Microbial and Industrial

Biochemistry
AIMMSCR, 4th Semester, B.Sc.(H) Biochemistry

Dr Sonia Kapoor
Steps In Designing a Fermentation Process

Microbial Strain selection

Fermentor selection
 Fermentation Media Selection
Fermentation process
 Upstream processing
 Downstream processing
Quality Control & Assurance
Product Recovery
Packaging
Steps In Designing a Fermentation Process

Microbial Strain selection

Fermentor selection
 Fermentation Media Selection
Fermentation process
 Upstream processing
 Downstream processing
Quality Control & Assurance
Product Recovery
Packaging
• Detailed investigation is needed to establish the most suitable
medium for an industrial fermentation process, but certain
basic requirements must be met by any such medium.

• Most fermentations require liquid media, often referred to as

broth; although some solid substrate fermentations (SSF)
are operated.

• All microorganism requires water, source of energy, C, N,

Mineral, vitamins, oxygen (aerobic).

• On a small scale it is easy to maintain but on large scale very

difficult to maintain the satisfactory growth.
Fermentation media must satisfy all the nutritional requirements of the
microorganism and fulfil the technical objectives of the process.

The nutrients should be formulated to promote the synthesis of the target

product, either cell biomass or a specific metabolite.

In most industrial fermentation processes there are several stages where

media are required. They may include several inoculum (starter culture)
propagation steps, pilot scale fermentations and the main production
fermentation. The technical objectives of inoculum propagation and the main
fermentation are often very different, which may be reflected in differences in
their media formulations. Where biomass or primary metabolites are the
target product, the objective is to provide a production medium that allows
optimal growth of the microorganism. For secondary metabolite production,
such as antibiotics, biosynthesis is not growth related. Therefore, media are
designed to provide an initial period of cell growth, followed by conditions
optimized for secondary metabolite production.
The main factors that affect the final choice of individual raw materials are as
follows:

1 Cost and availability: ideally, materials should be inexpensive and of consistent

quality and year round availability.

2 Ease of handling in solid or liquid forms, along with associated transport and storage
costs, e.g., requirements for temperature control.

3 Sterilization requirements and any potential denaturation problems.

4 Formulation, mixing, complexing and viscosity characteristics that may influence

agitation, aeration and foaming during fermentation and downstream processing stages.

5 The concentration of target product to be attained, its rate of formation and yield per
gram of substrate utilized.

6 The levels and range of impurities and the potential for generating further undesired
products during the process.

7 Overall health and safety implications.

On a large scale one must use nutrient sources to create a medium which will
meet as many of the following criteria:

1.Produce the maximum yield of product or biomass per gram substrate used.

2 Produce the maximum concentration of product or biomass.

3 Permit the maximum rate of product formation.

4 Minimum yield of undesired product.

5 Consistent in quality and readily available throughout the year.

6 Cause minimal problems during media making and sterilization.

7 Cause minimal problems during other aspects of the production process,

aeration and agitation, extraction, purification and waste treatment.
• The problem of developing a process from the laboratory to the pilot scale
& subsequently to the industrial scale must also be considered.

• Lab medium may not be ideal in a large fermenter with a low gas transfer
pattern.

• A medium with high viscosity will also need a higher power input for
effective stirring.

• pH variation, foam formation, oxidation reduction potential & the

morphological form of the organism, metabolic inhibitors – these are also
very important.

• Medium will also affect product recovery & effluent treatment.

• Historically – undefined complex natural materials used –
although they are cheaper but unpredictable biomass/yield
problems.
• Product recovery very difficult, residual components my
interfere with the recovery and contribute to the BOD of the
effluent.
• Although defined media is more expensive but it give more
predictable yield then undefined media.
• Control of pH and foam during growth in a fermenter were
indentified as two important parameters.
• Molasses would normally be used as the cheapest
carbohydrate to grow yeast biomass in a large scale process.
But this is not acceptable for Recombinant protein production
because of difficulties and cost for purification.
 Medium Formulation
• Medium formulation is an essential stage in the design of successful laboratory
experiments, pilot – scale development and manufacturing process.
• The initial step in media formulation is the examination of the overall process based
on the stoichiometry for growth and product formation. Thus for an aerobic
fermentation:

• The constituent of the medium must satisfy the elemental requirements for cell
biomass & metabolite production and there must be an adequate supply of energy
for biosynthesis.
• This equation should be expressed in quantitative terms, which is important in the
economical design of media if component wastage is to be minimal.

