Semester - V
Course MI-304
Unit 1: Introduction to fermentation technology
Fermentation technology
It is a field which involves the use of microorganisms and enzymes for
production of compounds that have applications in the energy, material,
pharmaceutical, chemical and food industries. The basic principle involved in
the industrial fermentation technology is that organisms are grown under
suitable conditions, by providing raw materials meeting all the necessary
requirements such as carbon, nitrogen, salts, trace elements and vitamins.
The basic principle involved in the industrial fermentation technology is that
organisms are grown under suitable conditions, by providing raw materials
meeting all the necessary requirements such as carbon, nitrogen, salts, trace
elements and vitamins. The end products formed as a result of their metabolism
during their life span are released into the media, which are extracted for use by
human being and that have a high commercial value. The major products of
fermentation technology produced economically on a large scale industrial basis
are wine, beer, cider, vinegar, ethanol, cheese, hormones, antibiotics, complete
proteins, enzymes and other useful products.
fermentation
The term fermentation is derived from the Latin verb fervere ,to boil ,thus
describing the appearance of the action of yeast on extracts of fruit or malted
grain. The boiling appearance is due to the production of carbon dioxide
bubbles caused by the anaerobic catabolism of the sugars present in the extract.
However, fermentation has come to have different meanings to biochemists and
to industrial microbiologists .It’s biochemical meaning relates to the generation
of energy by the catabolism of organic compounds.
Fermentation is defined as a process in which chemical changes occur in an
organic substrate through the action of enzymes produced by microorganisms. It
is one of the ancient food processing technologies. Fermentation is one of the
ancient food processing technologies. For example, yeast enzymes convert
sugars and starches into alcohol, while proteins are converted to peptides/amino
acids. Fermentation takes place in the lack of oxygen that produces ATP
(energy).It turns NADH and pyruvate produced in the glycolysis step into
NAD+ and various small molecules depending on the type of fermentation. The
fermenting microorganisms mainly involve L.A.B. like Enterococcus,
Streptococcus, Leuconostoc,Lactobacillus, and Pediococcus and yeasts and
molds like Debaryomyces, Kluyveromyces, Saccharomyces, Geotrichium,
Mucor, Penicillium, and Rhizopus species.
The main principle of fermentation is to derive energy from carbohydrates in
the absence of oxygen. Glucose is first partially oxidized to pyruvate by
glycolysis. Then pyruvate is converted to alcohol or acid along with
regeneration of NAD+ which can take part in glycolysis to produce more ATP.
Fig.1 Generalized pathways for the production of some fermentation end
from glucose by various organisms.
The catabolism of sugars is an oxidative process which results in the production
of reduced pyridine nucleotides which must be reoxidized for the process to
continue. Under aerobic conditions, reoxidation of reduced pyridine nucleotide
occurs by electron transfer, via cytochrome system, with oxygen acting as the
terminal electron .However, under anaerobic conditions, reduced pyridine
nucleotide oxidation is coupled with the reduction of an organic compound,
which is often a subsequent product of the catabolic pathway.
The oxidation of the substrate, which occurs through the Embden–Meyerhoff
(EMP) or Entner–Doudoroff(ED) pathways, results in the production of
pyruvate, ATP, and NAD (P) H. In the absence of external electron acceptors,
the pyruvate undergoes reduction with the regeneration of NAD+(P).This step is
essential for the fermentation process to progress and it leads to the production
of products (ethanol and organic acids).ATP is the main product of
fermentation, and it is generated by phosphorylation at the substrate level.
NADH is then re-oxidized, reverting to NAD+ in the second phase of
fermentation that reduces pyruvate to the fermentation product, such as
ethanol and lactate. For example, in the fermentation of glucose
by Streptococcus lactis, the pyruvate is converted to lactic acid to reform
NAD+ coenzymes so two ATP molecules are produced. In yeasts
like Saccharomyces, when pyruvate is converted to ethyl alcohol (ethanol),
NAD+ is reformed.
The Range of Fermentation Process
There are five major groups of commercially important fermentations:
1. Microbial biomass
2. Microbial enzymes
3. Microbial metabolites
4. Recombinant products
5. Transformation process
1.Microbial biomass
The commercial production of microbial biomass may be divided into two
main processes 1.The production of yeast to be used in the baking industry
and 2. The production of microbial cells to be used as human or animal food
which called single cell proteins. The microbial cells including algae, bacteria,
yeasts, moulds and mushrooms are dried and used as a good source of a
complete protein. SCP produced by some microorganisms is lysine rich whereas
those produced by others are methionine rich. The substrates used by the
microbes producing the single cell protein range from carbohydrates to
hydrocarbons and petrochemicals. Other organisms use the gases—methane,
ethane, propane, n-butane etc as substrate for fermentation. Baker’s yeast has
been produced on a large scale since the early 1900s and yeast was produced as
human food in Germany during the First World War. A few large scale
continuous processes for animal feed production were based on hydro-
hydrocarbon feed stocks which could not complete against other high protein
animal feeds, resulting in their closure in the late 1980s. However, the demise of
the animal feed biomass fermentations was balanced by ICI plc and Rank Hovis
McDougal establishing a process for the production of fungal biomass for
human food, this process was based on a more stable economic platform and
appears to have a promising future.
