 The use of microorganisms to produce
commercially valuable products
 Industrial microbiology includes many areas,
including:
 food production,
 pharmaceuticals,
 fuel, bioremediation,
 Biomining
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Bacteria
Fungi
Yeast
Protozoa

Using scientific methods with organisms to
produce new products or new forms of
organisms
 Biotechnology

use of an organism’s biochemical and
metabolic pathways for industrial production
The term can be used in two contexts
 In its broad context, “fermentation” means the
growth of microorganisms for the purpose of
manufacturing a product
 In its narrow context, “fermentation” refers to a
specific set of metabolic pathways in which
pyruvic acid is reduced to form reduced waste
products, with the regeneration of NAD for
glycolysis
 The medium or growth substrate on which the
microorganism is grown
 Some processes may use crude organic
components as media; others may required more
purified substrates
 Individual media components should provide a
readily available, inexpensive nutrient,
maximising production
 Other considerations include energy
requirements, inhibitory substances, state of
microbe etc
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

Nitrogen source-urea, corn steep liquor,
yeast extract, soy flour
Ammonia- ammonium sulphate
Buffering capacity- calcium carbonate,
phosphates
Antifoam-soy oil, vegetable oil, polypropylene
glycol
 Nitrate- nitrate salts
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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.
Fin=Fout=0
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dX/dT=µXi
X=biomass
T=time
µ=specific growth rate
Monod equation
Limiting substrate
µ=µMAX[S]
Ks+S
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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
Fin=Fout
New growth, stimulated by the added medium, and
depends on specific growth rate (µ)
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 (D)
D=F/V
 @ Steady state
 D=µ
If µ<D, Biomass overflows
If µ>D, Biomass increases
The steady state will be re-established.
 Hence, a chemostat is a nutrient limited selfbalancing culture system, which may be
maintained in a steady state over a wide range of
sub-maximum specific growth rates.
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In the fed-batch system, a fresh aliquot of the medium is
continuously or periodically added, without the removal of
the culture fluid. The system is always at a quasi-steady
state.
Fixed volume or Variable volume
Fin>0, Fout=0
Advantages:
a) maintaining conditions
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
Production of baker's yeast is mostly by fed-batch culture,
where biomass is the desired product.
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Temperature
pH
Mass transfer
1.Mass transfer step
2. Catalytic step
Oxygen transfer
OTR=kLa(C*-C)
Agitation
Inflow rate
Oxygen enrichement
Producer microorganism
◦ The organism used for a particular process
◦ Some are naturally occurring strains; others have
been modified through genetic manipulation
Trophophase
◦ The period of active growth of a microbe
◦ Equivalent to the logarithmic (exponential)
growth period
Figure 28.11a
Idiophase
 The period following trophophase, during which
microbial biomass production has peaked and
no new net biomass is produced
 Equivalent to stationary phase
Primary metabolites
 Microbial products produced during trophophase
 Examples include amino acids, nucleotides,
fermentation end products, and many types of
enzymes
Figure 28.11b

Products produced during idiophase
Examples include many antibiotics and
mycotoxins
 not essential for growth
 dependent on growth conditions (repression)
 over-production often achievable
(not growth related)
 often produced as a series of closely related
compds
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
After an organism producing a valuable product
is identified, it becomes necessary to increase the
product yield from fermentation to minimise
production costs. Product yields can be increased
by
(i) developing a suitable medium for
fermentation,
(ii) refining the fermentation process and
(iii) improving the productivity of the strain.

