some microorganism cannot synthesize a full complement of cell component - thus required pre-formed compounds • Ex: Vitamin, specific amino acid, fatty acid, sterols. • Many natural carbon and nitrogen sources contain some required growth factors.

32. Buffers • Functions: • Agent for control pH • As nutrient source • Ex: Calcium carbonate (pH 7), Phosphate, balance of carbon and nitrogen source.

33. Precursors • Some chemical when added to certain fermentation are directly incorporated into the desired product. • Ex: Phenylacetic acid (precursor in Penicillin production).

34. Inhibitors • Functions: • Production of specific products and inhibit undesirable product. • Accumulation of metabolic intermediate. • Example:

35. Inducers • Majority enzymes which are of industrial interest are inducible. • Induced enzymes are synthesized only in response to the presence of an inducer, often substrate. • Ex: pulllulan for pullulanase, pectin for pectinase

36. MEDIUM STERILIZATION • Steam is used almost universally for the sterilization of fermentation media. • Problem occur to the medium in a result of heat treatment: • Interaction of the nutrient component in the medium. Ex: Mallard type browning reaction results in the lost of medium colour and nutrient. • Destruction of component that are heat sensitive ex vitamin and amino acid • Therefore, need to design sterilization based on: • Batch sterilization process • Continuous sterilization process * Involve in specific calculation, will not covered in this course

37. SCALLING UP VS SCALLING DOWN PROCESS

38. SCALLING UP • Scalling up fermentation process is carried out at the laboratory or even pilot plant scale fermentors to yield data that could be used to extrapolate and build the large scale industrial fermentors with sufficient confidence it will function properly with all its behaviours anticipated. • Increasing the scale of fermenter. • Increase in volume • Problem of process scale up are due to different ways in which process parameters are affected by the size of the unit. • Ex: Laboratory scale pilot scale production scale

39. Upstream Processing Equipment Lab-Scale Bioreactor 3 liters Large-Scale Bioreactor 25,000 liters

40. SCALLING DOWN • Scalling down fermentation process is carried out studies on smaller bioreactors in order to gain data and confidence and predict the behaviour how things actually will behave in large production fermentor. • Scalling down fermentation process also will be performed to troubleshoot or trying to optimize the industrial scale fermentation. • This method is called the fermentation monitoring experiment (mimic the industrial conditions). • This approach important in the development of: • New product. • Improvement of an existing full-scale fermentation.

41. Downstream Processing Equipment Lab Scale Chromatography System Large Scale Chromatography System