Industrial fermentation

Batch Culture
The Batch Culture Growth Curve
Advantages of Batch Culture

• Simplicity of use.
• Operability and reliability
• production of secondary metabolites that are not growth-related
• fewer possibilities of contamination
• It is easy to assign a unique batch number to each run

Disadvantages of Batch Culture

• Culture ageing
• build up of toxic metabolites can restrict cell growth and product formation
• batch-to-batch variability
• the use of batch cultures in industrial systems can lead to an increased nonproductive
period due to down time required for cleaning, resterilisation, filling and cooling of
equipment
• cellular autolysis may occur during the decline and stationary phase, affecting the
amount of product
• initial substrate concentrations may have to be limited due to problems with inhibition
and repression effects, therefore affecting the amount of product that can be obtained
from such simple systems
2. Fed-batch Culture
3. Continuous Culture
Advantages of Continuous Culture
• Productivity and growth rate can be optimised by changing the feed flow rate during
Production

• Longer periods of productivity with less down time.

• It can take advantage of cell immobilization, which allows the maintenance of high cell
concentrations in the bioreactor at low substrate concentrations.

• The effects of environmental or physical factors are more easily analysed in a

continuous system, where any changes in the constant steady state are observed and
can be attributed solely to the change in those factors.

• Evolution in these cultures can be readily studied.

Disadvantages of Continuous Culture

• The US Food and Drug Administration (FDA) does not accept continuous culture in
the production of therapeutic products as a Current Good Manufacturing Practice
(cGMP).

• Not all products are produced optimally in continuous processes, e.g. some fermented
foods and beverages require cellular products released from different phases of batch
culture growth for full flavour development.
• Contamination can be a major problem in continuous cultivation, and can result in the
wash out of the desired organism and therefore a loss of product.

• Culture mutation can easily occur in continuous processes, often resulting in the
microorganism ‘shedding’ genes required for product formation, or in the case of
genetically engineered organisms, a ‘back mutation’ can occur, causing the slower-
growing recombinant population to wash out of the vessel.

For example, Clostridium acetobutylicum loses its ability to make acetone butanol in
continous culture and instead makes acetate and butyrate.
Solid-substrate fermentations
 SSF is successfully employed in the production of Coniothyrium minitans spores for
the biocontrol of the fungal plant pathogen, Sclerotinia sclerotiorum.

1 monocultures, as in mushroom production,

e.g. Agaricus bisporus;

2 dual cultures,
e.g. straw bioconversion using Chaetomium cellulilyticum and Candida tropicalis; and

3 mixed cultures, as used in composting and the preparation of silage, where the
microorganisms may be indigenous or added mixed starter cultures (inoculants).
Microbial growth kinetics
 For unicellular organisms such as bacteria, growth can be measured in terms of two
different parameters: changes in cell mass and changes in cell numbers.

Growth Kinetics and Specific Growth Rate

When bacteria are grown in a closed system (also called a batch culture) such as a
test tube, the population of cells almost always exhibits these growth dynamics:1. Lag phase,
2. Log phase, 3. Stationary phase, 4. Death phase.

Growth of cells in a microbial culture under a steady state or balanced growth can be
compared to a chemical reaction, in which the substrate is getting converted into products.

In a microbial culture under a steady state the substrate is the nutrients and the product is the
cell biomass. In such a reaction the rate of growth will be proportional to the cell biomass
present in the culture.

When the microbial culture is under a steady state the rate of increase in cell biomass
dX/dt is equal to the product of specific growth and cell concentration (biomass
concentration).
DX/dt = μX……………… (1)
where X is the cell concentration (gm/L) and μ is the specific growth rate (in hour–1).
The specific growth rate μ = dX/dt × 1/X, which is an index of rate of growth of cells in those
particular conditions.

It is possible to calculate the generation time or the doubling time of the organism or
bacterial cell, if we know the initial and final cell concentration of the culture and the specific
growth rate.

