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/ 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: 1. High volumetric productivity 2. Relatively high concentration of product 3. Less effluent generated 4. 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 m 3 (industrial applications)

View looking down into a 125m

3

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

 substrate requirement of cells nutritional, hormonal, and • In most cases, the medium is independent of the bioreactor design and process parameters •

Type of medium:

(mineral medium) complex and synthetic 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:

vitamins) (trace elements/ metals, •

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: 2. continuous: stirred tank chemostat, fluidized-bed 3. 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