Strain improvement is based on the following
three approaches:
(i) mutant selection,
(ii) recombination, and
(iii) recombinant DNA technology.
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Periodic transfer + refrigeration
Mineral oil slant + refrigeration
Washed culture + refrigeration
Freezing
Freezing with 50% glycerol
Drying
Lyophilization (freeze drying)
Ultracold freezing

complex chemical transformations. Microbes
become biocatalysts
Figure 28.12

Biosensors
◦ Devices in which a biospecific molecule (e.g., a
monoclonal antibody or a hormone receptor
protein) is attached to a “transducer” (often a
piezoelectrically-active quartz chip)
◦ When the biosensor binds to its target, it slighty
“twists” the transducer, creating a small electrical
current that can be amplified, detected, and
measured
Fermenter
 A vessel in which fermentation is carried out
 The fermenter must include systems to regulate
key growth requirements, such as nutrient
addition, temperature, oxygen, and pH
Upstream processing
 Components of the production system that occur
prior to fermentation
 Includes cleaning, formulation of the medium,
sterilization of the vessel and medium, adding
the medium and organism to the vessel, etc.
 Downstream processing
 Components of the production system that occur
after fermentation
 Includes harvesting and purification of the
product, disposal or the waste, etc.
 Some products are intracellular, which means
that the cells have to be harvested and lysed to
release the product
 Other products are secreted into the medium,
from which they may be purified
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Amino acids
Citric acid
Enzymes
Vitamins
Antibiotics
Steroids
UN 28.1
Pharmaceuticals
1.Antibiotics, alkaloids, steroids, vaccines
2.Recombinant human proteins, such as insulin,
growth hormone, and interferon
Microbial enzymes
1.“Bulk”enzymes, such as hydrolytic enzymes, can
be used with minimal DSP in partially purified
form
2.Other enzymes are highly purified for
specialized purposes, such as restriction
endonucleases
Figure 28.10
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Bioreactor
Vessel
Impeller
Spanger
Baffles
Water jacket
Basic features(CSTR)
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1. Activated sludge (CSTR)
2.Air Lift Bioreactor
3.Bubble column Bioreactor
4. Photobioreactor Bioreactor
5, Hybrid Bioreactor
5.Mist Bioreactor
6. Packed Bed Bioreactor
7.Rotating Bioreactor
8. Tower Bioreactor
9.Trickling Film Bioreactor
Large-scale industrial fermentation presents several
engineering problems while the microbial process must be
continuously monitored to ensure yield and no
contamination.
 Fermentation scale-up is an art in itself –optimization of
laboratory scale to production scale.
 Industrial fermentations will be anaerobic and aerobic
processes
 aeration in the latter being critical, and requires special
attention to stirring (impellor)/bubbling (sparger).
 Both require heat removal systems.
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A) Solid-Liquid separation
Flotation, Flocculation, Filtration
B) Cell disruption techniques
1. Physical- ultrsonication, Osmotic shock,
heat shock, Homogenisation
2. Chemical- alkali,organic solvents,
detergents
3.Enzymatic- Lysozyme, Glucanase
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C) Concentration
Evaporation
Liquid-liquid separation
Membrane filtration
Precipitation
D) Purification
Chromatography –ion exchange, affinity
chromatography
E.) Formulation
Mantainance and stabilisation
Spray drying and freeze drying
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Brew house processes-milling, mashing in,
lautering, kettle boiling and wort cooling
Fermentation processes-primary
fermentation and secondary fermentation.
Storage ,conditioning, maturation and
filtration
Packaging-beer filling , pasteurization and
labelling

Raw materials
Starch source-ranges from barely malt,
cornstarch, sorghum
Hops, water
Adjuncts-calcium sulphate, lactic acid at times
sugar
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Malting is the process where the barley grain
is made ready for brewing.
Malting is broken down into three steps
1.Steeping,
2.Germination
3.Kilning which help to release the starches in
the barley
Milling-The next step in the brewing process
is milling. This is when the grains that are
going to be used in a batch of beer are
cracked.
 Milling the grains makes it easier for them to
absorb the water that they are mixed with
and which extracts sugars from the malt
leftover sugar rich water is then strained
through the bottom of the mash in a process
known as lautering
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Boiling-evaporation of excess water
Centrifugal separation-separate residual
leftover spent grain
Transfer to ferment-through pipeline
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Primary fermentationFermentation in brewing is the conversion of
carbohydrates to alcohols and carbon dioxide
or organic acids using yeasts, bacteria, or a
combination thereof, under anaerobic
conditions.
A more restricted definition of fermentation
is the chemical conversion of sugars into
ethanol

