Fermentation Technology

PAMANTASAN NG LUNGSOD NG MAYNILA
University of the City of Manila

College of Engineering and Technology
Fermentation Technology
Introduction
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. Its biochemical meaning relates to the generation
of energy by the catabolism of organic compounds, whereas its meaning in industrial
microbiology tends to be much broader. Fermentation is a word that has many meanings for the
microbiologist:
1 Any process involving the mass culture of microorganisims, either aerobic or
anaerobic.
2 Any biological process that occurs in the absence of O2.
3 Food spoilage.
4 The production of alcoholic beverages.
FERMENTATION TECHNOLOGY
(Barros DS., Maninang C., Sangalang, KP.) Page 1

5 Use of an organic substrate as the electron donor and acceptor.

6 Use of an organic substrate as a reductant, and of the same partially degraded organic
substrate as an oxidant.
7 Growth dependant on substrate-level phosphorylation
Fermentation technology is a field which involves the use of microorganisms and
enzymes for production of compounds which have application in the energy, material,
pharmaceutical, chemical and the food industry. Though fermentation processes are used for
generations for the requirement for sustainable production of materials and energy is
demanding creation and advancement of novel fermentation processes.
Efforts are directed both to the advancement of cell factories and enzymes as well as of
design of new processes, concepts and technologies for fermentation process.
Through fermentation, we can produce enzymes for industrial purposes. Process of
Fermentation includes the use of microorganisms,
like yeast and bacteria for the production of
enzymes.
Mainly, there are two methods of
fermentation which are used to produce enzymes.
First is submerged fermentation and second is solid-state fermentation. In Submerged
fermentation, the production of enzymes is done by microorganisms in a liquid nutrient media.
Whereas in Solid- fermentation is carried out by cultivation of microorganisms and production of
enzyme is done on a solid substrate. Compounds containing carbon in or on the substrate are
busted down by the microorganisms thus producing the enzymes either extracellular or
intracellular. The enzy mes are isolated by various methods such as centrifugation, and for
extracellular produced enzymes and lying
of cells
for intracellular enzymes.
Various
industries
rely
on
enzymes for the production of their
dependent products. Industries which are
enzymes generated by fermentation are
wine making, brewing, cheese making
baking.
enzyme
used for
the
and

TYPES OF FERMENTATION CULTURE

A. BATCH PROCESSING OR CULTURE
At about the onset of the stationary phase, the culture is disbanded for the recovery of its
biomass (cells, organism) or the compounds that accumulated in the medium (alcohol, amino
acids), and a new batch is set up. This is batch processing or batch culture.
The best advantage of batch processing is the optimum levels of product recovery. The
disadvantages are the wastage of unused nutrients, the peaked input of labour and the time lost
between batches.
B. CONTINUOUS PROCESSING OR CULTURE
The culture medium may be designed such that
growth is limited by the availability of one or two
components of the medium. When the initial quantity of
this component is exhausted, growth ceases and a
steady state is reached, but growth is renewed by the
addition of the limiting component. A certain amount of
the whole culture medium (aliquot) can also be added
periodically, at the time when steady state sets in. The
addition of nutrients will increase the volume of the
medium in the fermentation vessel. It is so arranged that
the increased volume will drain off as an overflow, which
is collected and used for recovery of products. At each step of addition of the medium, the
medium becomes dilute both in terms of the concentration of the biomass and the products.
New growth, stimulated by the added medium, will increase the biomass and the products, till
another steady state sets in; and another aliquot of medium will reverse the process.

This is continuous culture or processing. Since the growth of the organism is controlled
by the availability of growth limiting chemical component of the medium, this system is called a
chemostat. The rate at which aliquots are added is the dilution rate that is in effect the factor that
dictates the rate of growth.
The events in a continuous culture are:
a) the growth rate of cells will be less than the dilution rate and they will be washed out
of the vessel at a rate greater than they are being produced, resulting in a decrease of
biomass concentration both within the vessel and in the overflow;
b) the substrate concentration in the vessel will rise because fewer cells are left in the
vessel to consume it;
c) the increased substrate concentration in the vessel will result in the cells growing at a
rate greater than the dilution rate and biomass concentration will increase; and
d) the steady state will be re-established.
Hence, a chemostat is a nutrient limited self-balancing culture system, which may be
maintained in a steady state over a wide range of sub-maximum specific growth rates.
The continuous processing offers the most control over the growth of cells.
Commercial adaptation of continuous processing is confined to biomass production, and
to a limited extent to the production of potable and industrial alcohol.
The steady state of continuous processing is advantageous as the system is far easier
to control. During batch processing, heat output, acid or alkali production, and oxygen
consumption will range from very low rates at the start to very high rates during the late
exponential phase. The control of the environmental factors of the system becomes difficult. In
the continuous processing, the rates of consumption of nutrients and those of the output
chemicals are maintainable at optimal levels. Besides, the labour demand is also more uniform.
Continuous processing may suffer from contamination, both from within and outside. The
fermenter design, along with strict operational control, should actually take care of this problem.
The production of growth associated products like ethanol is more efficient in continuous
processing, particularly for industrial use.
Continuous culturing is highly selective and favours the propagation of the best-adapted
organism in culture.

A commercial organism is highly mutated such that it will produce very high amounts of
the desired product. But physiologically such strains are inefficient and give way in culture to
inferior producers--a kind of contamination from within.
C. FED-BATCH CULTURE OR PROCESSING
In the fed-batch system, a fresh aliquot of the medium is continuously or periodically
added, without the removal of the culture fluid. The fermenter is designed to accommodate the
increasing volumes. The system is always at a quasi-steady state.
Fed-batch achieved some appreciable degree of process and product control.
A low but constantly replenished medium has the following advantages:
a) maintaining conditions in the culture within the aeration capacity of the fermenter;
b) removing the repressive effects of medium components such as rapidly used carbon
and nitrogen sources and phosphate;
c) avoiding the toxic effects of a medium component; and
d) providing limiting level of a required nutrient for an auxotrophic strain.
Production of baker's yeast is mostly by fed-batch culture, where biomass is the desired
product. Diluting the culture with a batch of fresh medium prevents the production of ethanol, at
the expense of biomass; the moment traces of ethanol were detected in the exhaust gas.
The production of penicillin, a secondary metabolite, is also by fed-batch method.
Penicillin process has two stages: an initial growth phase followed by the production phase
called the 'idiophase'. The culture is maintained at low levels of biomass and phenyl acetic acid,
the precursor of penicillin, is fed into the fermenter continuously, but at a low rate, as the
precursor is toxic to the organism at higher concentrations.
Nutritional Requirements and Media for Fermentation Processes

Specific nutritional requirements of microorganisms used in industrial fermentation
processes are as complex and varied as the microorganisms in question. Not only are the types
of microorganisms diverse (bacteria, molds and yeast, normally), but the species and strains
become very specific as to their requirements. Microorganisms obtain energy for support of
biosynthesis and growth from their environment in a variety of ways.

Organisms that use CO, as the principal carbon source are defined as autotrophic;
organisms that use organic compounds as the principal carbon source are defined as
heterotrophic. A combination ofthese two criteria leads to the establishment of four principal
categories: (i) photoautotrophic, (ii) photoheterotrophic, (iii) chemoautotrophic and (iv)
chemoheterotrophic organisms.
Photoautotrophic organisms are dependent on light as an energy source and employ
CO, as the principal carbon source. This category includes higher plants, eucaryotic algae, blue
green algae, and certain photosynthetic bacteria (the purple and green sulfur bacteria).
Photoheterotrophic organisms are also dependent on the light as an energy source and
employ organic compounds as the principal carbon source. The principal representatives of this
category are a group of photosynthetic bacteria known as the purple non-sulfur bacteria; a few
eucaryotic algae also belong to it.
Chemoautotrophic organisms depend on chemical energy sources and employ CO, as a
principal carbon source. The use of CO, as a principle carbon source by chemotrophs is always
associated with the ability to use reduced inorganic compounds as energy sources. This ability
is confined to bacteria and occurs in a number of specialized groups that can use reduced
nitrogen compounds (NH,, NO,), ferrous iron, reduced sulfur compounds @I,S, S, S,03,-), or H,
as oxidizable energy sources.
Chemoheterotrophic organisms are also dependent on chemical energy sources and
employ organic compounds as the principle carbon source. It is characteristic of this category
that both energy and carbon requirements are supplied at the expense of an organic compound.
Its members are numerous and diverse, including fungi and the great majority of the bacteria.
The chemoheterotrophs are of great commercial importance. This category may be
subdivided into respiratory organisms, which couple the oxidation of organic substrates with the
reduction of an inorganic oxidizing agent (electron acceptor, usually O,), and fermentative
organisms, in which the energy yielding metabolism of organic substrates is not so coupled. In
addition to an energy source and a carbon source, the microorganisms require nutritional
factors coupled with essential and trace elements that combine in various ways to form cellular
material and products.
Since photosynthetic organisms (and chemoautotrophes) are the only net producers of
organic matter on earth, it is they that ultimately provide, either directly or indirectly, the organic
forms of energy required by all other organisms.

