Fermentation Technology PDF

FERMENTATION TECHNOLOGY BY DR. ADEBAYO ISMAIL ABIOLA (PhD USM Penang) INTRODUCTION  Fermentation is a chemical process where by organic macromolecules such as glucose are broken down to give a desired end products. For examples, antibiotics, wine, yoghurt, and beer are produ...

FERMENTATION TECHNOLOGY BY DR. ADEBAYO ISMAIL ABIOLA (PhD USM Penang) INTRODUCTION  Fermentation is a chemical process where by organic macromolecules such as glucose are broken down to give a desired end products. For examples, antibiotics, wine, yoghurt, and beer are produced by fermentation process. A fermenter is needed for fermentation to take place industrially. The main function of a fermenter is to provide a suitable environment for microorganisms to yield or produce the desired products metabolites and cell biomass.  The performance of any fermenter depends on many physical and chemical factors. Laboratory fermentation can be performed in bottles or conical flasks, which are usually shaking for aeration. These vessels are usually cover with cotton wool to prevent contamination However, this can lead to restriction in exchange of gases and evaporation loss. Hence, vessels specifically constructed for fermentation are usually preferred. In industries, fermenters with capacities to contain several thousands of litters are used. INDUSTRIALLY IMPORTANT MICROORGANISMS The microorganisms that are used in industry for the production of certain useful and important products have some characteristics which are essential for them to be used in the industry. The characteristics are: 1. They grow vigorously and rapidly in the medium. 2. they must not produce toxic end-products or undesirable materials, especially when the products are meant for human consumption. 3. they must have significant physiological and genetic stability. 4. they should be able to use carbon sources that are cheap and readily available for growth. 5. it is desirous when the microorganisms have their optimum productivity at low pH and high temperature environment where there is less competition for growth medium. 6. the organisms should be able to produce agents that are inhibitory to competitors. 7. the organisms should be resistant to predators such as bacteriophages. Examples of some microorganisms that are used in Industry microorganisms Products Uses Antibiotic productions Penicillium chrysogenum, Penicillin Anti-fungal Penicillium griseofulvum Bacillus polymyxa Polymyxin B Anti-Gram-negative bacterial Streptomyces erythreus Erythromycin Anti-Gram-positive bacterial Streptomyces fradiae Neomycin Broad spectrum antimicrobial Streptomyces griseus Streptomycin Anti-Gram-negative bacterial Streptomyces rimosus Tetracycline Broad spectrum antimicrobial Streptomyces mediterranei Rifamycin Anti-tuberculosis Organic Acids production Microorganisms Organic acids Lactobacillus delbrueckii Lactic acid Acetobacter with ethanol solution Acetic acid Rhizopus nigricans in sugar-based Fumaric acid medium Aspergillus niger in molasses-based Citric acid medium Aspergillus niger in glucose-mineral salts Gluconic acid medium Biosurfactant Production Biosurfactants are agents or chemicals that are used for emulsification, solubilization and phase dispersion of solutions. They are used in food, beverages and soap industries. These biosurfactants are produced by bacteria, yeasts and fungi extracellularly and they form parts of their cell membrane where they perform vital functions. Examples of biosurfactants are fatty acids, phospholipids, glycolipids, and lipopeptides. Below are examples of biosurfactants that are produced by microorganisms: Microorganisms Surfactant produced Candida bombicola, Candida apicola Sophorolipids Pseudomonas aeruginosa Rhamnolipids Bacillus subtilis Surfactin Acinetobacter calcoaceticus Emulsan Other microorganisms that are used in Industry Microorganisms Uses Saccharomyces cerevisiae (yeast) Production of bread, beer, cheese, wine, spirits and other diary products Acetobacter species Production of vinegar Brevibacterium lactofermentum Production of lysine (amino acid) Bacillus subtilis Production of α-amylase Pseudomonas denitrificans Production of Vitamin B12 (cyanocobalamin) Salmonella typhi Production of vacine Isolation and screening of industrially important microorganisms from the environment The microorganisms that are industrially important are usually isolated by two methods: the ‘shotgun method’ and the ‘objective method. In the shotgun method, microorganisms are isolated from samples from biofilms, free living organisms and other microflora communities in plants and animals, water, sewage, soil, natural and man-made habitats and environments. In the objective approach, microorganisms are isolated from a specific and definite sites where they are considered to be the likely natural habitat of the target microorganisms. For example, samples can be taken from oil spillage environment or field to search for and isolate microorganisms that can biodegrade the oil and its by products. Once the microorganisms have been purely isolated by