fermentation systems

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Feb 20, 2013 (4 years and 3 months ago)

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Fermentor
system
s

November 12

2012

SUBMITTED TO: DR ZAFFAR MEHMOOD

SUBMITTED BY: TAHIRA KHAN


HASSAN CHAUDHRY


MADIHA HAMID


KANWAL SHAHEEN


AISHA NAEEM


Food
Biotechnology



2


TABLE OF CONTENTS:

Introduction to fermentor systems
…………………………………………... 1

Fermentor design
……………………………………………………………... 3

Types of fermentor systems………………………………………………….. 7

Submerged culture fermentor……………………………………………….. 9

Solid state fermentor…………………………………………………………. 15

Applications and types of Fermentors used in food industry……………… 2
0

Murree brewery an industrial model fermentor……………………………. 26

References
…………………………………………………………………….


37



3


FERMENTER SYSTEM
S

Fermentation is carried out in vessels known as Fermente
rs.


A f
ermenter can be a simple vessel but if it is c
onnected to complex integrated
system of
automated control
,

then
it is
termed as fermenter system.

HISTORY OF FERMENTATION

Divided into four stages:

1.

Pre 1900

2.

1900
-
1940

3.

1940
-
date

4.

1964
-
date

5.

1979

date

1.

First Stage:

Wooden vessels were used. These wooden
vessels had the capacity of approximately 1500
barrels. In the later years the trend of carrying out fermentation in copper vessels was seen.

2.

Second Stage:

Steel vessels were used which had the ample capacity of 200m3. Such vessels were mainly used
for ace
tone or butanol fermentation. Air spargers were also introduced in the second stage for the
aeration of yeast and mechanical stirring also came into practice for small vessels.

3.

Third Stage:

Mechanical aerated vessels are among the salient features of the
third stage. True fermenters i.e.
the ones which are operated aseptically also came into practice.

4.

Fourth Stage:


4

Development of pressure cycle and pressure jet vessels occurred in fourth stage which led to the
exclusion of some major problems like gas exch
ange and heat exchange.

5.

Fifth Stage:

Fermenters actually developed in the third and fourth stage. Along with fermenters animal cell
reactors were also developed.

FERMENTER VS BIOREACTOR

Fermenter system is used for the growth and maintenance of a populatio
n of bacterial or fungal
cells. While
,

a bioreactor is used for the growth and maintenance of a population of mammalian
or insect cells.

FERMENTER DESIGN

1.

MATERIALS USED IN A FERMENTER
:

Due to the strict aseptic environment, the materials used in fermenter
should be able to withstand
repeated sterilizations usually done with steam. Secondly the use of appropriate material depends
mainly upon the scale. For a small scale, it is a common practice to use glass or stainless steel.
Glass because it has a smooth s
urface, it is non
-
toxic and corrosion proof and most importantly it
is easy to examine the interior of the vessel. Pilot
-
scale and industrial scale vessels are normally
constructed of stainless steel or at least have a stainless
-
steel cladding to limit cor
rosion.

2.

CONDITIONS FOR A FERMENTER:

Following conditions should be met in order to make a proper fermenter and for

it to work in an
efficient way.
To achieve these
,

the fermenter should have:



Heat and oxygen transfer configuration



Impeccable sterilization

procedures



Foam control



Fast and thorough cleaning system


5



Proper monitoring and control system



Productivity and yield



Fermenter operability and reliability



Product purification



Water management




Energy requirements



Waste treatment

Other few significant things to be taken in
account include
:



Design in features so that process control will be possible over reasonable ranges of
process variables.



Operation should be reliable



Operation should be contamination free

3.

STERILIZATION:

A.

Sterilization Of The Fermente
r
:

Fermenters are designed in such a way so that it may be steam sterilized with pressure when
necessary. Moreover the medium also needs to be sterilized either in the vessel or outside the
vessel before it is added into the ve
ssel aseptically.

B.

