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.

1. submerged fermentation

2. 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

• 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/ Solid-state fermentation Cont….

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:

1. High volumetric productivity2. Relatively high concentration of product3. Less effluent generated4. 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

product

heat

O2

Microbes

Overview of bioreactors for submerged system

- Classification:

operation modes:

1. batch: stirred tank2. continuous: chemostat, fluidized-bed3. modified types of the above modes: fed-batch, chemostat with

recycle, multi-stage continuous reactors

Oxygen supply:- aerobic: airlift- anaerobic

Form of biocatalyst: free cell (enzyme) immobilized cell (enzyme)packed-bed, membrane reactor

Industrial Bioreactor

World's Largest Industrial Fermenter (Chem. Eng. News,10-Apr-78)

The fermenter is 200' high and 25 ft diam

The End