The cell biomass at the starting of exponential growth is X and after time, t, it
is 2X. Time required for doubling the biomass or the generation time of the cell can
be calculated by following the above equation (Equation No. 1).

ln 2X/t = μX
ln 2X/X = μt
ln 2 = μt
Therefore, t = ln 2/μ
t = 0.693/μ

Here t is the generation time or the doubling time of the cells. If the specific
growth ‘μ’ of the cells is calculated it can be substituted in the above equation to
determine the generation time or the doubling time. From this it is very clear that
the doubling time and specific growth rate are inversely related. As the doubling
time increases, the specific growth rate decreases.
The cell numbers of the microbial culture at different time intervals can be counted
and this data can be used for calculating the specific growth rate as follows:
ln 2X/X = μt or ln 2X – X = μ (t – t0)
on converting natural logarithm to logarithm to the base 10
log10 2X – log10 X = μ/2.303 (t – t0)

2X and X represent the amount of microbial cells at time t and t0.

Suppose if the culture medium contains 104 cells/ml at time t0, and after 4
hours (t) the number of cells is 108 cells/ml, then the specific growth rate of the
culture is
μ = (t – t0) 2.303/log10 (108 – 104) = (8 – 4) 2.303/4
= 4 × 2.303/4 = 2.303 hr–1
Thus, the specific growth rate is 2.303 hr–1
From equation 2, t = 0.693/μ
t = 0.693/2.303
t = 0.3 hrs.
Stoichiometry [Greek rsoijgeiom, element] is the quantitative relationship between
reactants and products in a chemical reaction. In microbiology, stoichiometry stands for
a quantitative relationship between substrates and products of microbial processes,
including biomass formation

kinetic and stoichiometry are tightly linked to each other, but stoichiometry mainly
addresses problems of a static nature (how much? in what proportion?), whereas
kinetics considers the dynamics questions (at what rate? by which mechanism?).

Mass-balance equations
The universal principle (Lavoisier principle) that matter cannot be created or destroyed
(unless there is a nuclear reaction) holds in biochemical systems. In any closed system
the total mass of every element, C, N, O, H, P, etc, is constant over time.
Measurement of Bioreactor
KLa
Most industrial microbial processes are aerobic, and are mostly carried out in
aqueous medium containing salts and organic substances; usually these broths are
viscous, showing a non-Newtonian behavior. In these processes, oxygen is an
important nutrient that is used by microorganisms for growth, maintenance and
metabolite production, and scarcity of oxygen affects the process performance.
Therefore, it is important to ensure an adequate delivery of oxygen from a gas
stream to the culture broth. Consequently, accurate estimation of the oxygen
transfer rate (OTR) at different scales and under different operational conditions has
a relevant role for the prediction of the metabolic pathway for both growth and
production of any wished metabolite in the aerobic cultures it is of critical
importance for the selection, design and scale-up of bioreactors.

The bioprocesses are usually conducted under previously optimized conditions

(temperature, pH, pressure, mixing, concentrations of biomass and nutrients), with
an operational mode previously chosen (batch, fed-batch, resting cell, continuous).
The overall mass transfer rate is not easy to measure, because different phenomena
are simultaneously taking place; also the relative importance of these phenomena
changes with the scale, the type of bioreactor, etc. Therefore, the OTR is influenced
by a high number of parameters (physical properties of gas and liquid, operational
conditions, geometrical parameters of the bioreactor) and also by the presence of
biomass, that is, the consumption of oxygen by the cells.
Oxygen transfer is complex, as it involves a phase change from its gaseous phase to the
liquid phase, and is influenced by the following factors.
1. the prevailing physical conditions; temperature, pressure and surface area of
air/oxygen bubbles;
2.the chemical composition of the medium;
3. the volume of gas introduced per unit reactor volume per unit time;
4. the type of sparger system used to introduce air into the fermenter;
5. the speed of agitation; or
6. a combination of these factors.
• C* (saturation oxygen concentration; max
solubility of the gas in liquid)
- Constant at a given T and P
- Available in tables (see on-line lab manual)

• CL (C(t)) the oxygen concentration at a given

time during the run; what we measure
- {C*- CL} = “driving force”
 In order for oxygen to transfer from the gaseous phase to an individual
cell or site of reaction, it must pass through several points of resistance

1 resistance within the gas film to the phase

boundary;
2 penetration of the phase boundary between
the gas bubble and bulk liquid;
3 transfer from the phase boundary to the
bulk liquid;
4 movement within the liquid;
5 transfer to the surface of the cell;
6 entry into cell; and
7 transport to the site of reaction within the
cell.
 Types of Bioreactors (fermenter)
(often depends on shear sensitivity)
• Stirred tank
– Aerobic or Anaerobic (air-sparged if aerobic)
– Most common for bacterial cells
• Bubble or airlift column
– Good for shear-sensitive cells
• Fixed bed systems
– Trickle beds, hollow membrane fiber (mammalian
cells), etc.
Industrial Stirred Fermenter
Transport in Bioprocess Systems
 Why is KLa Important?
• Dissolved oxygen is an important substrate in aerobic
fermentations. Since oxygen is sparingly soluble in water, it
may be the growth-limiting substrate in these fermentations.
For bacteria and yeast cultures, the critical oxygen
concentration is about 10% to 50% of the saturated DO
(dissolved oxygen concentration).