repressing the growth or killing the common microorganisms to allow the uncommon and target microorganisms to thrive, the microorganisms should be screened. The microbial isolates will be primarily screened for certain desired properties which is the production of the specific products such as antibiotics, inhibitory compound, enzymes etc. There will also be secondary screening for the microorganisms which will include the screening for the stability and non-toxicity of the microorganisms and their end-products. Preservation of cultures The cultures of industrially important microorganisms are generally preserved in culture collections. These culture collections housed microorganisms that are of past, present, and future interest. In the UK, the most common culture collection is the Nation Culture Collection (UKNCC) and in the US, the most common culture collection is the American Type Culture Collection (ATCC). These two collections hold and preserved all type of known microorganisms and they could supply any research institution the microorganisms if needed. The common methods of culture preservation are the cryopreservation, freeze drying, and a more convenient and better method which involves adsorption of cells in to glass beads. Cryopreservation involves the preservation of the microorganism in cryovial in a frozen condition. Freeze drying involves the act of freezing the microorganisms into dryness such that their biochemical activities are inactivated. The adsorption method involves the adsorption of microorganisms in to beads in a glass such that when the microorganisms are needed, 1-2 beads could be taking for thawing rather than thawing the whole sample. Strain improvement To maximise the production of desirable end-product by the microorganisms, the essential characteristics of the industrial microorganisms earlier stated could be enhanced or improved. The major approach employed is the use genetic manipulations which are the natural DNA recombination, mutagenesis, and genetic engineering. DNA recombination Mutagenesis Genetic engineering Media formulation Carbon source Carbon is an essential element for biosynthetic reactions that lead to reproduction, cell maintenance, and product formation. In fermentation, it is the primary energy source. The carbon requirements for growth and production of desired end products vary from one microorganisms to another. Traditionally, carbohydrate is the energy and carbon source for microbial fermentation, however, other sources such as alkanes, alcohol, organic acids, plant oils and animal fat could be used as the main carbon source or supplements. Molasses are preferred to pure glucose and sucrose as carbon source in fermentation industry because the two latter carbohydrates are relatively expensive. Molasses is a by-product of beet and cane sugar production which is cheaper. It is a viscous syrup with dark colour. It has about 60 % (w/w) carbohydrates which are majorly sucrose, 2 % (w/w) nitrogenous substances, and the remaining composition are vitamins and minerals. Another similar carbon source is hydro molasses, which is the by-product of maize starch processing and it primarily contains glucose. Yeast, filamentous fungi and actinomycetes are usually cultured in an industrial scale using malt extract as carbon source. The malt extract is a form of syrup that are made from aqueous extract of malted barley. The malt extract composed of 90 % carbohydrates, of which 55 % are disaccharides (maltose and sucrose), 20 % are hexoses (glucose and fructose), and 10 % are trisaccharide (maltotriose). The malt extract also contains amino acids, peptides, proteins, nitrogenous substances and vitamins. It should be noted that malt extract must not be over-heated during sterilization as it could results to loss of fermentable products, inhibition of microbial growth and change in colour of growth medium. Filamentous fungi that are amylase-producing microorganisms can directly metabolize polysaccharides such as starch and dextrins to produce a mixture of monosaccharides and disaccharides (maltose, glucose and maltotriose) which can then be used as carbon sources. Sulphite waste liquor which has hexoses and pentoses are also used for the yeast cultivation in industry. In solid-state fermentation, cellulose are used as substrate for mushrooms production. Cellulose is found in plant cell wall and it is usually gotten from industrial, agricultural, forestry and domestic waste. Lactose can also be sourced for from whey. Whey is the by-products of diary industry. However, fewer microorganisms such as Saccharomyces cerevisiae can use lactose as carbon source. Long chained Alkanes and methanol, ethanol are readily metabolized by certain microorganisms as carbon sources, however, they are very expensive and not cost effective for industrial scale production. Ethanol can be bio-transformed by Lactic acid bacteria to acetic acid and the method is still in use in the industry. Fat and Oils In antibiotic productions, plant oils from maize, linseed, cotton seed, rape seed, palm and soya, and also fish oil