2.Microbial enzymes
Enzymes have been produced commercially from plant, animal and
different microbial sources .However; microbial enzymes have the
enormous advantage of being able to be produced in large quantities by
established fermentation techniques. When microbes are cultured, they secrete
some enzymes into the media and these enzymes are extracted and widely used
in several industries like detergent, food processing, brewing and
pharmaceutical. They are also used for diagnostic, scientific and analytical
purposes. Biotechnological methods are used to engineer microbial cells so as to
induce them to produce enzymes like renin by E. coli and amylases by Bacillus
stearothermophillis. The advent of recombinant DNA technology has enabled
enzymes of animal origin to be synthesized by microorganisms. The uses to
which microbial enzymes have been put are summarized in below fig.2
The enzymes produced by fermenting microbes are—Glucose oxidase, protease
glucoamylase, amylase, glucose isomerase, rennin, pectinase, superoxide
dismutase, cellulase, invertase, lactase and lipase. Some thermophile bacteria
produce enzymes that are thermo-stable and which can be used in industrial
processes at high temperatures, ex. glyceraldehyde-3-phosphate dehydrogenase,
phosphofructo-kinase, alcohol dehydrogenase, superoxide dismutase and
restriction endonucleases, which are produced by Bacillus stearothermophillis
thermoysia. Further, genes for thermophilic enzymes are introduced into E. coli,
which is cultured for producing the thermophilic enzymes.
3.Microbial metabolites
The growth of microbial culture can be divided into number of stages.
Immediately after inoculation, there is no increase in the numbers of the
microbial cells for some time and this period is called lag phase or may be
considered as a period of adaptation .A period during the growth rate of the
cells gradually increases the cell grow at a constant, maximum rate and this
period is called as the log or exponential phase .Eventually ,the growth ceases
and the cells enter the new phase called stationary phase .after a further period
of time the viable cell number declines as culture enters the death phase.
During the log phase of growth the products produced are essential to the
growth of the cells and include amino acids, nucleotides, proteins, lipids etc.
These products are referred to as the primary products of metabolisms and the
the phase in which they are produced (equivalent to the log, or exponential
phase) as the trophophase.
Many products of primary metabolism are considerable economic importance
and are being produced by fermentation, as mentioned in below fig.3.
The synthesis of primary metabolites by wild type microorganisms is such that
their production is sufficient to meet the requirements of the organism.
During the deceleration and stationary phases some microbial cultures
synthesize compounds which are not produced during the trophophase and
which do not appear to have any obvious function in cell metabolism. These
compounds are referred to as the secondary compounds of metabolism and the
phase in which they are produced as knows as idiophase.it is important to
realize that secondary metabolism may occur in continuous cultures at low
growth rates and is a property of slow growing, as well as non-growing cells.
The inter relationship between primary and secondary metabolism is shown in
below fig.4
Primary products are shown in dark /heavy lines and secondary products are
shown in thin lines.
Secondary metabolites tend to be elaborated from the intermediates and
products of primary metabolism. Although the primary biosynthetic routes
are common to the vast majority of microorganisms, each secondary product
would be synthesized by only a very few different microbial species.
The physiological role of secondary metabolism, many secondary
metabolites have antimicrobial activity, others are specific enzyme
inhibitors, some are growth promoters and may have pharmacological
properties .Thus, the products of secondary metabolism have formed the
basis of number of fermentation processes. As is the case for primary
metabolites, wild type microorganisms tend to produced only low
concentration of secondary metabolites, their synthesis being controlled by
induction, catabolic repression and feedback systems.
4.Recombinant products
The advent of recombinant DNA technology has extended the range of
potential fermentation products. Genes from higher organisms may be
introduced into microbial cells such that the recipients are capable of
synthesizing ‘foreign’ proteins. A wide range of microbial cells have been
used as hosts for such systems including E.coli ,S.cerevisiae and
filamentous fungi. Products produced by genetically engineered organisms
include interferon, insulin, human serum albumin, factors VIII and IX,
epidermal growth factor, calf chymosin and bovine somato statin. Important
factors in the design of these processes include the secretion of the product,
minimization of the degradation of the product and control of onset of
synthesis during the fermentation, as well as maximizing the expression of
the foreign genes.