Lager yeast (Saccharomyces cerevisae )
typically undergoes primary fermentation at
7–12 °C (45–54 °F) (the fermentation phase),
and then is given a long secondary
fermentation at 0–4 °C (32–39 °F) (the
lagering phase).
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Ethanol fermentation
Ethanol fermentation (performed by yeast and
some types of bacteria breaks the pyruvate
down into ethanol and carbon dioxide.
in alcohol production, the carbon dioxide is
released into the atmosphere or used for
carbonating the beverage.
chemical equation
C6H12O6 → 2C2H5OH + 2CO2
Before fermentation takes place, one glucose
molecule is broken down into two pyruvate
molecules. This is known as glycolysis.


Top-Fermenting Yeast
Ale yeast strains are best used at temperatures ranging
from 10 to 25°C, though some strains will not actively
ferment below 12°C (33).
Ale yeasts are generally regarded as top-fermenting
yeasts since they rise to the surface during fermentation,
creating a very thick, rich yeast head. That is why the term
"top-fermenting" is associated with ale yeasts.
Fermentation by ale yeasts at these relatively warmer
temperatures produces a beer high in esters, which many
regard as a distinctive character of ale beers.
Top-fermenting yeasts are used for brewing ales, porters,
stouts, and wheat beers.
.
Bottom-Fermenting Yeast
◦ Lager yeast strains are best used at temperatures
ranging from 7 to 15°C. At these temperatures,
lager yeasts grow less rapidly than ale yeasts,
◦ less surface foam they tend to settle out to the
bottom of the fermenter as fermentation nears
completion. This is why they are often referred to
as "bottom" yeasts.
◦ The final flavour of the beer will depend a great
deal on the strain of lager yeast and the
temperatures at which it was fermented.
Table 28.5
Yeast cryovial(5ml) are used at the onset of
yeast propagation in the lab
 In the lab
5ml is transferred to 15ml then to 100ml to
200ml then 10l
In yeast plant ascetically transferred into
Small pot(10hl) then to medium pot (100hl)
then to large pot(300HL) then into
fermentation vessel

less
than
3%abv
14%
abv
20% abv
Repitch
WITH
CHAMPAG
NE YEAST
55% abv
by the
freezedistilling
process
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Carbon dioxide
Ethanol
Flavour compounds
Secondary fermentation-temporary storage in
storage vessel, with controlled temperatures
and infeed of sterile air
 Beer maturation-flavour development,
clarification
,standardization ,
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When the sugars in the fermenting beer have been almost
completely digested, the fermentation slows down and the
yeast starts to settle to the bottom of the tank.
At this stage, the beer is cooled to around freezing, which
encourages settling of the yeast, and causes proteins to
coagulate and settle out with the yeast. Unpleasant
flavours such as phenolic compounds become insoluble in
the cold beer, and the beer's flavour becomes smoother.
During this time pressure is maintained on the tanks to
prevent the beer from going flat.
Conditioning can take from 2 to 4 weeks, sometimes
longer, depending on the type of beer. . This cold aging
serves to reduce sulfur compounds produced by the
bottom-fermenting yeast and to produce a cleaner tasting
final product with fewer esters.
This is where aging occurs.
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Filtering
Filtering the beer stabilizes the flavour, and
gives beer its polished shine and brilliance
Many use pre-made filtration media such as
sheets or candles, while others use a fine
powder made of, for example, diatomaceous
earth, also called kieselguhr, which is
introduced into the beer and recirculated past
screens to form a filtration bed.
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Overview of packaging process
Depalletiser
Decrator
Washer
filler
Pasteuriser
Full’s sighter
Labeller
Crator
palletiser
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The most common species of wine grape is Vitis
vinifera
Red wine is made from the must (pulp) of red or
black grapes that undergo fermentation together with
the grape skins
White wine is made by fermenting juice which is
made by pressing crushed grapes to extract a
the Saccharomyces cerevisiae (also known as "sugar
yeast") species is used for winemaking.
Typically white wine is fermented between 18-20 °C
Red wine is typically fermented at higher
temperatures up to 29 °C.
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Factors affecting microbial spoilage
Acidity of pH
Sugar content
Concentration of alcohol
Concentration of tannins
Amount of sulfur dioxide present
Storage temp
Availability of air
Made from the finest
Red Grapes
 Harvested when they
are red juicy and ripe
 Fermented and
matured according to
Brewer’s taste.

