Fermentation KC

FERMENTATION TECHNOLOGY
Name of the Teacher: Ketaki Joshi Class: SYBT Subject: Biotechnology Paper: VI – Bioprocess technology
Bioprocessing
https://www.youtube.com/watch?v=SsozxmGX6cM
CAREER OPTIONS IN FERMENTATION TECHNOLOGY
• Students who are interested in conducting research in industrial fermentation can ﬁnd jobs in university, government,
and industry laboratories.
Following is the list of prospective job opportunities in this field:
• Quality Control/QC (the most common one)
• Analytical testing (a step ahead of QC)
• Production (the operation handler, working on big reactors, packaging units, and sterilizers)
• Research & Development ( many options: Therapeutic research, GMOs, Biopesticides, Biofertilizers, Flavor research
etc)
• Marketing
• process development
• technical services
• Some industrial fermentation specialists may be considered as
• • genetic engineers (using DNA techniques to modify living organisms)
• Bioprocess or chemical engineers (optimizing bioreactors and biochemical pathways for the desired product).
• When choosing a career in industrial fermentation, one should be prepared for an interdisciplinary science. Students
should obtain skills in microbiology, molecular biology; bioengineering, plant biology, organic chemistry, biochemistry,
agriculture, bioprocess engineering, and chemical engineering.
The industry works on 2 rules:
❑ Less cost, time, capital

❑ Maximum product
TOPICS BRIEFLY GROUPED AS:
• Fermentation requirements- Stanbury ed 3

• Media sterilization- Stanbury ed 3
• Design of a typical fermenter- Brock
• Types of fermenters
• Parameters at which fermenters are run
• Ethanol fermentation
• Penicillin fermentation
•
https://www.chhs.colostate.edu/fshn/programs-and-degrees/b-s-in-fermentation-science-and-t
echnology/
https://www.youtube.com/watch?v=JamupccmtjU
MEDIA FOR INDUSTRIAL FERMENTATIONS
• What basic requirements are present in the media?

• What you think might be present in fermentation media?
• The first step to consider is an equation based on the stoichiometry for growth and product formation. Thus for an aerobic
fermentation:
MICROBIAL MEDIA COMPO FERMENTATION MEDIA COMPO
WATER
Water is the major component of almost all fermentation media, and is needed in many of the ancillary services
such as heating, cooling, cleaning, and rinsing.
When assessing the suitability of a water supply it is important to consider pH, dissolved salts, and effluent
contamination. The mineral content of the water is very important in brewing, and most critical in the
mashing process, and historically influenced the siting of breweries and the types of beer produced.
Hard waters containing high CaSO4 concentrations are better for the English Burton bitter beers and
Pilsen type lagers, while waters with a high carbonate content are better for the darker beers such as stouts.
Laboratory tests to simulate the process showed that the Methylophilus methylotrophus could be grown
successfully with 86% continuous recycling of supernatant with additions to make up depleted nutrients. This
approach was therefore adopted in the full scale process to reduce capital and operating costs and it was estimated
that water used on a once through basis without any recycling would have increased water costs by 50% and
effluent treatment costs 10-fold
ENERGY SOURCES
• Most industrial microorganisms are chemoorganotrophs, therefore the commonest source of energy will be the carbon source
such as carbohydrates, lipids, and proteins.
CARBON SOURCES
• It is now recognized that the rate at which the carbon 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.
• Upto ten different carbon sources have been or are being used by Pfizer Ltd for an antibiotic production process depending on the
geographical location of the production site and prevailing economics. The purity of the carbon source may also affect the choice
of substrate.
• For example, metallic ions must be removed from carbohydrate sources used in some citric acid processes.
• There are similar laws applying to beer production in Germany. Scotch malt whisky may be made only from barley malt, water,
and yeast. Within France, many wines may be called by a certain name only if the producing vineyard is within a limited
geographical locality
The use of cane molasses, beet molasses, cereal grains, starch, glucose, sucrose, and lactose as carbon sources, and ammonium
salts, urea, nitrates, corn steep liquor, soya bean meal, slaughter-house waste, and fermentation residues as nitrogen sources, have
tended to meet most of the above criteria for production media because they are cheap substrates.
https://dir.indiamart.com/impcat/sugarcane-molasses.html
ANTIFOAMS
Pros
• Oils were first used as carriers for antifoams in antibiotic processes. Vegetable oils (olive, maize, cotton seed, linseed, soya bean, etc.)
may also be used as carbon substrates, particularly for their content of the fatty acids: oleic, linoleic, and linolenic acid, because costs
are competitive with those of carbohydrates.
• 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 dm3 of soya bean oil to add 10 kcal of energy to a fermenter, whereas it would take 5 dm3 of glucose or sucrose
assuming that they are being added as 50% w/w solutions.
• Oil based fed-batch fermentations permit this procedure to operate more successfully than those using carbohydrate feeds where a
larger spare capacity must be catered for to allow for responses to a sudden reduction in the residual nutrient level.
Cons
• However, residual (unutilized) oil can increase broth viscosity and hence decrease oxygen transfer efficiency and can create problems in
some downstream processing operations.
https://www.youtube.com/watch?v=2HILvF0YRw0
https://beermaverick.com/anti-foaming-agents-fermcap-s-five-star-defoamer-others/
BRIDGING-STRETCHING MECHANISM
• The stretching of this bridge in a radial direction

