Industrial Biotechnology

• Use of micro-organisms for large scale industrial processes

• Oldest form of microbiology and biotechnology which was used to make wine, beer, sake, bread with use of bacteria and yeasts without knowing scientific basis

• Production of ethanol, lactic acid, butanol using microbes and Enzymes like amylase, protease, invertase were used during early 20th century

• Pencillin was produced during WWII and other amino acids, nucleotides, enzymes were also produced later

OLD

• Now use in biotransformations of chemicals, genetic engineering of MO for non microbial products (insulin, interferons, HGH, vaccines)

• Microbial Fermentations (organic acids, amino acids, vitamins, antibiotics, enzymes) and fermented foods (dairy, meat, plant, breads, alcoholic beverages)

• Use in increase of crop productivity

• Biofertilizers and Biopesticides

• Use Microbes as food: single cell protein

• Bioenergy and Bioremediation

• Mining and metallurgy

MODERNWhite Biotechnology

Isolation and culturing Micro-organisms

Sources

• Soils, lakes, oceans, river, plant, animal, air, non living objects

Isolation methods

• Serial dilution, spread plate, gradient pour plate, streak plate

• Filtration, centrifugation• Importance of eliminating other organisms

(antibiotics, heating)

Isolation and culturing Micro-organisms

Growth Media

• MO require nutrients (C, N, P, Minerals), O2 requirement, temp, pH, salinity etc

• Synthetic media• Semi synthetic media• Natural media

• Media needs to be economical for large scale productions, consistent quality and available throughout. Raw material can be pre-treated if required

• Cheap C and N2 sources can be used

Isolation and culturing Micro-organisms

Sources of nutrition

Carbon: sugarcane molasses, beet molasses, vegetable oil, starch, cereal grains, whey, glucose, sucrose, lactose, malt, hydrocarbons

Nitrogen: corn steep liquor, slaughter house waste, urea, ammonium salts, nitrate, peanut granules, soyabean meal, yeast extract etc

Growth factors: vitamins and amino acids are added when MO cannot synthesize them

Isolation and culturing Micro-organisms

Sources of nutrition

• Trace elements: Zn, Mo, Mn, Cu, Co required for metabolism or in metallo-enzymes or in proteins (Hb)

• Inducers, precursors, repressors: for enzymes to function in metabolic processes inducers are required. Sometimes presence of presursors enhances production of a secondary metabolite or production an enzyme can be repressed due to repressors. Eg streptomycin is nduced by yeast extract, Sec metabolites can be repressed due to some cpds.

• Antifoams: sunflower oil, olive oil to prevent foaming

• Water: clean water of consistent composition, dissolved chemicals, pH is measured. Also required for cleaning, washing, rinsing, cooling, heating etc.

Culturing methods for Micro-organisms

Sterilization: devoid of MO (aseptic conditions)

Contamination free seed culture

Sterilization of equipment, media and air

Moist heat (121oC/15psi/20min), radiation, ultrasonic treatment,

chemicals, mechanical, gases (ozone), filtration for sterilizing air

CULTURING

Avoidance of contamination can be achieved by

• Use pure inoculum to start fermentation• Sterilize the media• Sterilize fermenter vessel• Sterilize all materials to be added to the fermentation during the process• Maintaining aseptic conditions during the fermentation

Control of environmental conditions for Microbial growth

TemperaturepHAgitationO2 conc

To be carefully monitored and maintained

Acidic pH: fungi and yeastPsychrophiles, acidophiles etc

Sterilization:

Elimination of threads and welding of pipes and tubes to reduce contamination

Fermenters have pipes which flush steam into the system

Media along with fermenter is sterilized

Among the several factors that influence killing are temperature, pH, osmotic pressure, shear, mass transport, and concentrations of extraneous substances that also react with the killing agent. These factors operate synergistically, and temperature plays roles other than simply affecting the kinetics of a reaction

Aeration and Mixing

Shake culture: flasks are kept on a shaker for required rotations

Fermenters: Stirrers for O2 mixing and baffles for increasing turbulence

V shaped notch

• Incrs turbulence• Incr eff of O2 transfer• Improves growth of MO

Fermentation

Any process for the production of useful products through mass culture of MOIn Microbiology

In Biochemistry

The numerous O-R reactions in which organic compounds used as carbon and energy act as acceptors and donors of H2 ion. The organic cpd gives rise to various products of fermentation which accumulate in the growth medium

Takes place in absence of O2

Now term industrial fermentation for large scale cultivation of micro-organisms…most of them is aerobic

Bioprocess technology (plants and animal cells) replaces fermentation technology (microbial use)………..not rigid

Fervere: to boil

Fermenter or bioreactor

• A biorector is a device in which the organisms are cultivated and motivated to form a desired product

• Closed vessel or containment designed to give a right environment for optimal growth and metabolic activity of the organism

• Fermenter: for microbes/ Bioreactor : for eukaryotic cells• Size variable ranging from 20-250 million litres or more.

• Large scale production (10-100L to1000-million L capacity)

• Helps to meet requirements of:pHtempaerationagitationdrain or overflowcontrol systemssensorscooling to achieve maximum microbial yield

What is fermentation technique (1)?