STERILIZATION OF AIR SUPPLY:

Aerobic fermentation processes an ample amount of air is needed and the air should be sterilized
before entering the fermenter. There are two conventional ways of sterilizing air i.e. is by heating
and by filt
ration. Heat is discouraged because of its being too costly to implement in full
-
scale
operation.


6

C.

STERILIZATION OF THE EXHAUST GAS FROM THE
FERMENTER:

Exhaust gas can be sterilized by using filters of 0.2 µm placed on the exhaust pipe. Usually the
exhaust
gas contains moisture and solid particles leading to aerosol formation. This can be
avoided by either using a cyclone operator before solids or the coalesce for liquids before the
exhaust pipe opening in series. To make sure that no viable cells are leavin
g the exhaust pipe.
The filter needs to be constantly checked.

4.

SENSOR PROBES
:

Glass electrodes are used as sensor probes but in order to seal these probes rings are used
commonly known as Double ‘O’ rings. These work as an aseptic seal and allow minimum
re
lease of micro
-
organisms and if in any case a leakage is inevitable then there are simple
disinfection protocols to deal with it.

Pre
-
installed back up probes are also necessary because if in a case a probe fails then there is a
chance of leakage of broth
if a retractable probe housing is used during the fermentation.

5.

AGITATOR
:

Also known as impeller. It is required for the purpose of mixing, e.g. broth mixing, transfer of
oxygen & heat, solid particles suspension and for maintaining a constant environment
in the
fermenting vessel.

6.

SPARGER
:

Sparger is also called aeration system. It is a device for introducing air into the fermenter broth.
There are three standard types of spargers namely:



Porous sparger.



Orifice sparger. A perforated pipe.



Nozzle sparger. A

partially closed pipe.

7.

TEMPERATURE CONTROL
:


7

Usually the heat produced by microbial activity and mechanical mixing is not enough and
external must be provided or the heat is too much and it must be removed from the system. This
can be done by silicone heat
ing coils or the heating jacket through which the water is circulated.
For the large scale fermenters, internal coils and cold water circulation are preferred because of
the increase in surface area.

OPERATION OF A FERMENTER SYSTEM

Industrial fermentation
processes may be carried out as batch fermentations
, fed
-
batch operations
or

continuous fermentations.

Batch and fed
-
batch operations are quite common, continuous
fermentations being rare.

The mode of operation is, to a large extent, dictated by
the type o
f
product being produced.


BATCH FERMENTATION

Batch
fermentation system

is a closed culture system which contains an initial, limited amount of
nutrient.

In batch processing, a batch of culture medium in a
fermenter

is inoculated with a
microorganism (the
‘starter culture’)

and
incubation

is allowed to proceed under optimal
physiological conditions. In the course of the entire

fermentation,
nothing is added except
oxygen, antifoam and
acid
/base

to

control

the

pH.

Composition

of

the

culture

medium,

biomass,

and

metabolite

concentration

change

constantly

as

a

result

of

the
cell

metabolism.

The fermentation proceeds for
a certain duration (
the ‘fermentation time’ or ‘batch time’) and the product is
harvested.

z
The inocu
lated culture
will

pass through a number of phases, as

shown in figure
. After
inoculation there is a

phase during
which it appears that no growth takes

place; this
period is
referred to as the lag phase and

considered as a time of adap
tation.

Following a period during
which the growth

rate of the
cells gradually increases, the cells grow at
maximum

rate and this
period is known as

log phase
or exponential, phase.

After

the substrate i
s exhausted
,

the growth
ceases and this

is called stationary phase. The
depletion
of substrate for maintenance and the
presence of

toxic

substances cause the cell death,
called death phase.

Batch fermentations typically extend

over 4
-
5 days, but some traditional food fermentations may
last month
s.

Most beer breweries use batch processes commercially.