• kLa is defined as the volumetric mass-transfer coefficient that

describes the efficiency with which oxygen can be delivered to
a bioreactor for a given set of operating conditions.
 Equation for Transport
Oxygen transfer is usually limited by the liquid film surrounding the gas
bubbles:

mO2 k L a C *  C L 

where mO2 is the rate of oxygen transfer per volume of bioreactor (mass O2/ L3
t), kL is the oxygen transport coefficient, [=]L/t, a is the gas-liquid interfacial area
per volume of reactor [=] L2/L3, kLa is the volumetric oxygen transfer coefficient
[=]1/t, C* is saturated DO (dissolved oxygen) concentration [=] m/L3 (approx. 7
mg/l at 25 deg. C and 1 atm.), CL is the actual DO concentration in the liquid [=]
m/L3
Downstream processing
Decanter centrifuge
 Product recovery

1. Salting Out
2. liquid–liquid extraction
3. Aqueous two-phase separation
4. Chromatography
5. Dialysis and electrodialysis
6. Distillation
 Finishing steps

Crystallization
Drying
Industrial production of Citric acid
Citric acid is widely used in the food industry as an acidulant and flavouring agent in
beverages.

It has roles in maintaining metals in solution for electroplating, as a cleaning and

‘pickling’ agent for metals, and as a replacement for polyphosphates in the detergent
industry, along with several pharmaceutical uses.

Citric acid has become one of the world’s major fermentation products, with an
annual production of over 550 000 tonnes and a value approaching US$800 million.
The demand for citric acid is still increasing, particularly for beverage applications.
Industrial production of l-Glutamic acid
 l-Glutamic acid

Corynebacterium glutamicum,
Brevibacterium flavum
Cobalamin (vitamin B12)
The two-phase industrial production process employs the bacteria Propionibacterium
shermanii or Pseudomonas denitrificans.

B12 precursor, 5,6-dimethylbenzimidazole,

intermediate, cobinamide
Industrial production of Penicillin
Industrial production of Streptomycin
Streptomycin, an antibiotic produced by
Streptomyces griseus.

It is composed of three moieties:

streptidine, streptose, and N-methyl-L-
glucosamine, joined by glycosidic bonds.

Streptose, the central moiety, is a C-3-

formyl derivative of 5-deoxy-Llyxose.
include the following steps
[l] : (a) transfer of dihydrostreptose to streptidine 6-phosphate to form 0-a-L-
dihydrostreptose(1 -+ 4)streptidine 6-phosphate (Fig. 1)

[2]; (b) transfer of N-methyl-L-glucosamine to the pseudodisaccharide with the

formation of dihydrostreptomycin 6-phosphate ; (c) oxidation of
dihydrostreptomycin 6-phosphate to streptomycin 6-phosphate

[3] ; (d) hydrolysis of streptomycin 6-phosphate to streptomycin

Immobilization of enzymes and cells
Asp206, Glu230 and Asp297
participate in catalysis
Applications

Dr. J. Takamine established the first industrial production of a-amylase from A. oryzae
known as Taka diastase, which was used as a digestive aid.

The global market for enzymes was about $2 billion in 2004. The share of carbohydrases
comprising amylases, isomerases, pectinases and cellulases is about 40 %.

The food and beverage sectors utilize 90 % of the carbohydrases produced. The annual
sale of a-amylases in global market is estimated to be $11 million.

The world production of a-amylases from B. licheniformis and Aspergillus sp. was about
300 tonnes of pure enzyme protein per year.
Single cell protein production
BIO PLASTIC PRODUCTION
BIO PESTICIDE production
Spores and bipyramidal crystals of
Bacillus thuringiensis
Bio fuel production