• Thus, to calculate the minimal quantities of nutrients which will be needed to

produce a specific amount of biomass, we can calculate substrate concentration
necessary to produce required product yields.
 Major medium components

 Carbon and Energy Source

 Nitrogen Source
 Water
 Minerals
 Growth factors
 Buffers
 Precursors, inducers and Inhibitors
 Oxygen
 Anti-foams
 Water
• Water: required in - Heating , cooling, cleaning & rinsing. It is required in
large quantities,

• pH, Dissolved salts and effluent contamination, Mineral content of water is

very important in brewing & most critical in the mashing process.
• Hard water containing CaSO4 concentration are better for the “English
Burton bitter beer” while water with high CO3 contents are better for the
darker beers i.e stouts.
• Nowadays, water may be treated by deionization, salt added or pH
adjusted, depend upon the type of breweries. Reuse or efficient use of
water is normally of high priority.
• SCP produced 60,000 tones by ICI, used huge amount of water on single
use bases, (30g biomass (dw) dm-3 would require 2700 x 10 6 dm3 of water
per annum.
• Eg. Methylophilus methlotrophus could be grown successfully with 86%
continuous recycling of supernatant with addition to make up depleted
nutrients. It results in reduced capital cost and operating cost by 50%.
 Energy Sources
• Energy comes either from oxidation of medium components
or from light.
• Most industrial microorganism are chemo-organotrophs,
therefore the commonest source of energy will be the carbon
(CHO), lipids & protein.
• Some micro-organisms can also use hydrocarbons or methanol
as carbon & energy sources.
 Carbon Sources
Factor influencing the choice of carbon source:
• Rate at which the C source is metabolized can often influence the formation of
biomass or production of primary or secondary metabolites. (Fast growth due to
high concentrations of rapidly metabolized sugars is often associated with low
productivity of secondary metabolites. Some use less readily metabolized sugar i.e
lactose but many process need continuous or semi continuous supply of sugar.)

Growth yield (Ycarbon) on minimal medium plus various carbon and energy sources
 Carbon Sources
Factor influencing the choice of carbon source:

• Main product of fermentation process depend on the choice of C source. Eg. Ethanol, or SCP -
60-77% of the production cost depend on cost of C source. it is the part of Company
development program – to determine the alternative C source to determine the yield.

• The purity of the C source may also affect the choice of substance.

• The method of media preparation, particularly sterilization may affect the suitability of CHO
for individual fermentation process. It is often best to sterilize sugar separately as they may
react with NH4 ions & AA to from black N containing compounds which will inhibit the
growth of many microorganism. Starch when sterilize it gelatinizes give rise to very viscous
liquid.

• The choice of substrate may also be influenced by Govt rules, eg. Beet sugar and molasses is
encouraged. Scotch malt whisky may be made only from barley malt, water and yeast.
 Carbon Sources

 Carbohydrates MOLASSES, MALT EXTRACT, STARCH AND

DEXTRINS, SULPHITE WASTE LIQUOR, CELLULOSE, WHEY

 Oils and fats: Hard animal fat, plant oils (primarily from cotton seed,
linseed, maize, olive, palm, rape seed and soya) and fish oil