All micro-organisms require water, sources of energy, carbon, nitrogen, mineral elements
and possibly vitamins plus oxygen if aerobic. On a small scale it is relatively simple to devise a
medium containing pure compounds, but the resulting medium, although supporting satisfactory
growth, may be unsuitable for use in a large scale process.
On a large scale one must normally use sources of nutrients to create a medium which
will meet as many as possible of the following criteria:
1. It will produce the maximum yield of product or biomass per gram of substrate used.
2. It will produce the maximum concentration of product or biomass.
3. It will permit the maximum rate of product formation.
4. There will be the minimum yield of undesired products.
5. It will be of a consistent quality and be readily available throughout the year.
6. It will cause minimal problems during media making and sterilization.
7. It will cause minimal problems in other aspects of the production process particularly
aeration and agitation, extraction, purification and waste treatment.
MEDIUM FORMULATION
Medium formulation is an essential stage in design of successful laboratory experiments,
Pilot-scale development and manufacturing processes. The constituents of a medium must
satisfy the elemental requirements for cell biomass and metabolite production and there must
be an adequate supply of energy biosynthesis and cell maintenance. The first step consider is
an equation based on the stoichiometry growth and product formation. Thus for an fermentation:
carbon + nitrogen + 02 + other -> and source require energy + source biomass +
products +
CO2 + H 20 + heat
This equation should be expressed in quantitative which is important in the economical
design of media component wastage is to be minimal. Thus, it should be possible to calculate
the minimal quantities of nutrients which will be needed to produce a specific amount of
biomass. Knowing that a certain amount of biomass is necessary to produce a defined amount
of product, it should be possible to calculate substrate concentrations necessary to produce
required product yields. There may be medium components which are needed for product
formation which are not required for biomass production. Unfortunately, it is not always easy to
quantify all the factors very precisely.

Knowledge of the elemental composition of a process micro-organism is required for the

solution of the elemental balance equation. This information may not be available so that data
which is given in Table 4.2 will serve as a guide to the absolute minimum quantities of N, S, P,
Mg and K to include in an initial medium recipe. Trace elements (Fe, Zn, Cu, Mn, Co, Mo, B)
may also be needed in smaller quantities.
WATER
Water is the major component of all fermentation media, and is needed in many of the
ancillary services such as heating, cooling, cleaning and rinsing. Clean water of consistent
composition is therefore required in large quantities from reliable permanent sources. 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 v ery 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 CaS04 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. Nowadays,
the water may be treated by deionization or
other techniques and salts added, or the
pH adjusted, to favour different beers so
that breweries are not so dependent on the
local water source. Detailed information is
given by Hough et al. (1971) and Sentfen (1989).
The reuse or efficient use of water is normally of high priority. When ICI pic and John
Brown Engineering developed a continuous-culture single cell protein (SCP) process at a
production scale of 60,000 tonnes per year it was realized that very high costs would be
incurred if fresh purified water was used on a oncethrough basis, since operating at a cell
concentration of 30 g biomass (dw) dm- 3 would require 2700 X 106 dm3 of water per annum
(Ashley and Rodgers, 1986; Sharp, 1989). 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 lO-fold.
ENERGY SOURCES
Energy for growth comes from either the oxidation of medium components or from light.
Most industrial micro-organisms are chemo-organotrophs, therefore the commonest source of
energy will be the carbon source such as carbohydrates, lipids and proteins. Some microorganisms can also use hydrocarbons or methanol as carbon and energy sources.
CARBON SOURCES

CARBOHYDRATES It is common practice to use carbohydrates as the carbon source in

microbial fermentation processes. The most widely available carbohydrate is starch obtained
from maize grain. It is also obtained from other cereals, potatoes and cassava.
Barley grains may be partially germinated and heat treated to give the material known as
malt, which contains a variety of sugars besides starch.
Malt is the main substrate for brewing beer and lager in
many countries. Malt extracts may also be prepared
from malted grain.
Sucrose is obtained from sugar cane and 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 solutions in
sugar refining. Molasses is used in the production of
high-volumeflow-value products such as ethanol, SCP, organic and amino acids and some
microbial gums.
OILS AND FATS Oils were first used as carriers for

antifoams in antibiotic processes (Solomons, 1969).
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.
HYDROCARBONS AND THEIR DERIVATIVES There
has been considerable interest in hydrocarbons. Development work has been done using nalkanes for production of organic acids, amino acids, vitamins and co-factors, nucleic acids,
antibiotics, enzymes and proteins (Fukui and Tanaka, 1980). Methane, methanol and n-alkanes
have all been used as substrates for biomass production (Hamer, 1979; Levi et at., 1979; Drozd,
1987; Sharp. 1989). Drozd (1987) discussed the advantages and disadvantages of

hydrocarbons and their derivatives as fermentation substrates, particularly with reference to

cost, process aspects and purity. In processes where the feedstock costs are an appreciable
fraction of the total manufacturing cost, cheap carbon sources are important.
NITROGEN SOURCES
Most industrially used micro-organisms can utilize inorganic or organic sources of
nitrogen. Inorganic nitrogen may be supplied as ammonia gas, ammonium salts or nitrates
(Hunter, 1972). 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
ceriuisiae (Collins, 1990). Ammonium salts such as ammonium sulphate 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 and MacMillan, 1954).
Organic nitrogen may be supplied as amino acid, protein or urea. 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 non-homogeneous, 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, soya meal, peanut meal, cotton-seed 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.
Chemically defined amino acid media devoid of protein are necessary in the production
of certain vaccines when they are intended for human use.
Factors influencing the choice of nitrogen source
Control mechanisms exist by which nitrate reductase, an enzyme involved in the
conversion of nitrate to ammonium ion, is repressed in the presence of ammonia (Brown et al.,

1974). For this reason ammonia or ammonium ion is the preferred nitrogen source. In fungi that
have been investigated, ammonium ion represses uptake of amino acids by general and
specific amino acid permeases (Whitaker, 1976). In Aspergillus nidulans, ammonia also
regulates the production of alkaline and neutral proteases (Cohen, 1973). Therefore, in mixtures
of nitrogen sources, individual nitrogen components may influence metabolic regulation so that
there is preferential assimilation of one component until its concentration has diminished.
MINERALS
All micro-organisms require certain mineral elements for growth and metabolism
(Hughes and Poole, 1989, 1991). In many media, magnesium, phosphorus, potassium, sulphur,
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. There is obviously a need for batch analysis of media components to ensure that
this assumption can be justified, otherwise there may be deficiencies or excesses in different
batches of media.