Figure 28.9
1.
Harvest
-ripe grapes are picked
2.
Destemmer and Crusher
-removes the stems, and crushes the grapes
3.
Maceration
-the must is allowed to sit,
Fermentation
4.
-sugar
5.
carbon dioxide and (ethanol)
Pumping over
-"cap" is punched down, or must is pumped
from the bottom of the tank over the cap.
6.
Drain free-run juice
-the juice portion of the must is removed
and pomace is sent to the press.
7.
Press
-squeezes the remaining juice out of the
pomace.
8.
Cold settle juice
-juice (wine) needs to settle.
9.
Racking
-wine moved from one tank to another
10.
Malolactic fermentation
-secondary fermentation, turns the tart malic
acid into the softer lactic acid.
11.
Barrel aging
-usually aged in barrels for 6 months
12.
Fining and stabilisation
-removal of anything that may be making
wine cloudy.
13.
Filtering
-removal of any fining agents, or other
undesirable elements
14.
Bottling
-wine should not come in contact with air.
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Sparkling wines
Carbonated (artifitially)
Dry wines
Sweet wines
Fortified wines
Table wines
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The industrial production of L-glutamate (LGln) started with its fermentation in the late
1960s.
Obtained by using Corynebacterium
glutamicum
it is now manufactured for use as
pharmaceuticals and health foods at the
worldwide annual production of 2000 metric
tons.
CO2
glucose
Phosphoenol
pyruvate
pyruvate
Acetyl
coA
CO2
oxaloacetate
Malate
citrate
Succinyl
coA
CO2
Alpha
ketogluterate
Lglutamate
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Compared with wild-type strains, LGlutamate producing strains are weak and are
compromised in a contaminated
environment.
it is important to maintain the tank under
positive pressure by aeration during
fermentation to prevent contamination by
other microorganisms and external materials.
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Glucose
Ammonia
Minerals (small amounts)
Vitamins (small amounts)
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pH
Temperature
Dissolved oxygen
Glucose concentration
Dissolved
nutrients
Sugar tank
Steriliser
B tank
Buffer
Buffer tank
Seed
fermentor
Sterile
air
pH
control
Steriliser
Production
fermentor
Harvesting tank
Anion
exchange
antifoam
NaoH
Monosodium
glutamate
formation
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The broth is centrifuged or filtered through
membrane filter to separate cells& debris.
The purpose of the isolation process is to
obtain crude L – glutamate with adequate
purity from the fermentation broth.
The final product is obtained as crystalline
powder.
Product is released doing quality tests.
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Vinegar is acetic acid which is produced by acetic
acid
bacteria (Gluconobacter and Acetobacter)
oxidizing an alcohol-containing fruit juice.
Oxidative fermentation
Bacteria of the genus Acetobacter
 Commonly used feeds include apple cider wine,
and fermented grain, malt, rice or potato
mashes.
Although a strict aerobe, Gluconobacter oxidizes
ethanol partially

