Biotechnology: integration of different branches of science to deals with living benign

Fermentation: Microorganisms are cultured under controlled conditions in large vessel called
bioreactor to produced desired product
Type of Fermentation
Aerobic
Bioreactors supply with sterile air. And have a mechanism for mixing the medium and cells.
(Antibiotics, enzymes, vitamins.)
Anaerobic
Air or oxygen is usually not needed.( Lactic acid, ethanol, wine.)
Methods of Fermentation

Submerged fermentation (SmF)

 Utilizes liquid substrates.

 The bioactive compounds are secreted into the fermentation broth.
 suitable for microorganisms that need high moisture content
 The substrates are utilized and need constantly supplied with nutrients.
 Purification of products is easier.
 Used in the production of secondary metabolites which used in liquid form.

Solid-state fermentation (SSF)

 Utilizes solid substrates, like Starch, Cellulose, Lignin, and paper pulp.
 suitable for fungi that need limited moisture content

Factor Liquid substrate fermentation Solid substrate fermentation

Substrates Soluble Substrates (sugars) Insoluble Substrates: Starch ,Cellulose, Lignin
Aseptic conditions controled aseptic conditions non-sterile conditions
Water High volumes of water consumed Limited volumes of water consumed
Aeration High level of air required high surface exchange air/substrate
Agitation Need good mixing Static conditions
Scale up available Need special Equipment
Energy High energy consuming Low energy consuming
Volume High volumes and high cost Low volumes and low costs
 Modes of fermentation
Batch system
 Closed system where all nutrients are present at the start of the fermentation
 Fixed volume. The only further additions may be acids or bases for pH control, or gases
(aeration).
fed-batch system
 Semi-closed system, where fresh medium are fed continuously
 The volume of the batch increases with time.
Continuous system
 Open systems where fresh medium is continuously fed into the fermentation vessel
 The volume remains constant as spent medium & cells are removed at the same rate.

Batch Fed-batch Continuous

All the medium components are Nutrients are systematically added. Nutrients are continually added to the
placed in the reactor at the start of bioreactor and product is removed at
Fermentation. the same time.

Closed system. Semi-closed system. Open system.

Discontinuous process. semi-continuous process Continuous process.

Microorganisms go through four- Microorganisms go through four- Microorganisms are maintained at lag
phase(Lag , Log, Stationary, Death) phase (Lag, Log, Stationary, Death) and log phases through the process.

The volume remains constant The volume increases. The volume remains constant

Rate is low. Rate is medium. rate is high

no chance of contamination a chance of contamination There is a chance of contamination.

Less control on microbial growth and More control on microbial growth More control on the microbial growth
product. and product. and product.

Simple to run. difficult to run and maintain Difficult to run and maintain.

Product yield is low Product yield is high. Product yield is high

Used for the production of secondary Used for the production of alcohol Production of primary metabolites
products such as antibiotics. and proteins. such as amino acids and organic acids.

Downstream is easy. Downstream is easy. Downstream is complicated.

 Microbial Growth curve

Lag phase
 Adaptation of microorganisms with environment condition before start dividing
Exponential phase (log phase)
 Cells divide and there is a gradual increase in the population.
 The cells divide at a constant rate in
 When log number of cells is plotted against time there is a straight line.
Stationary phase
 Growth rate decreases due to decrease of nutrients or production of toxic products.
 Reproduction rate equals to death rate.
Death phase
 The depletion of nutrients and accumulation of products like acids
 Death rate more than Reproduction rate

Types of bioreactor

1.Continuous Stirred Bioreactors:( stirred tank reactors ) (STRs)

 Cylindrical vessel with central motor that supports mechanically moving agitators (impellers)
 The vessel have Baffles are usually flat, vertical, with width about 1/10 of the vessel diameter
 The filtrated air is added to the culture medium under pressure through a sparger.
 The sparger with impellers enables better gas distribution throughout the vessel.
 The bubbles generated by sparger are broken down to smaller ones by impellers and transfer
through the medium.
 The heat transfer through the outer jacket which surrounding the vessel
 The vessel have sensors (PH , temp. oxygen conc.) to monitoring and control
 2. Bubble Column Bioreactors:

The air is introduced at the base of the vessel through sparger causes a turbulence to enable an
optimum gas exchange .and good mixing

3. Airlift Bioreactors:

Liquid movement by the injection of compressed air at the bottom of the internal
riser column

And the air bubbles expand in the riser causing the upward movement of liquid within the
fermenter
 4. Fluidized Bed Bioreactors:

Suspended biocatalysts in a continuous contact with the reaction contents.