as a result of uncompensated capillary pressures
at the oil-water and gas-water interfaces led to
the eventual formation of a thin unstable oil film
in the bridge center.
• The rupture of this oil film resulted in the
perforation of the entire foam lamella. This
mode of foam film rupture was termed the
bridging stretching mechanism.
NITROGEN SOURCES
• Most industrially used microorganisms can utilize inorganic or organic sources of nitrogen.
• Inorganic nitrogen may be supplied as ammonia gas, ammonium salts, or nitrates (Hutner, 1972).
Discuss about buffers.
• Ammonia has been used for pH control and as the major nitrogen source in a defined medium for the commercial production
of human serum albumin by Saccharomyces cerivisiae (Collins, 1990). Ammonium salts such as ammonium sulfate will
usually produce acid conditions as the ammonium ion is utilized and the free acid will be liberated.
• On the other hand nitrates will normally cause an alkaline drift as they are metabolized. Ammonium nitrate will first cause
an acid drift as the ammonium ion is utilized, and nitrate assimilation is repressed.
• When the ammonium ion has been exhausted, there is an alkaline drift as the nitrate is used as an alternative nitrogen source
(Morton & MacMillan, 1954).
BUFFERS
Buffer, as we have defined, is a mixture of a conjugate acid-base

pair that can resist changes in pH when small volumes of strong
acids or bases are added.
When a strong base is added, the acid present in the buffer

neutralizes the hydroxide ions.
When a strong acid is added, the base present in the buffer