1. Open vat (Orleans) – shallow vats /slime/ lots
of air
 2. Trickle Generator: beechwood shavings
covered by a trickle of alcoholic substrate, with
air entry at the base
 Recycle the product until 4% acetic acid is
obtained.
 3. Bubble method: Large scale fermentor with
massive air input (and heat removal).
Efficiency 90-98% conversion.
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Anaerobic fermentation
Clostridium, can convert sugars to acetic acid
directly, without using ethanol as an
intermediate
C6H12O6 → 3 CH3COOH
Clostridium bacteria are less acid-tolerant
than Acetobacter.
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Uses
In the form of vinegar, acetic acid solutions
(typically 4% to 18% acetic acid)
A condiment (flavor)
Food pickling

Production is using Aspergillus niger
pasteurization
Curd formation
Pressing/cheddaring
Maturation
/ripenning
Salting
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Pasteurisation
. Regular pasteurisation at 72 – 73°C for 15 –
20 seconds is most commonly applied.
to kill bacteria capable of affecting the quality
of the cheese,
coliforms, which can cause early “blowing”
and a disagreeable taste.
spore-forming microorganisms in the spore
state survive pasteurisation (Clostridium
tyrobutyricum)
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Starter cultures
Two principal types of culture are used in cheese
making:
mesophilic cultures with a temperature optimum
between 20 and 40°C
thermophilic cultures which develop at up to 45°C.
The most frequently used cultures are mixed strain
cultures
Three characteristics of starter cultures
ability to produce lactic acid in the curd
ability to break down the protein and, when
applicable,
ability to produce carbon dioxide (CO2).
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Rennet
The active principle in rennet is an enzyme
called chymosine
Coagulation of casein is the fundamental
process in cheese making
Transformation of casein to paracasein under
the influence of rennet
Precipitation of paracasein in the presence of
calcium ions.
A : during stirring
B : during cutting
C : during whey
drainage
D : during pressing
:
Cottage cheese 0.25 – 1. %
Emmenthal 0.4 – 1.2
Gouda 1.5 – 2.2
Cheddar 1.75 – 1.95
Limburger 2 – 3.5
Feta 3.5 – 7.0
Gorgonzola 3.5
Salting
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Types
Cheddar
Emmenthal
Smear-treated types of cheese – Tilsiter,
Havarti
Gouda
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Fed-batch fermentation
Fixed volume
Variable volume
Limiting substrates-sugars + nutrients
Fin>0, Fout =0
F out=0
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Medium engineering
Beet and cane molasses are commonly used as
raw material
Contain a mixture of sucrose, fructose and
glucose, are readily fermentable
Require certain minerals, vitamins and salts for
growth
Nitrogen is supplied in the form of ammonia and
phosphate is supplied in the form of phosphoric
acid
The sterilized molasses, is commonly referred to
as mash or wort,

Scaling up
Production
Seed tank
Frozen
culture
fermentor
• Nutrients and sugars in vessels
Set-batch
fermentation
• Inoculation
• Gradual addition of sugars and nutrients
Fed-batch
• Sterile conditions
• Centrifugation
Separators
• Yeast inoculate commercial fermentors
Fermention(fed-batch, aeration, pH 4.5)
Nozzle type centrifuge(separation)
Yeast cream
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Types
Cream yeast is essentially a suspension of yeast cells in
liquid, siphoned off from the growth medium. Its primary
use is in industrial bakeries with special high-volume
dispensing and mixing equipment, and it is not readily
available to small bakeries or home cooks.
Compressed yeast is essentially cream yeast with most of
the liquid removed. It is a soft solid, beige in color, and
arguably best known in the consumer form as small, foilwrapped cubes of cake yeast.
Rapid-rise yeast is a variety of dried yeast (usually a form
of instant yeast) that is of a smaller granular size, thus it
dissolves faster in dough, and it provides greater carbon
dioxide output to allow faster rising
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Edible mushroom varieties areAgaricus bisporus (button mushroom),
Pleurotus ostreatus (Oyster mushroom),
Volvariella volvacea