5. Packed Bed Bioreactors:

 Operation of Bioreactor

1. Sterilization
2. Inoculation and sampling
3. Aeration
4. Agitation
5. Control systems
6. Cleaning
Sterilization

 Bioreactor, tools, media and added air must be sterile to avoid any contamination
 Bioreactors sterilize by a method called In situ sterilization (sterilization by vapor)
 In sterilization process the volume of media must be not over 60% of the total vessel volume
to get the best heat distribution and the steam can reach to any point in media
 By increase the volume the time need to sterilization increased
 Some tools are sensitive to heat so that they sterilized by another method such as ethylene
oxide
 Some media ingredient may destroy by heat (sugar , urea , vitamins ) so that it sterilize before
add to bioreactor
 Air used in bioreactor sterilization by filtrated
 Material of bioreactor is Stainless steel 316 L or glass to be easy to sterilization

Inoculation and Sampling

The size of the inoculum is generally 1-10% of the total volume of the medium

Aeration
 Aerobic fermentation requires sterile air or oxygen
 Filtered air or oxygen usually enters the fermenter through a sparger system.
 The bioreactor has about 80% working volume.
 And about 20% of its volume as vaccum space
 The gases released during fermentation accumulate in the headspace then pass out through an
air outlet.

Agitation
 Good mixing (by agitation) produces one phase
  Creates optimal growth environment, and good product formation.
 preventing the accumulation of toxic metabolic byproducts .
 Increase the distribution of nutrients:
 produce homogeneous conditions
 Increase gas and heat transfer.
 Reduces O2 bubble size to increase the surface area of O2 transfer.
 Make Turbulence increase dissolved oxygen
 Suitable Agitation conditions during the fermentation is very important
 High Agitation: may damage sensitive cells, increase the temperature of the medium, and
increased foam formation
 Low Agitation: unwanted growth on surfaces (vessel walls & electrods)

Cleaning

Due to large size of the bioreactors, it is not possible to clean manually so that we use :

CIP cleaning in place

SIP sterilizing in place

Fermentation process factors

1. pH
 Most of the microorganisms grow between pH 5.5 and 8.5
 In the bioreactor, as the microorganisms grow, they release metabolites into the medium
which change pH.
 The pH of the medium should be continuously monitored and maintained at the optimum
level.
 all bioreactor having PH probe (Ph electrode ) to stabilized PH ratio

2. Temperature
 Heat generated in the fermentation due to metabolic activity of microorganisms and
mechanical agitation processes
 The reaction vessel must maintain at optimum temperature suitable to microorganisms
 To control it use a thermal jacket around bioreactor vessel
 3. Dissolved oxygen
 O2 is electron acceptor so it is the main factor in oxidation and reduction reaction
 O2 in air is 21% and the ratio of dissolved oxygen in water is 20%

That mean we have 4 DO2 molecule for 100 molecule of air pushed in water

 O2 dissolve in cold water more than hot water

When temperature Increase the gases accept energy and O2 become free and move up

 Microorganism classified into three main groups according to its need of oxygen:

Aerobic (required O2 to live)

Anaerobic (live without O2)

Obligate anaerobic (O2 killed it)

Facultative anaerobic (live with or without O2)

 Determine DO2 by using dissolved oxygen Electrode (ion selective electrode)

4. Viscosity
 Viscosity effect on temperature and dissolved oxygen
 calculate the biomass by turbidity

turbidity: is reflected light emission

Optical density: is the absorbed light

5. pressure
 Effect on gases dissolved and temperature
 By increasing pressure temperature and dissolved oxygen increased
 we increase or decrease pressure by air or pure O2
6. Nutrient concentration
 Added nutrient during fermentation occur systematically by using Peristaltic pump
 High nutrient concentrations inhibiting microbial growth.

7. Foam formation
 Antifoam chemicals are used to lower surface tension of the medium (silicone or vegetable
oils )
 mechanical foam breakers at the top of the bioreactor break the foam bubbles
 Fermentation broth
Fermentation broth contains all the necessary elements required for growth the microorganisms.

Generally, it contains a carbon source, nitrogen source, mineral source, precursors and
antifoaming agents

1. Carbon Source: carbohydrate ,glucose , molasses, or soy meal

Maltose : is (reducing sugar ) a combination of two molecules of glucose

Sucrose : is (non reducing sugar) a combination of glucose and fructose

Fructose : (non reducing sugar) ketone sugar end with CHO group start with OH group

Glucose (dextrose ): (reducing sugar ) start with CHO group

2. Nitrogen Source

Ammonium salts such as ammonium sulphate, ammonium acetate, ammonium lactate or ammonia

3. Mineral Source

Elements include phosphorus, sulphur, magnesium, zinc, iron, and copper which generally added in
the form of water soluble salts.

Molarity & Normality

Molarity : the number of moles present in the solution

Normality: the amount of gram equivalent of compound present in the solution

Chemical reaction and biochemical reaction

 Chemical reaction needs harsh condition more than biochemical reaction

 In Chemical reaction PH highly acidic or highly alkaline but in biochemical reaction PH is
slightly acidic or alkaline
 Yield in chemical reaction not pure
 Bio chemical reaction high specific more than Chemical reaction
 Bio chemical reaction Catalyzed by organisms which decrease the activation energy