 Hydrocarbons and their derivatives: ALKANES AND ALCOHOLS

 Carbohydrates
• Its common practice to use CHO in microbial fermentation e.g starch from maize
grain
• It may also be obtained from, others cereals, potatoes & cassava.
• Hydrolyzed cassava starch is used as a major C source for glutamic acid production.
• Syrups produced by acid hydrolysis may also contain toxic products which may
make them unsuitable for particular processes.
• Sucrose is obtained from sugar cane & sugar beet. It is commonly used in
fermentation media in a very impure form as beet or cane molasses which are the
residues left after crystallization of sugar soln in sugar refining.
• Molasses is used in the production of high volume/low value products such as
ethanol , SCP, organic and AA and some microbial gums.
• Molasses or sucrose also may be used for production of higher
value/low bulk products such as antibiotics, specially
enzymes, vaccines & fine chemicals.
• However, molasses based fermentation will often need a more
expensive & complicated extraction / purification procedure to
remove impurities & effluents which make the procedure
costly.
• Corn steep liquor is a by product after starch extraction from
maize. Although primarily used as a N source, it does contain
lactic acid, small amounts of reducing sugars & complex
polysaccharides. (Table 4.7)
• In Soyabean & pharmamedia, along with N, it also contain
small but significant amount of CHO.
 Oils and Fats
• Oils were first used as carriers for antifoams in antibiotics processes.
• Vegetables oils may also be used as C substrates, particularly for their content of
the fatty acid, oleic, linoleci and linolenic acid b/c of cost are competitive with
those of CHO.
• Bader 1984 – factors favouring the use of oils instead of CHO. A typical oil
contains approximately 2.4 times the energy of glucose on a per weight basis.
• Oils also have a volume advantage as it would take 1.24 dm 3 of soya bean oil to
add 10KCal energy to a fermenter whereas it would take 5dm 3 of glucose or
sucrose assuming that they are being added 50%w/w solutions.
• Ideally in any fermenter, max working capacity should be used.
• Oil based fed-batch fermenter permit this procedure to operate more successfully.
• Oil also has antifoam properties which may make downstream processing simpler.
• Glycerol trioleat is known to be used in some fermentation where substrate purity is
an important consideration.
• Methly oleate has been used as the sole C substrate in cephalosporin production
 Nitrogen sources
• Most industrially used microorganism can utilize inorganic or
organic sources of nitrogen.
• Inorganic N may be supplied as NH3 gas, NH4 salts or nitrates. NH3
has been used for pH control & as the major N source in a defined
medium for the commercial production of human serum albumin
by yeast.
• Organic N may be supplied as Amino Acids, protein or urea. Other
proteinaceous N compounds serving as sources of AA include corn-
steep liquor, soyasmeal, peanut meal, cotton seed.
• In storage these products may be affected by moisture, temp, &
ageing.
 Minerals
• All micro-organism requires
certain mineral elements for
growth & metabolism.

• In many media, Mg, P, K, S, Ca

& Cl are essential components,
Co, Cu, Zn, Fe, Mn, Mb & Zn
are also essential

• P is used as a Buffer to minimize •Ca salts is used to ppt the excess inorganic
pH changes when external pH is PO4 so indirectly improve the yield of
not being used. streptomycin.
•It also influence the production of bactricin,
citric acid, ergot, monomycin,novobiocin,
oxytetracycline, polyene, ristomycin,
rifamycin, streptomycin, vacomycin &
viomycin
• Liras 1990 – enzymes were repressed by PO4, a PO4 control sequence has
also been isolated & characterized from the PO4 regulated promoter that
control biosynthesis of candicidin.
• Wienberg 1970 – nine trace element proved to be critical, conc of Mn & Zn
are the most critical in secondary metabolism.
• Cl does not appear to play a nutritional rule in the metabolism of fungi but
however required by some of the halophilic bacteria. Eg. Chlortetracycline
& griseofulvin.
• Other Cl containing metabolites are caldriomycin, nornidulin & mollisin.
 Chelators
• Many media can’t be prepared or autoclaved without the
formation of a visible precipitate of insoluble metal PO4 .
• The problem of insoluble metal PO4 may be eliminated by
incorporating low concentration of chelating agents i.e EDTA,
citric acid,polyphosphate etc
• These chelating agents form complexes with the metal ions in
a medium. The metal ions then may be gradually utilized by
the micro-organism.
• Chelating agent should not cause inhibition of growth.
 Growth Factors

• Vitamins, specific AA, fatty acids or sterols.

• Many of the natural C & N sources used in media
formulation contain all or some of the required growth
factors.
• If only one vitamin is required it may be more economical
to add a pure vitamin instead of large bulk of cheaper
multiple vitamins source. E.g Ca pantothenate has been
used in one medium formulation for vinegar production.
• In glutamic acid, limited conc of biotin must be present in
the medium, some requires thiamin.
 Buffers
• The control of pH may be extremely important if optimal
productivity is to be achieved.
• Many media are buffered at about pH 7.0 by the incorporation
of CaCO3, if pH decreases the CO3 is decomposed.
• PO4 which are the part of many media also play an important
role in buffering. High PO4 conc are critical in the production
of many secondary metabolites.
• C & N sources will also a basis for pH control as buffering
capacity can be provided by the protein, peptides & AA such
as in corn steep liquor.
• The pH may also be controlled externally by addition of NH3
or NaOH & H2SO4.
 The addition of precursors & metabolic regulators to media
Some components of a fermentation medium help to regulate the
production of the product rather than support the growth of the
micro-organism. Such additives include, precursors, inhibitors, and
inducers