Chelators
Many media cannot be prepared or autoclaved without the formation of a visible
precipitate of insoluble metal phosphates. The problem of insoluble metal phosphate(s) may
eliminated by incorporating low concentrations chelating agents such as ethylene diamine
tetraa,cetic acid (EDTA), citric acid, polyphosphates, etc., into medium. These chelating agents
preferentially complexes with the metal ions in a medium. The ions then may be gradually
utilized by the organism (Hughes and Poole, 1991).
In many media, particularly those commonly used in large scale processes, there may
not be a need to add a chelating agent as complex ingredients such as yeast extracts or
proteose peptones will complex with metal ions and ensure gradual release of them during
growth (Ramamoorthy and Kushner, 1975).
GROWTH FACTORS
Some micro-organisms 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 and Mavituna, 1991a). When there
is a vitamin deficiency it can often be eliminated by careful blending of materials (Rhodes and
Fletcher, 1966). It is important to remember that if only one vitamin is required it may be

occasionally more economical to add the pure vitamin, instead of using a larger bulk of a
cheaper multiple vitamin source. 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. Some production
strains may also require thiamine (Kinoshita and Tanaka, 1972).
BUFFERS
The control of pH may be extremely important if optimal productivity is to be achieved. A
compound may be added to the medium to serve specifically as a buffer, or may also be used
as a nutrient source. Many media are buffered at about pH 7.0 by the incorporation of calcium
carbonate (as chalk). If the pH decreases the carbonate is decomposed. Obviously, phosphates
which are part of many media also play an important role in buffering. However, high phosphate
concentrations are critical in the production of many secondary metabolites.
The balanced use of the carbon and nitrogen sources will also form a basis for pH
control as buffering capacity can be provided by the proteins, peptides and amino acids, such
as in corn-steep liquor. The pH may also be controlled externally by addition of ammonia or
sodium hydroxide and sulphuric acid.
OXYGEN REQUIREMENTS
It is sometimes forgotten that oxygen, although not added to an initial medium as such,
is nevertheless a very important component of the medium in many processes, and its
availability can be extremely important in controlling growth rate and metabolite production.
The medium may influence the oxygen availability in a number of ways including the following:
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.
Nutritional factors can alter the oxygen demand the culture. Penicillium chrysogenum will
utilize glucosemore rapidly than lactose or sucrose, and it therefore has a higher specific
oxygen uptake rate when glucose is the main carbon source (Johnson, 1946). Therefore,
when there is the possibility of oxygen limitation due to fast metabolism, it may be overcome
by reducing the initial concentration 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 carbohydrates (lactose, starch, etc.) and proteins which are not very
rapidly metabolized and do not support such a large specific oxygen uptake rate.
2. Rheology. The individual components of the medium can influence the viscosity of the final
medium and its subsequent behaviour with respect to aeration and agitation.
3. Antifoams. Many of the antifoams in use will as surface active agents and reduce the
transfer rate.
In most microbiological processes, foaming is a probIt may be due to a component in the
medium or factor produced by the micro-organism. The most common cause of foaming is due
to proteins in the medium, such as corn-steep liquor, Pharmamedia, meal, soybean meal, yeast
extract or meat ex- (Schugerl, 1985). These proteins may denature at the air-broth interface and
form a skin which does not rupture readily. The foaming can cause removal of cells from the
medium which will lead to autolysis and the further release of microbial cell proteins will
probably increase the stability of the foam. If uncontrolled, then numerous changes may occur
and physical and biological problems may be created. These include reduction in the working
volume of the fermenter due to oxygenexhausted gas bubbles circulating in the system (Lee
and Tyman, 1988), changes in bubble size, lower mass and heat transfer rates, invalid process
data due to interference at sensing electrodes and incorrect monitoring and control (VardarSukan, 1992). The biological pn)blemls include deposition of cells in upper parts of fermenter,
problems of sterile operation with the air filter exits of the fermenter becoming wet, and there is
danger of microbial infection and the possibility of siphoning leading to loss of product.
Nutrient sources for industrial fermentation
Nutrient
Raw material
Source
Carbon
Glucose
Corn sugar, Starch, Cellulose
Sucrose
Sugarcane, Sugar beet molasses
Lactose
Milk whey
Fats
Vegetable oils
Hydrocarbons
Nitrogen
Protein
Petroleum fractions
Soybean meal, Cornsteep liquor, Distillers'
solubles

Ammonia
Nitrate
Nitrogen
Phosphorous
Pure ammonia or ammonium salts

Nitrate salts
Air
Phosphate salts



BIOCHEMICAL ENGINEERING
I.
KREBS CYCLE
All cells must produce energy to survive. Hans Adolf Krebs first elucidated the process
of cells converting food into energy, the Citric Acid Cycle, in 1937. Krebs proposed a specific
metabolic pathway within the cells to account for the oxidation of the basic components of food
carbohydrates, protein and fats for energy. The Krebs cycle takes place inside the
mitochondria or power plant of cells and provides energy required for the organism to function.
Mitochondria are found in all cells in the human body, with the exception of mature red
blood cells. The primary function of these tiny organelles (each cell contains between 500 and
2,000 mitochondria) is to convert energy found in nutrient molecules and store it in the form of
adenosine triphosphate (ATP). ATP is the universal energy-yielding molecule used by
enzymes to perform a wide range of cellular functions. Humans cannot survive, even for a
second, without a constant supply of ATP.
The citric acid cycle also known as the tricarboxylic acid (TCA) cycle or the Krebs
cycle is a series of chemical reactions used by all aerobic organisms to generate energy
through the oxidation of acetyl-CoA derived from carbohydrates, fats and proteins into carbon
dioxide and chemical energy in the form of guanosine triphosphate (GTP). The name of this
metabolic pathway is derived from citric acid (a type of tricarboxylic acid) that is consumed and
then regenerated by this sequence of reactions to complete the cycle. In addition, the cycle
consumes acetate (in the form of acetyl-CoA) and water, reduces NAD+ to NADH, and
produces carbon dioxide as a waste byproduct. The NADH generated by the TCA cycle is fed
into the oxidative phosphorylation (electron transport) pathway. The net result of these two
closely linked pathways is the oxidation of nutrients to produce usable chemical energy in the
form of ATP.
In eukaryotic cells, the citric acid cycle occurs in the matrix of the mitochondrion. In
prokaryotic cells, such as bacteria which lack mitochondria, the TCA reaction sequence is
performed in the cytosol with the proton gradient for ATP production being across the cell's
surface (plasma membrane) rather than the inner membrane of the mitochondrion.
The citric acid cycle is a key metabolic pathway that unifies carbohydrate, fat, and
protein metabolism. The reactions of the cycle are carried out by 8 enzymes that completely
oxidize acetate, in the form of acetyl-CoA, into two molecules each of carbon dioxide and water.

The 8 Enzymes of the Krebs Cycle:
Citrate synthase
Aconitase
Isocitrate dehydrogenase
-ketoglutarate dehydrogenase
Succinyl coenzyme A synthetase

Succinate dehydrogenase
Fumarase
Malate dehydrogenase
Through catabolism of sugars, fats, and proteins, a two-carbon organic product

acetate in the form of acetyl-CoA is produced which enters the citric acid cycle. The reactions of
the cycle also convert three equivalents of nicotinamide adenine dinucleotide (NAD+) into three
equivalents of reduced NAD+ (NADH), one equivalent of flavin adenine dinucleotide (FAD) into
one equivalent of FADH2, and one equivalent each of guanosine diphosphate (GDP) and
inorganic phosphate (Pi) into one equivalent of guanosine triphosphate (GTP). The NADH and
FADH2 generated by the citric acid cycle are in turn used by the oxidative phosphorylation
pathway to generate energy-rich adenosine triphosphate (ATP).
Process Diagram:

The Net Equation:
Acetyl CoA + 3 NAD + FAD + ADP + HPO4-2 > 2 CO2 + CoA + 3 NADH+ +
FADH+ + ATP
Reaction 1: Formation of Citrate
Its a testament to the importance of the citric acid cycle that it has not one, not two, but
three different names in common usage today. The name we'll primarily use here, the
citric acid cycle, refers to the first molecule that forms during the cycle's reactions
citrate, or, in its protonated form, citric acid.
The first reaction of the cycle is the condensation of acetyl-CoA with oxaloacetate to
form citrate, catalyzed by citrate synthase. Once oxaloacetate is joined with acetyl-

CoA, a water molecule attacks the acetyl leading to the release of coenzyme A from the
complex.
Reaction 2: Formation of Isocitrate
The citrate is
rearranged
to
form
an
isomeric
form, isocitrate by
an
enzyme acontinase. In this reaction, a water molecule is removed from the citric acid
and then put back on in another location. The overall effect of this conversion is that the
OH group is moved from the 3 to the 4 position on the molecule. This transformation
yields the molecule isocitrate.
Reaction 3: Oxidation of Isocitrate to -Ketoglutarate

In
this
step,
isocitrate
dehydrogenase
catalyzes
oxidative
decarboxylation
of isocitrate to form -ketoglutarate. In the reaction, generation of NADH from NAD is

seen. The enzyme isocitrate dehydrogenase catalyzes the oxidation of the OH group
at the 4 position of isocitrate to yield an intermediate which then has a carbon dioxide
molecule removed from it to yield alpha-ketoglutarate.
Reaction 4: Oxidation of -Ketoglutarate to Succinyl-CoA
Alpha-ketoglutarate is oxidized, carbon dioxide is removed, and coenzyme A is added

to form the 4-carbon compound succinyl-CoA. During this oxidation, NAD+ is reduced
to NADH + H+. The enzyme that catalyzes this reaction is alpha-ketoglutarate
dehydrogenase.

