Production Of Enzymes By Fermentation Method

What is fermentation? Pasteur’s definition: “life without air”, anaerobe redox reactions in organisms. New definition: a form of metabolism in which the end products could be further oxidized. For example: a yeast cell obtains 2 molecules of ATP per molecule of glucose when it ferments it to ethanol.

What is fermentation? Microorganisms, typically grown on a large scale, to produce valuable commercial products or to carry out important chemical transformations. This process is commonly referred to as Fermentation.

Types of fermentation process There are two methods of fermentation used to produce enzymes. submerged fermentation solid-state fermentation.

Submerged fermentation/ Solid-state fermentation Submerged fermentation involves the production of enzymes by microorganisms (e.g. bacteria, yeast) in a liquid nutrient media (water content of the media: > 95%) Solid-state fermentation is the cultivation of microorganisms on a solid substrate. Carbon containing compounds in or on the substrate are broken down by the micro organisms, which produce the enzymes either intra-cellularly or extra-cellularly.

Submerged fermentation/ Solid-state fermentation Cont…. The enzymes are recovered by methods such as centrifugation, for extra cellularly produced enzymes and lysing of cells for intracellular enzymes. Many industries are dependent on enzymes for the production of their goods. Industries that use enzymes generated by fermentation are the brewing, wine making, baking and cheese making.

Submerged Fermentation Cont… Advantages: Measure of process parameters is easier than with solid-state fermentation. Bacterial and yeast cells are evenly distributed throughout the medium. There is a high water content which is ideal for bacteria. Disadvantages: High costs due to the expensive media.

2. Solid State Fermentation Solid-state fermentation (SSF) is another method used for the production of enzymes. In solid state, water content is 40~ 80%. Solid-state fermentation involves the cultivation of microorganisms on a solid substrate, such as rice husk, wheat bran, sugar beet pulp, wheat and corn flour.

Solid State Fermentation Cont… Advantages: SSF has many advantages over submerged fermentation. These include: High volumetric productivity Relatively high concentration of product Less effluent generated Simple fermentation equipment.

Fermentation Aerobic Anaerobic

Aerobic fermentation Adequate aeration. Bioreactors- adequate supply of sterile air. In addition, these fermentors may have a mechanism for stirring and mixing of the medium and cells . eg. Antibiotics, enzymes, vitamins.

Anaerobic fermentation In anaerobic fermentation, a provision for aeration is usually not needed. e.g. Lactic acid, ethanol, wine When referring to fermentation regarding food, there are no distinctions between anaerobic and aerobic metabolism.

Cont…. Fermentation changes the characteristics of the food by the action of the enzymes produced by bacteria, mould and yeasts, which can occur in aerobic or anaerobic conditions

Cont…. The process of fermentation requires a food source (e.g. glucose); enzymes form bacteria or yeast and (depending on the product) anaerobic or aerobic conditions.

Cont.. Of all the microbial products manufactured commercially, antibiotics are the most important. Antibiotics are chemical substances produced by microorganisms to kill other microorganisms. They are used in the treatment of infectious diseases.

RANGES OF FERMENTATION PROCESS Microbial cell (Biomass) ….Yeast Microbial enzymes ….Glucose isomerase Microbial metabolites ….Penicillin Food products ….Cheese, yoghurt, vinegar Vitamins ….B12, riboflavin Transformation reactions ….Steroid biotransformation

Fermenter The heart of the fermentation process is the fermentor. In general: Stirred vessel, H/D  3 Volume 1-1000 m3 (80 % filled) Biomass up to 100 kg dry weight/m3 Product 10 mg/l –200 g/l

Types of fermentor Simple fermentors (batch and continuous) Fed batch fermentor Air-lift or bubble fermentor Cyclone column fermentor Tower fermentor Other more advanced systems, etc The size is few liters (laboratory use) - >500 m3 (industrial applications)

View looking down into a 125m3 stainless steel fermentor

Cross section of a fermenter for Penicillin production

Cross section of a fermenter for Penicillin production

Fermentation could be: Batch mode Fed batch mode (continuous)

Batch fermentation Most fermentations are batch processes Nutrients and the inoculum are added to the sterile fermentor and left to get on with it! Anti-foaming agent may be added. Once the desired amount of product is present in the fermentor the contents are drained off and the product is extracted. After emptying, the tank is cleaned & prepared for a new batch.

Continuous fermentation Some products are made by a continuous culture system. Sterile medium is added to the fermentation with a balancing withdrawal of broth for product extraction.

Fermentation Medium Definition of medium  nutritional, hormonal, and substrate requirement of cells In most cases, the medium is independent of the bioreactor design and process parameters Type of medium: complex and synthetic medium (mineral medium) Even small modifications in the medium could change cell line stability, product quality, yield, operational parameters, and downstream processing.

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

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)

Required value generation in fermentors as a function of size and productivity

Microbial rates of consumption or production C, N, P, S source H2O H+ biomass CO2 O2 product heat

Microbes

Overview of bioreactors for submerged system Classification: operation modes: 1. batch: stirred tank 2. continuous: chemostat, fluidized-bed 3. modified types of the above modes: fed-batch, chemostat with recycle, multi-stage continuous reactors Form of biocatalyst: free cell (enzyme) immobilized cell (enzyme) packed-bed, membrane reactor Oxygen supply: - aerobic: airlift - anaerobic

Industrial Bioreactor

World's Largest Industrial Fermenter (Chem. Eng. News,10-Apr-78) The fermenter is 200' high and 25 ft diam

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