neutralizes the hydronium ions.
https://www.youtube.com/watch?v=8U5tP6GL9wM
• Organic nitrogen may be supplied as amino acid, protein or urea, or in a complex media as yeast extract.
• In many instances growth will be faster with a supply of organic nitrogen, and a few microorganisms have an absolute
requirement for amino acids. It might be thought that the main industrial need for pure amino acids would be in the deliberate
addition to amino acid requiring mutants used in amino acid production.
• However, amino acids are more commonly added as complex organic nitrogen sources, which are nonhomogeneous, cheaper,
and readily available.
• In lysine production, methionine and threonine are obtained from soybean hydrolysate since it would be too expensive to use
the pure amino acids (Nakayama, 1972a).
• Other proteinaceous nitrogen compounds serving as sources of amino acids include corn-steep liquor (see also carbon
sources), soya meal, peanut meal, cottonseed meal (Pharmamedia, Table 4.8; and Proflo), Distillers’ solubles meal, and yeast
extract.
• Analysis of many of these products which include amino acids, vitamins, and minerals are given by Miller and Churchill
(1986) and Atkinson and Mavituna (1991a). In storage these products may be affected by moisture, temperature changes, and
ageing.
Corn steep liquor(CSL) is a by-product of corn wet-milling. A viscous concentrate of corn solubles which contains amino
acids, vitamins and minerals, it is an important constituent of some growth media. It was used in the culturing of
Penicillium during research into penicillin by American microbiologist Andrew J. Moyer.
MINERALS
• All microorganisms require certain mineral elements for growth and metabolism (Hughes & Poole, 1989, 1991).
• In many media, magnesium, phosphorus, potassium, sulfur, calcium, and chlorine are essential components, and
because of the concentrations required, they must be added as distinct components. Others such as cobalt, copper,
iron, manganese, molybdenum, and zinc are also essential but are usually present as impurities in other major
ingredients.
• The concentration of phosphate in a medium, particularly laboratory media in shake flasks, is often much higher
than that of other mineral components. Part of this phosphate is being used as a buffer to minimize pH changes
when external control of the pH is not being used.
• In a review of antibiotic biosynthesis, Liras, Asturias, and Martin (1990) recognized target enzymes which were
(1) repressed by phosphate, (2) inhibited by phosphate, or (3) repression of an enzyme occurs but phosphate
repression is not clearly proved.
• A phosphate control sequence has also been isolated and characterized from the phosphate regulated promoter
that controls the biosynthesis of candicidin. Weinberg (1970) has reviewed the nine trace elements of biological
interest (Atomic numbers 23–30, 42).
• Of these nine, the concentrations of manganese, iron, and zinc are the most critical in secondary metabolism
• Chlorine does not appear to play a nutritional role in the metabolism of fungi (Foster, 1949). It is, however, required by
some of the halophilic bacteria (Larsen, 1962).
• Obviously, in those fermentations where a chlorine-containing metabolite is to be produced, the synthesis will have to be
directed to ensure that the nonchloro-derivative is not formed. T
• The most important compounds are chlortetracycline and griseofulvin. In griseofulvin production, adequate available
chloride is provided by the inclusion of at least 0.1% KCl (Rhodes et al., 1955), as well as the chloride provided by the
complex organic materials included as nitrogen sources.
• Other chlorine containing metabolites are caldriomycin, nornidulin, and mollisin.
CHELATORS
• Many media cannot be prepared or autoclaved without the formation of a visible precipitate of insoluble metal
phosphates. Gaunt, Trinci, and Lynch (1984) demonstrated that when the medium of Mandels and Weber (1969) was
autoclaved, a white precipitate of metal ions formed, containing all the iron and most of the calcium, manganese, and
zinc present in the medium.
• The problem of insoluble metal phosphate(s) may be eliminated by incorporating low concentrations of chelating agents
such as ethylene diamine tetraacetic acid ,EDTA , citric acid, polyphosphates, etc., into the medium.
• These chelating agents preferentially form complexes with the metal ions in a medium. The metal ions then may be
gradually utilized by the microorganism (Hughes & Poole, 1991).
• Gaunt et al. (1984) were able to show that the precipitate was eliminated from Mandels and Weber’s medium by the
addition of EDTA at 25 mg dm–3. It is important to check that a chelating agent does not cause inhibition of growth of
the microorganism which is being cultured.
GROWTH FACTORS
• Some microorganisms cannot synthesize a full complement of cell components and therefore require preformed
compounds called growth factors.
• The growth factors most commonly required are vitamins, but there may also be a need for specific amino acids, fatty
acids, or sterols. Many of the natural carbon and nitrogen sources used in media formulations contain all or some of
the required growth factors (Atkinson & Mavituna, 1991
• Calcium pantothenate has been used in one medium formulation for vinegar production (Beaman, 1967). In processes
used for the production of glutamic acid, limited concentrations of biotin must be present in the medium (Chapter 3
OXYGEN REQUIREMENTS
The medium may influence the oxygen availability in a number of ways including the following:
1. 1. Fast metabolism. The culture may become oxygen limited because sufficient oxygen cannot be
made available in the fermenter if certain substrates, such as rapidly metabolized sugars which lead to
a high oxygen demand, are available in high concentrations.
2. 2. Rheology. The individual components of the medium can influence the viscosity of the final
medium and its subsequent behavior with respect to aeration and agitation.
3. 3. Antifoams. Many of the antifoams in use will act as surface active agents and reduce the oxygen
transfer rate.
MEDIUM OPTIMIZATION
• Medium optimization by the classical method of changing one independent variable (nutrient, antifoam, pH, temperature,
etc.) while fixing all the others at a certain level can be extremely time consuming and expensive for a large number of
variables.
• To make a full factorial search which would examine each possible combination of independent variable at appropriate
levels could require a large number of experiments, x’, where x is the number of levels and n is the number of variables.
• Industrially the aim is to perform the minimum number of experiments to determine optimal conditions.
• Other alternative strategies must therefore be considered which allow more than one variable to be changed at a time.
• Upstream processes are those in which biological materials are either obtained from an outside source or inoculated
and grown in culture, under controlled conditions, to manufacture certain types of products.
• Downstream processes are those in which the products are harvested, tested, purified and packaged.
TYPES OF FERMENTATIONS
• batch and contionous sterilization/fermentation
• Log D value, log 50%
• Stanbury, pg 329
Name of the Teacher: Ketaki Joshi Class: SYBT Subject: Biotechnology Paper: VI – Bioprocess
technology
RESTART THE UNIT
• https://www.youtube.com/watch?v=xDxCk2lfn3U
•
https://www.chhs.colostate.edu/fshn/programs-and-degrees/b-s-in-fermentation-science-and-tech
nology/
DESIGN OF A TYPICAL FERMENTER
TYPES OF FERMENTATION TECHNIQUES
TYPES OF CONTINOUS FERMENTERS
http://www.davidmoore.org.uk/
21st_century_guidebook_to_fu
ngi_platinum/Ch17_11.htm
WHAT IS FERMENTATION ?
https://socratic.org/questions/how-do-fermentation-and-anaerobic-respiration-differ
• https://www.youtube.com/watch?v=6-D1oes63_U
• https://www.youtube.com/watch?v=FYClCHVT00M
Fedback, feedback, steady state, turbidostat, chemostat, surface fermentation,
Nduka odafur-pg 192 solid state fermentations
technology
SUBMERGED FERMENTER
• Fermentations may be liquid, also known as submerged, or solid state, also known as surface. Most fermentations used in the
industry are submerged processes, because the fermentors used in submerged fermentation save space and are more amenable to
engineering control and design.
• Most enzyme production is carried out in deep submerged fermentation; a few are best produced in semi-solid media.
technology
SOLID STATE FERMENTER
Pretreatment using acid hydrolysis: H2SO4 and amylolytic enzymes.
Penicillin fermentation- Alexander Fleming
Also used in world war 2