Techniques for large-scale production of microbial products. It must both provide an optimum environment for the microbial synthesis of the desired product and be economically feasible on a large scale. They can be divided into surface (emersion) and submersion techniques. The latter may be run in batch, fed batch, continuous reactors

In the surface techniques, the microorganisms are cultivated on the surface of a liquid or solid substrate. These techniques are very complicated and rarely used in industry

What is fermentation technique (2)?

In the submersion processes, the microorganisms grow in a liquid medium. Except in traditional beer and wine fermentation, the medium is held in fermenters and stirred to obtain a homogeneous distribution of cells and medium. Most processes are aerobic, and for these the medium must be vigorously aerated. All important industrial processes (production of biomass and protein, antibiotics, enzymes and sewage treatment) are carried out by submersion processes.

Some important fermentation products

Product Organism Use

Ethanol Saccharomyces cerevisiae

Industrial solvents, beverages

Glycerol Saccharomyces cerevisiae

Production of explosives

Lactic acid Lactobacillus bulgaricus

Food and pharmaceutical

Acetone and butanol

Clostridium acetobutylicum

Solvents

-amylase Bacillus subtilis Starch hydrolysis

16

Some important fermentation products

Some important fermentation products

Some important fermentation products

19

Winemaking fermenter

General Aspects of Fermentation Processes

Fermenter

The heart of the fermentation process is the fermenter.

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

Component parts of a fermenter

1. Formulation of media to be used in culturing the organism during development of inoculum and in the production fermenter

2. Sterilization of the medium, fermenter and ancillary equipment

3. Production of an active, pure culture in sufficient quantity to inoculate the production vessel

4. The growth of the organism in the production fermenter under optimum conditions for product formation

5. The extraction of the product and its purification

6. Disposal of effluents produced by the process

DOWNSTREAM PROCESSING

Culturefluid

Cellseparation

Biomass

Cell freesupernatant

Productextraction

Product purification

Product packaging

Effluent treatment

Productionfermenter

Medium raw material

Medium FORMULATION

Medium STERILIZATION

Stockculture

Shakeflask

Seedfermenter

Cross section of a fermenter for Penicillin production ( Copyright: http://web.ukonline.co.uk/webwise/spinneret/microbes/penici.htm)

 

25Cross section of a fermenter for Penicillin production ( Copyright: http://web.ukonline.co.uk/webwise/spinneret/microbes/penici.htm )

Flow sheet of a multipurpose fermenter and its auxiliary equipment

Basic modes of operations of a fermenter

1. Batch cultureBatch fermentation refers to • a partially closed system in which most of the materials

required are loaded onto the fermentor, decontaminated before the process starts and then, removed at the end.

• The only material added and removed during the course of a batch fermentation is the gas exchange and pH control solutions.

• In this mode of operation, conditions are continuously changing with time, and the fermentor is an unsteady-state system, although in a well-mixed reactor, conditions are supposed to be uniform throughout the reactor at any instant time.

The principal disadvantage of batch processing is the high proportion of unproductive time (down-time) between batches, comprising the charge and discharge of the fermenter vessel, the cleaning, sterilization and re-start process

Basic modes of operations of a fermenter

2. Continuous cultureContinuous culture is a technique involving feeding the microorganism used for the fermentation with fresh nutrients and, at the same time, removing spent medium plus cells from the system

A unique feature of the continuous culture is that a time-independent steady-state can be attained which enables one to determine the relations between microbial behavior (genetic and phenotypic expression) and the environmental conditions.

Basic modes of operations of a fermenter

3. Fed-batch processes

The fed-batch technique was originally devised by yeast producers in the early 1900s to regulate the growth in batch culture of Saccharomyces Yeast producers observed that in the presence of high concentrations of malt, a by-product - ethanol - was produced, while in low concentrations of malt, the yeast growth was restricted. The problem was then solved by a controlled feeding regime, so that yeast growth remained substrate limited.The concept was then extended to the production of other products, such as some enzymes, antibiotics, growth hormones, microbial cells, vitamins, amino acids and other organic acids.

Basically, cells are grown under a batch regime for some time, usually until close to the end of the exponential growth phase.

At this point, the reactor is fed with a solution of substrates, without the removal of culture fluid.

This feed should be balanced enough to keep the growth of the microorganisms at a desired specific growth rate and reducing simultaneously the production of by-products (that can be growth or product production inhibitory and make the system not as effective).

3. Fed-batch processes

By products may lead to cell death

A fed-batch is useful in achieving high concentration of products as a result of high concentration of cells for a relative large span of time.

Two cases can be considered: the production of a growth associated product and the production of a non-growth associated product. In the first case, it is desirable to extend the growth phase as much as possible, minimizing the changes in the fermenter as far as specific growth rate, production of the product of interest and avoiding the production of by-products.

For non-growth associated products, the fed-batch would be having two phases: a growth phase in which the cells are grown to the required concentration and then a production phase in which carbon source and other requirements for production are fed to the fermenter.This case is also of particular interest for recombinant inducible systems: the cells are grown to high concentrations and then induced to express the recombinant product

• Simple fermenters (batch and continuous)• Fed batch fermenter• Air-lift or bubble fermenter• Cyclone column fermenter• Tower fermenter• Fluidized bed bioreactors• Packed bed bioreactor• photobioreactor

• Other more advanced systems, etc

The size is few liters (laboratory use) - >500 m3 (industrial applications)

Types of Bioreactors