Precursors:
• Some chemicals when added to certain fermentation are
directly incorporated into the desired product. E.g
Penicillin yields
A range of different side chains can be incorporated into the penicillin
moleucle, eg corn steep liquor increased the yield of penicillin form 20
units to 100 units.
• Corn steep liquor contain phenylethlamine when incorporated, it yield
benzyl penicillin.
 Phenylacetic acid

PenG

Phenoxy
acetic acid

PenV
• Include phenylacetic acid or phenylacetamide
added as side-chain precursors in penicillin
production.
• D-threonine is used as a precursor in l-
isoleucine production by Serratia marsescens,
and
• anthranillic acid additions are made to
fermentations of the yeast Hansenula
anomala during l-tryptophan production
 Inhibitors
• When certain inhibitors are added to fermentations more of specific product
may be produced, or a metabolic intermediate which is normally metabolized
may get accumulated
• Glycerol production depends on modifying the ethanol fermentation by
removing acetaldehyde. (sodium bisulphite is added)

Inhibitors have also been used to affect cell wall structure and increase the permeability for
release of metabolites. The best example is the use of penicillin & surfactants in glutamic acid
production.
 Inducers
• The majority of enzymes which are industrial interest are
inducible.
• Induced enzymes are synthesized only in response to the presence
in the environment of an inducer.
• Inducers are often substrate such as starch or dextrins for amylase,
maltose for pullulanase & pectin for pectinase.
• Most inducers which are included in microbial enzyme media are
substrate or substrate analogues but intermediates and products
may sometime be used as inducers.
• E.g maltodextrins will induce amylase & fatty acids induce lipase.
(use depend upon the cost)
• It is now possible to produce a no of heterologous protein (HLP)
in yeasts, fungi & bacteria. But HLP may show some degree of
toxicity to the host, it may restrict the growth and biomass.
 Oxygen requirement
Very important in controlling growth rate & metabolic
production. Medium may influence:

Fast metabolism: culture may become oxygen limited because sufficient

oxygen can’t be made available in the fermeter if certain substrate such as
rapidly metabolized sugars which lead to a high oxygen demand are
available in high concentration.

Rheology: the individual components of the medium can influence the

viscosity of the final medium & its subsequent behavior with respect to
aeration & agitation.

Antifoam: Many of the antifoams in use will act as surface active agents &
reduce the oxygen transfer rate.
 Fast Metabolism
• Nutritional factors can alter the Oxygen demand of the culture.
• Penicillium chrysogenum will utilize glucose more rapidly than lactose or sucrose, &
it therefore has a higher specific O 2 uptake rate when glucose is the main C source.
• Therefore, when possibility of O 2 limitation due to fast metabolism, it may be
overcome by reducing the initial conc of key substrates in the medium and adding
additional quantities of these substrates as a continuous or semi continuous feed
during the fermentation.
• It can also be overcome by changing the composition of the medium, incorporating
higher CHO (lactose, starch) & protein which are not very rapidly metabolized & do
not support such a large specific O uptake
Rheology:
There can be considerable variation in the viscosity of compounds that may be included in
fermentation media, eg. Polymer in solution ie polysaccharides
As polysaccharides aredegraded, rhelogical properties will change.
Antifoam:
In most microbiological process, foaming is a problem. It may be due to a
component in the medium or some factor produced by the microorganism. E.g
most common due to protein i.e corn steep liquor, pharmamedia, peanut,
soybean, yeast extract or meat extract
These proteins may denature at the air broth interface & form a skin which does
not rupture readily.
• The foaming can cause removal of cells from the
medium which will lead to autolysis & the further
release of microbial cell proteins will probably
increase the stability of the foam.

• If uncontrolled, then numerous changes may

occur & physical & biological problems may be
created.

• These include reduction in the working volume of

the fermenter,
• lower mass & heat transfer rates,
• Invalid process data due to interference at sensing
electrode & incorrect monitoring & control.

• Biological problem inlcude deposition of cells in

upper parts of the fermenter, problems of sterile
operation as the air filter at exit of the fermenter
become wet, so danger of microbial infections, so
loss of product.
Approaches to take care of excessive foaming

1. Use defined medium

2. Use antifoams

3. Use a mechanical foam breaker

 Reference

Principles of fermentation Technology, Stanbury, Whitaker, Chapter 4