5.Transformation process
Microbial cells may be used to convert a compound into a structurally
related, financially more valuable, compound. Because microorganisms can
behave as chiral catalysts with high positional specificity and stereo
specificity, microbial processes are more specific than purely chemical ones
and enable the addition, removal or modification of functional groups at
specific sites on a complex molecule without the use of chemical protection.
The reactions which may be catalysed include dehydrogenation, oxidation,
hydroxylation, dehydration, decarboxylation, amination and isomerization.
Microbial processes have the additional advantages over chemical reagents
of operating at relatively low temperatures and pressures without the
requirements for potentially polluting heavy metal catalysts. Although the
production of vinegar is the most well established microbial transformation
process the majority of these processes involve the production of high value
compounds including steroids, antibiotics and prostaglandins.
The anomaly of the transformation fermentation process is that a large
biomass has to be produced to catalyse a single reaction. Thus, many
processes have been streamlined by immobilizing either the whole cells, or
the isolated enzymes which catalyse the reactions, on an inert support. The
immobilized cells or enzymes may then be considered as catalyst which
may be reused many times.
The Chronological development of the fermentation industry
The chronological development of the fermentation industry may be
represented as five overlapping stages as mentioned in below fig.5
The development of the industry prior to 1900 is represented by stage 1,
where the products were confined to potable alcohols and vinegar. Vinegar
was originally produced by leaving wine in shallow bowls or partially filled
barrels where it was slowly oxidized to vinegar by the development of a
natural flora. The vinegar generator may be considered as the first aerobic
fermenter to be developed. Between the years, 1900 and 1940 the main new
products were yeast biomass, glycerol, citric acid, lactic acid, acetone and
butanol. Probably the most important advantages during this period were the
developments in the baker’s yeast and solvent fermentations. The
development of the acetone – butanol fermentation during the First World
War by the pioneering efforts of Weizmann led to the establishment of the
first truly aseptic fermentation.
During 1940s, the development of the fermentation industry arose as a result
of the wartime need to produce penicillin in submerged culture under
aseptic conditions. The production of penicillin is an aerobic process which
is very vulnerable to contamination. Penicillin was synthesized in very small
quantities by the initial isolates and this resulted in the establishment of
strain improvement programmes which became a dominant feature of the
industry in next years. The development of large scale extraction process for
the recovery of penicillin was another major advance at those times. Most
significant changes in fermentation technology took place resulting in the
establishment of many new processes over the period, including other
antibiotics, vitamins, amino acids, enzymes and steroid transformation.
During 1960s, Microbial products were screened for activities other than
simply antimicrobial properties and screens became more and more
sophisticated. These screens have evolved into those operating utilizing
miniaturized culture systems, robotic automation and elegant assays.
Hydrocarbons were considered as potential carbon sources which would
result in increased oxygen demands and high heat-outputs. The aseptic
operations of fermenters was achieved as a result of the high standards of
fermenter construction, the continuous sterilization of feed streams and the
utilization of computer systems to control the sterilization and operation
cycles, thus minimizing the possibility of human error.
During 1979, Genetic engineering approximately coincided with another
major development in biotechnology which influenced the progress of the
fermentation industry- the production of monoclonal antibodies as well as
production of heterologous proteins by Both microbial cells and animal
cells. The availability of monoclonal antibodies opened the door to
sophisticated analytical techniques and raised hopes for their use as
therapeutic agents. Thus, animal cell culture processes were established to
produce mono-clonals on a commercial scale. Animal cells were also used
as hosts for the production of human proteins, especially where post–
translational a modification was essential for protein activity.However,the
appreciation by the pharmaceutical industry that the activity of microbial
metabolites extended well beyond anti-bacterial has resulted in a number of
new microbial products reaching the marketplace in the late 1980s and
1990s.They listed four secondary metabolites which were launched in the
1980s A) Cyclosporine, an immunoregulant used to control rejection of
transplanted organs B) Imipenem, a modified carbapenem which has the
widest antimicrobials spectrum of any antibiotic C) Lovastatin, a drug used
for reducing cholesterol levels and D) Ivermectin, an anti-parasitic drug
which has been used to prevent ‘African River Blindness’ as well as in
veterinary practice.
The Component parts of Fermentation processes
The Component parts of fermentation process include:
1. The formulation of media to be used in culturing the process organism
during the development of the inoculums and in the production fermenter.
2. The sterilization of the medium, fermenters and ancillary equipment.
3. The production of an active, pure culture in sufficient quantity to
inoculate the production vessel.
4. The growth of the organisms in the production fermenter under optimum
conditions for product formation.
5. The extraction of the product and its purification.
6. The disposal of effluents produced by the process.
Interrelationships of six component parts of fermentation process are
mentioned in below fig.6