Reaction 5: Conversion of Succinyl-CoA to Succinate
CoA is removed from succinyl-CoA to produce succinate. The energy released is used
to make guanosine triphosphate (GTP) from guanosine diphosphate (GDP) and Pi by
substrate-level phosphorylation. GTP can then be used to make ATP. The enzyme
succinyl-CoA synthase catalyzes this reaction of the citric acid cycle.
Reaction 6: Oxidation of Succinate to Fumarate
Succinate is oxidized to fumarate. During this oxidation, FAD is reduced to FADH2. The
enzyme succinate dehydrogenase catalyzes the removal of two hydrogens from
succinate.

Reaction 7: Hydration of Fumarate to Malate
The reversible hydration of fumarate to L-malate is catalyzed by fumarase (fumarate

hydratase).
Fumarase continues
the
rearrangement
process
by
adding Hydrogen and Oxygen back into the substrate that had been previously
removed.
Reaction 8: Oxidation of Malate to Oxaloacetate
Malate is oxidized to produce oxaloacetate, the starting compound of the citric acid
cycle by malate dehydrogenase. During this oxidation, NAD+ is reduced to NADH +
H+.

ATP Generation
Total ATP = 12 ATP
3 NAD+ = 9 ATP
1 FAD = 2 ATP
1 ATP = 1 ATP
Reviewing the whole process, the Krebs cycle primarily transforms the acetyl group and
water, into carbon dioxide and energized forms of the other reactants.
Significance of Krebs Cycle:
1. Intermediate compounds formed during Krebs cycle are used for the synthesis of
biomolecules like amino acids, nucleotides, chlorophyll, cytochromes and fats etc.
2. Intermediate like succinyl CoA takes part in the formation of chlorophyll.
3. Amino Acids are formed from - Ketoglutaric acid, pyruvic acids and oxaloacetic acid.
4. Krebs cycle (citric Acid cycle) releases plenty of energy (ATP) required for various
metabolic activities of cell.
5. By this cycle, carbon skeleton are got, which are used in process of growth and for
maintaining the cells.

In an eight-step reaction sequence, the acetyl group of acetyl-CoA is oxidised

into two molecules of CO2 (see Figure). These reactions are catalysed by eight different
enzymes. Instead of producing high amounts of ATP, eight electrons were removed from the
acetyl group and transferred to the coenzymes NAD+ and FAD, which are reduced to NADH
and FADH2. They are therefore called electron carrier coenzymes and are used to transport
electrons from the Krebs cycle to the respiratory chain. Through a series of molecules, the
reduced coenzymes NADH and FADH2 are oxidised and the released electrons were used to
reduce O2. Finally, the electrons that are released in the Krebs Cycle and the transported to the
respiratory chain were used there to produce ATP out of ADP and Pi.
Example from Everyday Life:
Without vitamin C we cannot even sustain life. Vitamin C is an essential molecule

required for the most basic generation of energy in the body at the cellular level. This is known
as the Krebs Cycle, which is the complex chemical process whereby ATP is converted to ADP,
or adenosine triphosphate to adenosine diphosphate, releasing energy. All aerobic, oxygen
breathing, living beings that we know of depend on this Krebs Cycle to generate their most
basic energy for life.

II.
FERMENTATION PROCESSES
Fermentation is a metabolic process that converts sugar to acids, gases, or

alcohol. It occurs in yeast and bacteria, and also in oxygen-starved muscle cells, as in the case
of lactic acid fermentation. Fermentation is also used more broadly to refer to the bulk growth of
microorganisms on a growth medium, often with the goal of producing a specific chemical
product. French microbiologist Louis Pasteur is often remembered for his insights into
fermentation and its microbial causes. The science of fermentation is known as zymology.
6 Types of Fermentation:
Solid State Fermentation

Submerged Fermentation
Anaerobic Fermentation
Aerobic Fermentation
Immobilized Cell Bioreactors
Immobilized Enzyme Bioreactors
1. Solid State Fermentation
Solid-state fermentation has emerged as a potential technology for the

production of microbial products such as feed, fuel, food, industrial chemicals and
pharmaceutical products. Its application in bioprocesses such as bioleaching, biobeneficiation,
bioremediation, biopulping, etc. has offered several advantages. Utilisation of agro-industrial
residues as substrates in SSF processes provides an alternative avenue and value-addition to
these otherwise under- or non-utilised residues. Today with better understanding of biochemical
engineering aspects, particularly on mathematical modelling and design of bioreactors
(fermenters), it is possible to scale up SSF processes and some designs have been developed
for commercialisation. It is hoped that with continuity in current trends, SSF technology would be
well developed at par with submerged fermentation technology in times to come. Introduction
Solid-state (substrate) fermentation (SSF) has been defined as the fermentation process
occurring in the absence or near-absence of free water.
Solid state fermentation (SSF) is a biomolecule manufacturing process used in

the food, pharmaceutical, cosmetic, fuel and textile industries. These biomolecules are mostly
metabolites generated by microorganisms grown on a solid support selected for this purpose.
This technology for the culture of microorganisms is an alternative to liquid or submerged
fermentation, used predominantly for industrial purposes. This process consists of depositing a
solid culture substrate, such as rice or wheat bran, on flatbeds after seeding it with
microorganisms; the substrate is then left in a temperature-controlled room for several days.

At the beginning of the growth process, the substrates and solid culture
compounds are non-soluble compounds composed of very large, biochemically complex
molecules that the fungus will cut off to get essential C and N nutrients. To develop its natural
substrate, the fungal organism sets forth its entire genetic potential to produce the metabolites
necessary for its growth. The composition of the growth medium guides the microorganism's
metabolism towards the production of enzymes that release bio-available single molecules such
as sugars or amino acids by carving out macromolecules. Therefore, when selecting the
components of the growth medium it is possible to guide the cells towards the production of the
desired metabolite(s), mainly enzymes that transform polymers (cellulose, hemicellulose,
pectins, proteins) into single moieties in a very efficient and cost-effective manner.
Compared to submerged fermentation processes, solid state fermentation is

more cost-effective: smaller vessels, lower water consumption, reduced wastewater treatment
costs and lower energy consumption.
Solid substrates generally provide a good dwelling environment to the microbial

flora comprising bacteria, yeast and fungi. Among these, filamentous fungi are the best studied
for SSF due to their hyphal growth, which have the capability to not only grow on the surface of
the substrate particles but also penetrate through them.
2. Submerged Fermentation
Submerged fermentation is a method of manufacturing biomolecules in which

enzymes and other reactive compounds are submerged in a liquid such as alcohol, oil or a
nutrient broth. The process is used for a variety of purposes, mostly in industrial manufacturing.
It utilizes free flowing liquid substrates, such as corn steep liquor, molasses and nutrient broths.
The enzymes and bioactive compounds are secreted into the fermentation broth. The
substrates are utilized quite rapidly and hence need to be constantly supplemented with
nutrients. This fermentation technique is best suited for microorganisms such as bacteria that
require high moisture. Submerged fermentation takes place in large tanks that are filled with
liquid and then seeded with microorganisms that trigger the fermentation process. Fermentation
usually takes several days to complete, and a steady stream of oxygen must be circulated
through the tanks the entire time. Other variables that affect submerged fermentation include
the pH level of the liquid, temperature and ionic strength.