In this article we will discuss about:-
1. Introduction to Penicillin 2. Biosynthesis of Penicillin 3. Structure 4. Fermentation Process 5. Uses.
Chemically the natural penicillin is 6-amino penicillanic acid (6 – APA), which consists of thiazolidine ring with a condensed β-lactum ring. The
various penicillins differ primarily in the nature of R-side chain which are attached by an amido linkage to the chemical nucleus of the molecule.
Fleming’s original Penicillium notatum strain, when grown on his medium produced penicillin-F, which is known as 2-pentinyl penicillin.
Subsequently P. chrysogenum proved to be better fungus and more suitable for submerged fermentation.
So we have the classification of penicillin, we have natural or biosynthetic way. Penicillin that is harvested from the mould through the fermentation
we considered as a natural or biosynthetic penicillin-V, penicillin-G. Semi-synthetic derived from the penicillin because we add some kind of we
make some kind of chemical alteration here to get the ampicillin, oxacillin, carbenicillin and methicillin etcetera, resistant to stomach acid. And this
penicillin, are resistant to stomach acid we can take it in the form of capsule because then it will be quite stable that is not natural not pure natural
but semi-synthetic. Semi-synthetic means this is derived from the penicillin which is mostly it is derived from the penicillin-G that it is.
Mode of action of penicillin