could be used as primary or supplementary source of carbon. The oils contains oleic and linoleic acids and they have more energy per weight than carbohydrates. Nitrogen sources Both organic and inorganic nitrogen sources are used by industrial microbes for getting nitrogen. Nitrogen sources that are used in industry are usually supplied in crude forms, and they are by-products of other industries. The nitrogen sources are yeast extracts, soya meal, corn steep liquor and peptones. Purified amino acids are only used in special, exceptional situation as precursors for specific products. Water All fermentation processes with the exception of the solid-substrate fermentations, need a large quantity of water. Sometimes, trace elements are supplied into the growth media through the water. Prior to use, colloids, suspended solid materials and microorganisms must be removed from the water. If the water is hard, salts such as calcium carbonated should be removed. The water must be well-treated such that it meets the standards to be used in the fermentation process. Other substances and chemicals needed in the media includes minerals, vitamins, growth factors, inducers and elicitors, inhibitors, oxygen and cell permeability modifiers etc. Fermentation and its types In the context of industrial microbiology, fermentation is the process of growing large quantities of cells under aerobic or anaerobic conditions, within a bioreactor or fermenter. Fermentation can be aseptic or non- aseptic. The aseptic and non-aseptic fermentations can also be aerobic and non-aerobic. An example of aseptic, aerobic fermentation is the antibiotic production by microorganisms while the production of lactic acid by lactic acid bacteria is an aseptic, anaerobic fermentation process. Mushroom production is a non-aseptic, aerobic fermentation process and microbial diary fermentation is a non-aseptic, anaerobic fermentation process. The two major types of fermentations are the solid- substrate fermentation and submerged fermentation Solid state fermentation Solid state fermentation is a fermentation process that involves growing of microorganisms on solid materials, which are normally organic but could be inorganic, in the presence of little or no free water. It is a common method that is used for fermentation of foods in Asian countries. The substrates that are commonly used for this type of fermentations are legumes, bran, cereal grains and lignocellulosic materials such as straw and wood. This type of fermentation can be used for the production of cheese and mushrooms. Ethanol, enzymes and organic acids can also be produced by solid-substrate fermentation process. Even though, solid-substrate fermentation has its advantages and disadvantages, in certain cases, the method is inevitable. For instance, in the production where fungi sporulation is required, solid- substrate fermentation must be used because fungi can not form spores in submerged fermentation. The method is employed in the production of Coniothyrium minitans spores for the biocontrol of Sclerotinia sclerotiorum (a fungal plant pathogen).  Advantages and disadvantages of solid-substrate fermentation Advantages Disadvantages Cheap media usage Bacterial contamination Simple technology Difficulty in controlling substrate moisture level It has the ability to provides better Problems with heat build-up productivity Low-capital cost Difficulty often encountered on a scale- up Relatively low energy requirements Low waste-water output Absence of foaming problem Step-by-step process of solid-substrate fermentation Solid-substrate fermentation is usually performed in a sequential multistep processes, which are: 1.Pretreatment of a substrate by chemical, biological or mechanical processing 2. Hydrolysis of primarily polymeric substrates. E.g. proteins and polysaccharides 3. Utilization of the hydrolysis products 4. separation and purification of end-products. Environmental parameters that influence solid-substrate fermentation The environmental parameters that influence or affect solid-substrate fermentation are water activity, temperature, and aeration. Water is lost during fermentation through metabolic activity and evaporation. This water is usually replaced by addition of water or humidification. If excess water is added such that the moisture level is too high, the porosity of the substrate will be reduced, the diffusion rate of the oxygen will be lowered, rate of gaseous exchange will also be reduced, the rate of substrate degradation will be decreased, and there will be high risk of microbial contamination. On the other hand, if the moisture level is too low, the substrate does not swell and becomes less accessible, thus, it will results to reduction of microbial growth. Temperature level in solid-substrate fermentation is largely controlled by substrate agitation and aeration. Heat generated during the process can be more problematic and it has a major influence on the humidity within the fermentation. Aeration: most solid-substrate fermentations are aerobic but the oxygen requirements depends on the