3. Anaerobic Fermentation
Anaerobic fermentation is a method cells use to extract energy from

carbohydrates when oxygen or other electron acceptors are not available in the surrounding
environment. This differentiates it from anaerobic respiration, which doesnt use oxygen but
does use electron-accepting molecules that come from outside of the cell. The process can
follow glycolysis as the next step in the breakdown of glucose and other sugars to produce
molecules of adenosine triphosphate (ATP) that create an energy source for the cell.
Through this method, a cell is able to regenerate nicotinamide adenine

dinucleotide (NAD+) from the reduced form of nicotinamide adenine dinucleotide (NADH), a
molecule necessary to continue glycolysis. Anaerobic fermentation relies on enzymes to add a
phosphate group to an individual adenosine diphosphate (ADP) molecule to produce ATP, which
means it is a form of substrate-level phosphorylation. This contrasts with oxidative
phosphorylation, which uses energy from an established proton gradient to produce ATP.
There are two major types of anaerobic fermentation: ethanol fermentation and
lactic acid fermentation. Both restore NAD+ to allow a cell to continue generating ATP through
glycolysis.
Ethanol fermentation converts two pyruvate molecules, the products of

glycolysis, to two molecules of ethanol and two molecules of carbon dioxide. The reaction is a
two-step process in which pyruvate is converted to acetaldehyde and carbon dioxide first, by the
enzyme pyruvate decarboxylase. In the second step, alcohol dehydrogenase converts
acetaldehyde to ethanol. This metabolic process occurs in certain types of bacteria cells and in
yeast cells. This makes yeast popular for making bread, beer, and wine, by using either the
carbon dioxide or the ethanol from fermentation.
Lactic acid fermentation is another form of anaerobic fermentation, and is

commonly used by muscle cells during times of stress when not enough oxygen is available.
These cells convert the two molecules of pyruvate from glycolysis into two molecules of Llactate using the enzyme lactate dehydrogenase. This process is known as homolactic
fermentation, because two molecules of pyruvate undergo the same chemical reactions, and
this form of lactic acid fermentation occurs in animal muscle cells and red blood cells. In
heterolactic fermentation, the pyruvate molecules undergo different chemical reactions. One is
converted to lactate, while the other is converted to ethanol and carbon dioxide. This process
occurs in some species of anaerobic organisms.

4. Aerobic Fermentation
Aerobic fermentation occurs when the cells demand more energy than can be
produced from an oxygen reaction. Part of the cellular reaction still occurs, and some ATP is
formed. The term "aerobic" means in the presence of oxygen; for example, it is used in aerobic
exercise to show the body is using oxygen to burn sugars for energy. This process of burning
simple sugars to produce energy in cells is called aerobic respiration. Human and plant cells
can create some energy without oxygen being present, a process called aerobic fermentation.
This term is perhaps mis-named, because fermentation is normally an anaerobic process, or
produced without oxygen. Humans have used fermentation for thousands of years to produce
alcohol from various plants and grains. The process uses a reaction between yeast and plant
sugars to produce alcohol and a gas by-product, usually carbon dioxide. Decay of plant and
animal matter in swamps, marshes and trash landfills is also an anaerobic fermentation
process, which produces carbon dioxide, methane and other gases.
Aerobic Fermentation VS Anaerobic Fermentation:
AEROBIC FERMENTATION
Favors cell and microbial growth

Favors ATP production
Less biomass production
ANAEROBIC FERMENTATION
Favors biomass production

Minimal cell and microbial growth
5. Immobilized Cell Bioreactors
Organisms growing in bioreactors may be submerged in liquid medium or may be

attached to the surface of a solid medium. Submerged cultures may be suspended or
immobilized. Suspension bioreactors can use a wider variety of organisms, since special
attachment surfaces are not needed, and can operate at much larger scale than immobilized
cultures. However, in a continuously operated process the organisms will be removed from the
reactor with the effluent. Immobilization is a general term describing a wide variety of cell or
particle attachment or entrapment. It can be applied to basically all types of biocatalysis
including enzymes, cellular organelles, animal and plant cells. Immobilization is useful for
continuously operated processes, since the organisms will not be removed with the reactor
effluent, but is limited in scale because the microbes are only present on the surfaces of the
vessel.
Pamantasan ng Lungsod ng Maynila

Department of Chemical Engineering
An immobilized cell bioreactor is disclosed wherein the cells are harbored within
or upon an immobilization matrix including cell support sheets comprised of common textile
fabric. The cell support sheets are oriented in a vertical parallel layered array with a gas phase
substantially surrounding each sheet. The vertical orientation allows nutrient culture supply and
product recovery to be assisted by gravity. The vertical orientation also allows the sheets to
extend into unused vertical space, producing a space-efficient bioreactor, and it also allows a
series of bioreactors to be closely and efficiently organized within the laboratory space.
Many industries and research facilities grow living cells for use in their
manufacturing and research processes. Such cells may include microorganisms (such as
bacteria, fungi, etc.) or cells from human, animal, plant, or other tissues. The cells are generally
grown in vitro, and their environment is engineered to coerce the cells into producing some
desired cell product, generally acids, enzymes, proteins, vitamins, bacteria, fungi, or other
metabolic by-products, e.g., ethanol and penicillin, or more cells per se, e.g., yeast.
The prior art reveals various apparatuses for culturing cells and harvesting cell
products produced by these cells. These apparatuses, frequently referred to in the art as
bioreactors, include immobilized cell bioreactors wherein the cells to be cultured are
immobilized by affixing them to components of the bioreactor. The cells are usually permanently
or temporarily entrapped upon, within, or behind an immobilization matrix or membrane
containing cell support surfaces, usually porous surfaces or surfaces having a gel or an electric
field thereupon. A nutrient cell culture solution is supplied to the cell support surfaces so that the
cells may feed upon it. As the cells feed upon the nutrient cell culture solution, they produce the
desired cell product, generally either more cells or some cell byproduct. The nutrient cell culture
solution (or some other carrier medium) carries the cell product and other cell wastes away from
the cell support surfaces. The cell product may then be removed from the bioreactor in batches,
or it may be removed continuously by collecting the cell product from the outgoing nutrient
stream or from a separate cell product stream.
6. Immobilized Enzyme Bioreactors
In the last decade, the bioreactors with fixed bed of biocatalysts (packed-bed
bioreactors) became some of the most used type of bioreactors, because of their low costs of
exploitation and maintenance, easiness of scaling-up and of automatic controlling, generation of
significant lower shear forces, and, consequently, avoidance of the breakage of biocatalysts
particles. The packed-bed bioreactors are used for wastewater treatment, biosorption from
wastewater of different metallic ions, solvents and fuels, pharmaceuticals and fine chemicals
production. Immobilization of biocatalysts helps in their economic reuse and in the development
of continuous bioprocesses. Biocatalysts can be immobilized either using the isolated enzymes
Fermentation Technology | Barros, D., Maninang, C., Sangalang, K.
32

or the whole cells. Immobilization often stabilizes structure of the enzymes, thereby allowing
their applications even under harsh environmental conditions of pH, temperature and organic
solvents, and, thus enable their uses at high temperatures in nonaqueous enzymology, and in
the fabrication of biosensor probes. In the future, development of techniques for the
immobilization of multienzymes along with cofactor regeneration and retention system can be
gainfully exploited in developing biochemical processes involving complex chemical
conversions. The present review outlines some of the above aspects, and delineates the
present status and future potentials of immobilized enzymes and nonviable cells in the emerging
biotech industries.
Biotechnology is currently considered as a useful alternative to conventional

process technology in industrial and analytical fields. This is mainly because, unlike the
chemical catalysts, the biological systems have the advantages of accomplishing complex
chemical conversions under mild environmental conditions with high specificity and efficiency.
Biological systems help in ingredient substitution, processing aid substitution, more efficient
processing, less undesirable products, increased plant capacity, increased product yields, and
improved or unique products. The variety of chemical transformations catalysed by enzymes
has made these catalysts a prime target of exploitation by the emerging biotech industries.
Despite these advantages, the use of enzymes in industrial applications has