It has been reported that most of the high yielding strains of P. chrysogenum are genetically unstable. Genetic unstability increases with
the increase in the yield. However, it can be controlled to some extent by following suitable preservation methods.
1. A spore suspension is stored in a frozen state under liquid nitrogen.2. A spore suspension can be lyophilized in an appropriate medium.
3. A spore suspension is mixed with a sterile finely divided inert material like soil or sand and desiccated.
Fermentation Process of Penicillin:
Penicillin fermentation is an aerobic process with a volumetric oxygen absorption rate of 0.4 -0.8mm min -1. The required aeration rate
varies according to the strain, the type of fermenter used and on the impellor system. However, the aeration rate varies between 0.5 and
1.0 vvm. It is produced by fed batch submerged fermentation in a stirred tank fermenter. Corn steep liquor (CSL) is an important
component present in media.
This process can be described under following headings:
1. Strain development,
2. Inoculum production,
3. Inoculation,
4. Extraction and purification

The medium employed for penicillin production should be suitable to achieve:
1. An abundant growth of the mycelium.
2. Maximum accumulation of the antibiotic.
3. Easy and inexpensive extraction and purification of the antibiotic.
Carbon source is generally supplied in the form of lactose. Glucose, sucrose, glycerol and
sorbitol can also be employed as carbon source. Nitrogen source is generally supplied in the
form of ammonium sulphate or ammonium acetate or ammonium nitrate.
Potassium, phosphorus, magnesium, sulphur, zinc and copper are supplied in the form of salts.
Penicillin yields with time are linear from approximately 48 to 96 hours. The final penicillin yield is in the range of 3 to 5% which largely
depends upon the amount of carbohydrate consumed during fermentation process, which is approximately equal to 1500 international units
per milliliter.
Penicillin easily get carboxylated to form penicillianic acid which is biologically inactive by the action of enzyme penicillinase. The enzyme
penicillinase is widely distributed among different microorganisms. These organisms may enter into the fermenter at any stage and may
convert penicillin into penicillianic acid,
In the typical penicillin fermentation there is a growth of 10 hrs duration with a doubling time of 6 hrs during which the greater part of the cell
mass is formed. The oxygen supply in the growing culture is critical since the increasing viscosity hinders oxygen transfer. After growth phase,
the culture proceeds to actual penicillin production. The growth is sharply reduced by feeding with various culture medium components.
The penicillin is excreted into the medium and less than 1% remains as mycelium bound. Extraction of penicillin is carried out by employing
counter current extraction method. The pH of the liquid after separation of the mycelium is adjusted to 2.0 to 2.5 by adding phosphoric or
sulphuric acid. This treatment converts penicillin into anionic form.
The liquid is immediately extracted with an organic solvent such as amylacetate or butylacetate or methyl isobutyl ketone. This step has to be
carried out quickly because penicillin is quite unstable at low pH values.
Uses of Penicillin:
1. Most of the penicillin’s are active against Gram-positive bacteria, in which they inhibit the cell wall synthesis leading to the death of bacteria.
2. Used therapeutically in the treatment of infectious diseases of humans caused by Gram (+) positive bacteria.
https://www.youtube.com/watch?v=JRl7iJqFGiw

Fermentation KC

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FERMENTATION TECHNOLOGY

❑ Less cost, time, capital

• Fermentation requirements- Stanbury ed 3

• What basic requirements are present in the media?

• The stretching of this bridge in a radial direction

Buffer, as we have defined, is a mixture of a conjugate acid-base

When a strong base is added, the acid present in the buffer

When a strong acid is added, the base present in the buffer

Also used in world war 2

Mode of action of penicillin

Fermentation Process of Penicillin:

This process can be described under following headings:

4. Extraction and purification

1. An abundant growth of the mycelium.

2. Maximum accumulation of the antibiotic.

3. Easy and inexpensive extraction and purification of the antibiotic.

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