specific process and microorganisms used. The rate of oxygen transfer is determined by the size of substrate particles. Bioreactors used for solid-substrate fermentations The solid-substrate fermentations are usually batch processes, they are not continuous. Some processes do not require a bioreactor, in such processes, substrates are simply spread onto a suitable surface or floor. The bioreactors that are used in the solid state fermentation include: rotating drum fermenter, bed systems, tray fermenter, fluidized bed reactor, and column bioreactor. Rotating drum fermenter It comprises of a 100L capacity cylindrical vessel. Attached to the sides of vessel are two rollers that support and rotate the vessel. The fermenter is used in microorganisms biomass and enzyme production. The major demerit is that the drum can only be filled to 30 % capacity for efficient agitation. (the figure below was adopted from Waites et al., 2001) Tray fermenter Tray fermenter is used for the production of enzymes and fermented oriental foods. The substrates are spread onto each tray and the trays are stacked in aerated chamber that allows the circulation of humidified air. (the figure below was adopted from Waites et al., 2001) Bed system This type of fermenter is used in koji production. It consists a 1 m deep bed of substrate. Humidified air is continuously forced into the system from below the system. (the figure below was adopted from Waites et al., 2001)  A Typical Bioreactor/ Fermenter (the figure below was adopted from Waites et al., 2001) Wine production The alcoholic fermentation of the sugars of juices of fruits especially grapes produce wine. This alcoholic fermentation depends on microorganisms to occur. The varieties of several flavours and aromas of wine that are available are as a result of microbial transformation and processing that occur during fermentation and ageing. Ethanol Production In 1970s, Brazil and other countries used to produce ethanol by employing Saccharomyces cerevisiae to ferment sucrose to ethanol. Other substrates that can be fermented to produce ethanol are the simple sugars from diary by-products and plants. Hydrolysed starch from rice, maize, wheat, potato and cassava can serve as simple sugar substrate sources for fermentation to produce ethanol. Substrates can also be derived from lignocellulosic materials of plants. In maize processing, milling processing are used to separate starch from corn. The separated starch undergoes gelatinization, then saccharification, whereby heat-resistant amylases such as glucoamylases were used to convert starch to simple fermentable sugars such as glucose. S. cerevisiae ferments the sugars at pH of 4.5- 5.0 and temperature of 32-38 ˚C. The Gram-negative bacterial species that belong to the genus Zymomonas like Zymomonas mobilis are alternative microorganisms that can be used to ferment simple sugars such as sucrose, fructose and glucose to produce ethanol. They generate higher ethanol yield than S. cerevisiae but they are less tolerant to ethanol. Downstream processing The downstream processing after fermentation involves 5 key steps: Release of intracellular products (if needed) Solid-liquid separation Concentration of the separated liquid Chromatographic purification Formulation Release of intracellular products (if needed) Some products such as enzymes and vitamins are located within the cells. They are intracellular products that must be released out of the cells into the solution before they are isolated. To release these products, the microbial cells or other cells must be disrupted or disintegrated by chemical, physical or enzymatic methods. The suitable method to be used for the cell disruption depends on the nature of the cells because each of the methods has its merits and demerits. Solid-liquid separation The step involves the separation of the solid, insoluble substances and ingredients, including the cell biomass from the liquid culture broth. The several methods employs in this process include; 1. Filtration 2. Centrifugation 3. Flocculation 4. Flotation  Concentration The desired products are minor constituents of the filtrate. Usually, over 80% of the filtrate is water, therefore, the water must be removed to get the desired products in concentrated form. The methods of concentration that are used in the process are: 1. Membrane filtration 2. Liquid-liquid extraction 3. Adsorption 4. Precipitation 5. Evaporation The factors that determine the adequate method to be selected for the concentration process are the qualitative and the quantitative nature of the desired products, and the cost of the method. Chromatographic separation Formulation Formulation basically means the maintenance of the activity, efficacy and stability of the products during distribution and storage. For instance, the formulation of citric acids and antibiotics can be achieved by crystallization using salt. Proteins are formulated by adding stabilizers such as sucrose, lactose, glycerol, and sodium chloride etc. Proteins can be formulated in form of solution or powder. Thank you for listening