been limited by several factors, mainly the high cost of the enzymes, their instability, and
availability in small amounts. Also the enzymes are soluble in aqueous media and it is difficult
and expensive to recover them from reactor effluents at the end of the catalytic process. This
restricts the use of soluble enzymes to batch operations, followed by disposal of the spent
enzyme-containing solvent. Over the last few decades, intense research in the area of enzyme
technology has provided many approaches that facilitate their practical applications. Among
them, the newer technological developments in the field of immobilized biocatalysts can offer
the possibility of a wider and more economical exploitation of biocatalysts in industry, waste
treatment, medicine, and in the development of bioprocess monitoring devices like the
biosensor.
Immobilization means associating the biocatalysts with an insoluble matrix, so

that it can be retained in a proper reactor geometry for its economic reuse under stabilized
conditions. Immobilization thus allows, by essence, to decouple the enzyme location from the
flow of the liquid carrying the reagents and products. Immobilization helps in the development of
continuous processes allowing more economic organization of the operations, automation,
decrease of labour, and investment/capacity ratio. Immobilized biocatalysts offer several other
advantages, notable among them is the availability of the product in greater purity. Purity of the
product is very crucial in food processing and pharmaceutical industry since contamination
could cause serious toxicological, sensory, or immunological problems. The other major
advantages include greater control over enzymatic reaction as well as high volumetric
productivity with lower residence time, which are of great significance in the food industry,
33

especially in the treatment of perishable commodities as well as in other applications involving
labile substrates, intermediates or products
III.
Fermenter Design & Cell Kinetics
Fermenter Design:
A bioreactor is a device in which a substrate of low value is utilized by living cells

or enzymes to generate a product of higher value. Bioreactors are extensively used for food
processing, fermentation, waste treatment, etc. On the basis of the agent used, bioreactors are
grouped into the following two broad classes: (i) those based on living cells and, (ii) those
employing enzymes. But in terms of process requirements, they are of the following types: (i)
aerobic, (ii) anaerobic, (iii) solid state, and (iv) immobilized cell bioreactors. All bioreactors deal
with heterogeneous systems dealing with two or more phases, e.g., liquid, gas, solid. Therefore,
optimal conditions for fermentation necessitate efficient transfer of mass, heat and momentum
from one phase to the other. Chemical engineering principles are employed for design and
operation of bioreactors. But, in general, theoretical explanation usually lags behind technical
realization.
Agitation & Aeration:
In scaling up, both chemical (oxygen, pH, medium constituents and removal of
wastes) and physical (the configuration of bioreactor and power supplied to the reactor) factors
have to be optimised for good results.The medium must be suitably stirred to keep the cells in
suspension and to make the culture homogeneous; it becomes increasingly difficult with the
scaling up. Various types of stirrers range from simple magnetic stirrers, flat blade turbine
impellers, to marine impellers, to those using pneumatic energy, e.g., airlift fermenter, and those
using hydraulic energy, e.g., medium perfusion.
34

The basic objective of aeration is to provide microorganisms growing in

submerged cultures with adequate oxygen for their metabolic needs. Agitation, on the other
hand, aims to ensure a homogeneous distribution of microorganisms and the nutrients in the
broth. The type of aeration- agitation system used in the fermenter is dictated by the
characteristics of the fermentation process. For example, in processes based on low viscosity,
low total solids broths, agitation may not be needed as aeration itself would create the
necessary agitation.
1. Agitator (Impeller)
Agitators achieve the following

objectives; (a) bulk fluid and gas-phase mixing, (b)
air dispersion, (c) oxygen transfer, (d) heat transfer,
(e) suspension of solid particles, and (f)
maintenance of a uniform environment throughout
the vessel. These objectives are achieved by a
suitable combination of the most appropriate
agitator, air sparger and baffles, and the best
positions for nutrient feeds, acid or alkali for pH
control and antifoam addition. Agitators are of
several different types, e.g., (i) disc turbines, (ii)
vaned discs, (iii) open turbines of variable pitch and
(iv) propellers.
2. Stirrer Glands & Bearings
The satisfactory sealing of the stirrer

shaft assembly has been one of the most difficult
problems; this is very important for maintaining
aseptic conditions over long periods. Four basic
types of seal assembly have been used in
fermenters: (1) the stuffing box (packed- gland
seal), (2) the simple bush seal, (3) the mechanical seal and (4) the magnetic drive.<p>Most
modern fermenters use mechanical seals; these seals are more expensive, but they are more
durable and less prone to leakage or contaminant entry. Magnetic drives, although quite
expensive, are being used in some animal cell culture vessels. The mechanical seal consists of
two parts; one part remains stationery in the bearing housing, while the other rotates on the
shaft. The two components of the seal are pressed together by springs or expanding bellows.
Steam condensate is used to lubricate and cool the seals during operation and servers as a
contaminant barrier.
35

3. Baffles
Baffles are metal strips roughly one-tenth of the vessel diameter and attached radially to
the fermenter wall. They are normally used in fermenters having agitators to prevent vortex
formation and to improve aeration efficiency. Usually, four baffles are used, but larger
fermenters may have 6 or 8 baffles. Extra cooling coils may be attached to baffles to
improve cooling. Further, the baffles may be installed in such a way that a gap exists
between the baffles and the fermenter wall. This would lead to a scouring action around
and behind the baffles, which would minimise microbial growth on the baffles and the
fermenter wall.
4. Aeration System (Sparger)
The device used to introduce air into the fermenter broth is called sparger.
Spargers are of the following three basic types: (1) porous spargers, (2) orifice spargers and (3)
nozzle spargers. Porous spargers may be made of sintered glass, ceramics or a metal. They
are used primarily on a laboratory scale in non-agitated vessels. The bubble size from such
spargers is always 10 to 100 limes larger than the pore size of the sparger. These spargers
have low air throughput because pressure drops across the sparger, and the fine holes often
become blocked by microbial growth.
IV.
Batch Cultivation
Bacterial growth is the asexual reproduction, or cell division, of a bacterium into

two daughter cells, in a process called binary fission. Providing no mutational event occurs the
resulting daughter cells are genetically identical to the original cell. Hence, "local doubling" of
the bacterial population occurs. Both daughter cells from the division do not necessarily survive.
However, if the number surviving exceeds unity on average, the bacterial population undergoes
exponential growth. The measurement of an exponential bacterial growth curve in batch culture
was traditionally a part of the training of all microbiologists.
In autecological studies, the growth of bacteria (or other microorganisms,

as protozoa, microalgae or yeasts) in batch culture can be modeled with four different
phases: lag phase (A), log phase or exponential phase (B), stationary phase (C), and death
phase(D).
36

1. During lag phase, bacteria adapt themselves to growth conditions. It is the period where the
individual bacteria are maturing and not yet able to divide. During the lag phase of the
bacterial growth cycle, synthesis of RNA, enzymes and other molecules occurs.
2. The log phase (sometimes called the logarithmic phase or the exponential phase) is a
period characterized by cell doubling. The number of new bacteria appearing per unit time is
proportional to the present population. If growth is not limited, doubling will continue at a
constant rate so both the number of cells and the rate of population increase doubles with
each consecutive time period. For this type of exponential growth, plotting the natural
logarithm of cell number against time produces a straight line. The slope of this line is the
specific growth rate of the organism, which is a measure of the number of divisions per cell
per unit time. The actual rate of this growth (i.e. the slope of the line in the figure) depends
upon the growth conditions, which affect the frequency of cell division events and the
probability of both daughter cells surviving. Under controlled conditions, cyanobacteria can
double their population four times a day. Exponential growth cannot continue indefinitely,
however, because the medium is soon depleted of nutrients and enriched with wastes.
3. The stationary phase is often due to a growth-limiting factor such as the depletion of an
essential nutrient, and/or the formation of an inhibitory product such as an organic acid.
Stationary phase results from a situation in which growth rate and death rate are equal. The
number of new cells created is limited by the growth factor and as a result the rate of cell
growth matches the rate of cell death. The result is a smooth, horizontal linear part of the
curve during the stationary phase.
4. At death phase (decline phase), bacteria die. This could be caused by lack of nutrients,
environmental temperature above or below the tolerance band for the species, or other
injurious conditions.
37

This basic batch culture growth model draws out and emphasizes aspects of
bacterial growth which may differ from the growth of macrofauna. It emphasizes clonality,
asexual binary division, the short development time relative to replication itself, the seemingly
low death rate, the need to move from a dormant state to a reproductive state or to condition the
media, and finally, the tendency of lab adapted strains to exhaust their nutrients. In reality, even
in batch culture, the four phases are not well defined. The cells do not reproduce in synchrony
without explicit and continual prompting (as in experiments with stalked bacteria [6]) and their
exponential phase growth is often not ever a constant rate, but instead a slowly decaying rate, a
constant stochastic response to pressures both to reproduce and to go dormant in the face of
declining nutrient concentrations and increasing waste concentrations.
Batch culture is the most common laboratory growth method in which bacterial
growth is studied, but it is only one of many. It is ideally spatially unstructured and temporally
structured. The bacterial culture is incubated in a closed vessel with a single batch of medium.
In some experimental regimes, some of the bacterial culture is periodically removed and added
to fresh sterile medium. In the extreme case, this leads to the continual renewal of the nutrients.
Computation for Microbial Growth:
Bacterium
Medium
Generation Time
(minutes)
Escherichia coli
Glucose-salts
17
Bacillus megaterium
Sucrose-salts
25
Streptococcus lactis
Milk
26
Streptococcus lactis
Lactose broth
48
Staphylococcus aureus
Heart infusion broth
Lactobacillus acidophilus
Rhizobium japonicum
Mannitol-salts-yeast
extract
Mycobacterium
tuberculosis
Treponema pallidum
Milk
Synthetic
27-30
66-87
344-461
792-932
Rabbit testes
1980
38

G (generation time) = (time, in minutes or hours)/n(number of generations)
t = time interval in hours or minutes
B = number of bacteria at the beginning of a time interval
G = t/n
b = number of bacteria at the end of the time interval
n = number of generations (number of times the cell population doubles during the time
interval)
b = B x 2n (This equation is an expression of growth by binary fission)
Solve for n:
logb = logB + nlog2
n = logb - logB
log2
n = logb - logB
.301
n = 3.3 logb/B
G = t/n
39

Stirred Tank Fermenter
Microbial fermentations received prominence during 1940's namely for the production of life
saving antibiotics. Stirred tank reactor is the choice for many (more than 70%) though it is
not the best. Stirred tank reactors have the following functions: homogenization,
suspension of solids, dispersion of gas-liquid mixtures, aeration of liquid and heat
exchange. The Stirred tank reactor is provided with a baffle and a rotating stirrer is attached
either at the top or at the bottom of the bioreactor. The typical decision variables are: type,
size, location and the number of impellers; sparger size and location. These determine the
hydrodynamic pattern in the reactor, which in turn influence mixing times, mass and heat
transfer coefficients, shear rates etc. The conventional fermentation is carried out in a batch
mode. Since stirred tank reactors are commonly used for batch processes with slight
modifications, these reactors are simple in design and easier to operate. Many of the
industrial bioprocesses even today are being carried out in batch reactors though
significant developments have taken place in the recent years in reactor design, the
industry, still prefers stirred tanks because in case of contamination or any other
substandard product formation the loss is minimal. The batch stirred tanks generally suffer
due to their low volumetric productivity. The downtimes are quite large and unsteady state
fermentation imposes stress to the microbial cultures due to nutritional limitations. The fed
batch mode adopted in the recent years eliminates this limitation. The Stirred tank reactors
offer excellent mixing and reasonably good mass transfer rates. The cost of operation is
40

lower and the reactors can be used with a variety of microbial species. Since stirred tank
reactor is commonly used in chemical industry the mixing concepts are well developed.
Stirred tank reactor with immobilized cells is not favored generally due to attrition problems;
however by separating the zone of mixing from the zone of cell culturing one can
successfully operate the system.
Plug Flow Reactor
Plug Flow Reactor - In this type of continuous fermentation, the culture solution flows
through a tubular reactor without back mixing. The composition of the nutrient solution, the
number of cells, mass transfer, and productivity vary at different locations within the
system. At the entrance to the reactor, cells must be continuously added along with the
nutrient solution.
In a plug flow reactor, nutrients (and sometimes organisms) are introduced to the reactor
continuously and move through the reactor as a plug. The system may be either
contained (as in a water main, oil pipeline, or blood vessel) or open (as in a shower curtain,
stream, or canyon seep).
In an ideal plug flow reactor, it is assumed that there is no mixing of the medium along the
long axis (X-axis) of the reactor although there may be lateral mixing in the medium at any
point along the long axis (ie the Y-axis).
Due to the metabolic activity of the organisms in the biofilm, constituents of the reactor will
change as medium flows along the long axis due to consumption of nutrients and
elimination of waste products. The nutritional conditions at any given point along this long
axis should, however, remain constant in a stable reactor. On the other hand, as an
41

organism moves through the PFR it encounters constantly changing concentrations of
nutrients, oxygen and waste products.
In a PFR, one or more fluid reagents are pumped through a pipe or tube. The chemical
reaction proceeds as the reagents travel through the PFR. In this type of reactor, the
changing reaction rate creates a gradient with respect to distance traversed; at the inlet to
the PFR the rate is very high, but as the concentrations of the reagents decrease and the
concentration of the product(s) increases the reaction rate slows. Some important aspects
of the PFR:
All calculations performed with PFRs assume no upstream or downstream mixing, as

implied by the term "plug flow".
Reagents may be introduced into the PFR at locations in the reactor other than the inlet. In
this way, a higher efficiency may be obtained, or the size and cost of the PFR may be
reduced.
A PFR typically has a higher efficiency than a CSTR of the same volume. That is, given the
same space-time (or residence time), a reaction will typically proceed to a higher
percentage completion in a PFR than in a CSTR. This is not always true for reversible
reactions.
For most chemical reactions of industrial interest, it is impossible for the reaction to proceed
to 100% completion. The rate of reaction decreases as the reactants are consumed until
the point where the system reaches dynamic equilibrium (no net reaction, or change in
chemical species occurs). The equilibrium point for most systems is less than 100%
complete. For this reason a separation process, such as distillation, often follows a
chemical reactor in order to separate any remaining reagents or byproducts from the
desired product. These reagents may sometimes be reused at the beginning of the
process, such as in the Haber process. In some cases, very large reactors would be
necessary to approach equilibrium, and chemical engineers may choose to separate the
partially reacted mixture and recycle the leftover reactants.
Kind of Phases Represent: Gas Phase
Usage:
1. Large Scale
2. Fast Reactions
3. Homogeneous Reactions
42

4. Heterogeneous Reactions
5. Continuous Production
6. High Temperature
Advantages
1. High Conversion per Unit Volume
2. Low operating (labor) cost)
3. Continuous Operation
4. Good heat transfer
Disadvantages
1. Undesired thermal gradients may exist
2. Poor temperature control
3. Shutdown and cleaning may be expensive
Productivity in Fermentation Process
43

Figure 1. Fermentation rates and productivity
To avoid confusion, the term productivity has been recommended for the time-average
output of a process.9 The expression fermentation rate can then be reserved for the
instantaneous rate of change of any concentration factor sugar, product, cell weight, etc.
These distinctions are shown graphically in Fig. 1. Productivity is defined as the final
product concentration divided by the time from inoculation to delivery of the batch. It might
seem more reasonable to divide by the total process time from delivery of one batch to
delivery of the next. This would include many operational factors involved in turnover of a
tank, like cleaning, batching and filling, which have little or nothing to do with the actual
fermentation system. While it is essential for proper economic analysis of the plant, such an
overall productivity has little use in analyzing the fermentation process itself.
Productivity is:
The time-average output of a process
The final product concentration divided by the time from inoculation to delivery of the
batch
44

The higher the productivity, the smaller the fermenter volume that is needed for the
desired product
Techniques in Increasing Productivity
Cell Immobilization
Cell Recycle
Cell Immobilization
The cells are retained inside the reactor through:
Attachment to a surface
Entrapment within porous matrices
Containment behind a barrier
Self-aggregation
Cell Recycle
The cells are separated from the product by:
Filtration
Centrifugation
Settling,
followed by returning the cells back to the reactor
Multiple Fermenters in Series
The previous modeling clearly proves that the MHR acts globally as a CSTR
Nevertheless, the simultaneous analysis of permeate and recirculate samples allows

Verification that the recirculate and permeate concentrations were different whatever the
experimental conditions, product concentration was always higher in permeate than in
recirculate
and
the
ratio
n
the
In parallel with the global reactor analysis, it can IV proposed that the membrane pores
may act like a second CSTR although with a higher residence time (there is no recirculation
inside the membrane matrix. The main consequence will be the achievement of higher
conversion degrees, when compared with one unique CSIR. Since the increment in
permeate conversion was remarkable this may raise the possibility of having the inside of
the membrane acting as a PFR.
45

Both hypotheses require enzymes to be retained in the pores of the membrane. In each
series of reactors there is a distribution of cutinase on membrane surface and in the pores
accounting the conversions detected in the recirculate and in the permeate.
The two CSTRs model desrbie the results very well. When the now rate is low, recirculation
decreases, but by maintaining high recirculating flow rates, the MBR acts as a CSTR on the
hollow membrane side. The flux through the membrane pores significantly lower, and times
causing a deviation from CSTR characteristics. Therefore, the inside (matrix) of the
membrane
is
classified
as
a
second
CSTR.
Besides this reactor sequence being a good model for the experimental behavior of the
MBR, it also upholds the fact that two CSTRs are more efficient than a unique CSTR
Accordingly, in the design and analysis of biological reactors for process optimization, the
sequence of reactors plays an important role. Most of applications described in the
literature are related to fermenters. To obtain high final cell concentrations, the best
46

combinations are always multiple fermenters connected in senes (Lee, 1992). Bailey and
Ollis (1977) and Levenspiel (1972) also discussed the efficiency of reactor and that a
CSTR-PFR Sequence or a cascade of two CSTR maximize product yield and are suitable
for a wide variety of process kinetics and design
Analysis of stability on a series CSTR

From the aforementioned, the stability be determined for the two CSTRs. The
determination of half-life time is again based on conversion degree obtained in the
retentate and permeate, The enzyme on the membrane surface (if using the retentate
conversion degree evolution) has a raster deactivation (43 times faster) than that observed
for the enzymes entrapped in pore. The explanation for this is based on the exposition
conditions of the two portions of enzvme. The cutinase adsorbed onto the surface of the
membrane supports high shear forces caused by the fluid, which accelerates the
denaturing process. be understcxod bv the calculation of the linear velocity at the
membrane surface.
The velocity on the surface of the membrane is 10^5 times higher than inside the
membrane pores. These velocities have quite different effects on the properties of the fluid
circulation in the reactor.
References:
http://bioprocess-maulik.blogspot. com/2007/07/types-of-culture-systems.html
Principles of Fermentation Technology by Peter F. Stanbury,Allan Whitaker,Stephen J.
Hall
Bioprocess Technology and Fermentation referred fromPrinciples of Fermentation Technology, P.F. Stanbury & A. Whitaker, Pergamon Press.
Industrial Microbiology by Casida
Industrial Microbiology by Prescott
Lowenstein JM (1969). Methods in Enzymology, Volume 13: Citric Acid Cycle. Boston:
Academic Press.
Krebs HA, Weitzman PD (1987). Krebs' citric acid cycle: half a century and still turning.
London: Biochemical Society. p. 25.
Wagner, Andreas (2014). Arrival of the Fittest (first ed.). New York: Penguin Group. p.
100.
Lane, Nick (2009). Life Ascending: The Ten Great Inventions of Evolution. New York:
W.W. Norton & Co.
"The Nobel Prize in Physiology or Medicine 1937". The Nobel Foundation. Retrieved
2011-10-26.
Gest H (1987). "Evolutionary roots of the citric acid cycle in prokaryotes". Biochem. Soc.
Symp. 54: 316.
Lehninger Principles of Biochemistry. 5th Edition. Chapter 15 : The Citric Acid Cycle.
Clackamas Community College. Citric Acid Cycle.
Shmoop. Citric Acid Cycle.
47

Gary E. Kaiser. Community College of Baltimore County. The Citric (Krebs) Acid Cycle.
Newcastle University Teaching Server. School of Biomedical Sciences Wiki. Krebs
cycle.
University of California, Davis. Chem Wiki. Krebs Cycle.
Khan Academy. Krebs / citric acid cycle.
Tutor Vista. Biology. Citric Acid Cycle.
Study.com. The Citric Acid (Krebs) Cycle: Products and Steps.
Biesebeke, R.; Ruijter, G.; Rahardjo, Y.S.P.; Hoogschagen, M.J.; Heerikhuisen, M.;
Levin, A,; van Driel, K.G.A.; Schutyser, M.A.I.; Dijksterhuis, J.; Zhu, Y.; Weber, F.J.; de
Vos, W.M.; van den Hondel, K.A.M.J.J.; Rinzema, A.; Punt, PJ, (March 2002).
"Aspergillus oryzae in solid-state and submerged fermentations Progress report on a
multi-disciplinary project". FEMS Yeast Res. 2: 245248.
Capalbo; Valicente, F.H.; Moraes, I.O.; Pelizer, M.H. (August 2001). "Solid-state
fermentation of Bacillus thuringiensis tolworthi to control fall armyworm in maize".
Electronic J. Biotechnol 4 (2): 15.
Durand, A. (March 2003). "Bioreactor designs for solid state fermentation". Biochem.
Eng. J. 13 (23): 113125
Singhania, R.R.; Patel, A.K.; Soccol, C.R.; Pandey, A. (April 2009). "Recent Advances in
solid-state fermentation". Biochem. Eng. J. 44 (1): 1318.
48

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PAMANTASAN NG LUNGSOD NG MAYNILA

University of the City of Manila

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5 Use of an organic substrate as the electron donor and acceptor.

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TYPES OF FERMENTATION CULTURE

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Nutritional Requirements and Media for Fermentation Processes

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Knowledge of the elemental composition of a process micro-organism is required for the

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CARBOHYDRATES It is common practice to use carbohydrates as the carbon source in

OILS AND FATS Oils were first used as carriers for

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hydrocarbons and their derivatives as fermentation substrates, particularly with reference to

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Corn sugar, Starch, Cellulose

Sugarcane, Sugar beet molasses

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Pure ammonia or ammonium salts

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The 8 Enzymes of the Krebs Cycle:

Succinyl coenzyme A synthetase

Through catabolism of sugars, fats, and proteins, a two-carbon organic product

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The Net Equation:

Reaction 1: Formation of Citrate

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Reaction 2: Formation of Isocitrate

Reaction 3: Oxidation of Isocitrate to -Ketoglutarate

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of isocitrate to form -ketoglutarate. In the reaction, generation of NADH from NAD is

Reaction 4: Oxidation of -Ketoglutarate to Succinyl-CoA

Alpha-ketoglutarate is oxidized, carbon dioxide is removed, and coenzyme A is added

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Reaction 5: Conversion of Succinyl-CoA to Succinate

Reaction 6: Oxidation of Succinate to Fumarate

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Reaction 7: Hydration of Fumarate to Malate

The reversible hydration of fumarate to L-malate is catalyzed by fumarase (fumarate

Reaction 8: Oxidation of Malate to Oxaloacetate

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Total ATP = 12 ATP

Significance of Krebs Cycle:

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In an eight-step reaction sequence, the acetyl group of acetyl-CoA is oxidised

Example from Everyday Life:

Without vitamin C we cannot even sustain life. Vitamin C is an essential molecule

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Fermentation is a metabolic process that converts sugar to acids, gases, or

Solid State Fermentation

1. Solid State Fermentation

Solid-state fermentation has emerged as a potential technology for the