8

FED
-
BATCH FERMENTATION

A fed
-
batch is a biotechnological batch process which is based on feeding of a growth limiting
nutrient substrate to a culture. The fed
-
batch strategy is typically used in bio
-
industrial processes
to reach a high cell density in the bioreactor. Mostly the feed solution is highly concentrated to
avoid dilution of the bioreactor. The controlled addition of the nutrient directly affects the
growth rate of the culture and helps to a
void overflow metabolism (formation of side
metabolites, such as acetate for Escherichia coli, lactic acid in cell cultures, ethanol in
Saccharomyces cerevisiae), oxygen limitation (anaerobiosis)
.The volume of fermenting broth
increases with each addition
of the medium, and the
fermenter

is harvested after the batch time.

CONTINUOUS FERMENTATION

In continuous fermentations, sterile medium is fed continuously into a
fermenter

and fermented
product is continuously withdrawn
, so the fermentation

volume remains unchanged .Typically ,
continuous fermentations are started as batch cultures and feeding begins after the microbial
population has reached a certain concentration. In some continuous
fermentation
s
, a small part of
harvested culture may be
recycled, to continuously inoculate the sterile fee
d medium entering the
fermenter
.

‘Plug flow’ fermentation devices, such as long tubes that do not allow back mixing,
must be inoculated continuously . Elements of fluid moving along in a plug flow device b
ehave
like tiny batch
fermenter
. Hence, true batch fermentation processare relatively easily transformed
into continuous operations in plug flow
fermenter
s, especially if p H control and aeration are not
required. Continuous cultures are particularly susce
ptible to microbial contamination, but in
some cases the formation conditions may be selected (e.g. low pH, High Alcohol or salt content)
to favor the desired microorganisms compared to potential contaminants.


9


TYPES OF FERMENTER

SYSTEMS

Most commercially useful fermentations may be classified as either
submerged cultures

or
solid

state fermentations
.

These are the two basic ty
pes of
fermenter

systems
, which will be
discussed in detail.

Solid
-
state and submerged fermentations may be each s
ubdivided
-

into oxygen requiring aerobic
processes, and anaerobic that must be conducted in the absence of oxygen. Examples of
aerobic
fermentations

include submerged
-
culture citric acid production by Aspergillus Niger and solid
state Koji fermentation (us
ed in production of soya sauce). Fermented meat products such as
bologna sausage (polony) dry sausage, pepperoni and salami are produced by solid state
anaerobic fermenations

utilizing acid
-
forming bacteria, particularly lactobacillus, Pediococcus
and mirc
oococcus species.

A submerged culture anaerobic fermentation occur
s

in yog
urt
making.

SUBMERGED
CULTURE
FERMENTER

SYSTEM

Submerged fermentation is the cultivation of microorganisms in liquid nutrient broth.
Submerged
fermenter

systems may use a dissolved substrate e.g. sugar solution, or a solid substrate

10

suspended in a large amount of w
ater to form a
slurry. Submerged fermentations are used for
pickling vegetables, producing yoghurt, brewing beer and producing wine and soay sa
uce.

The process

involves growing carefully selected microorganisms in closed vessels containing a
rich broth of nutrients (the

fermentation medium) and a high concentration of oxygen. As the
microorganisms break down

the nutrients, they release the desire
d enzymes into solution.
Fermentation takes place in large
fermenter

with volumes of up to 1,000 cubic metres. The
fermentation media
sterilizes
nutrients based on renewable raw materials like maize, sugars and
soya. Parameters like temperature, pH, oxygen

consumption and carbon dioxide formation

are
measured and controlled to optimise the fermentation process. Firstly, in harvesting enzymes

from the fermentation medium one must remove insoluble products, e.g. microbial cells. This is

normally done by centr
ifugation. As most industrial enzymes are extracellular (secreted by cells

into the external environment), they remain in the fermented broth after the biomass has been

removed. The biomass can be recycled as a fertiliser, but first it must be treated with

lime to

inactivate the microorganisms and stabilise it during storage.

Advantages:



Measure of process parameters is easier than with solid
-
state fermentation.



Bacterial and yeast cells are evenly distributed throughout the medium.



There is a high water
content which is ideal for bacteria.

Disadvantages:



High costs due to the expensive media



Large reactors are needed and the behaviour of the organism cannot be predicted at times.



There is also a risk of contamination



11

A typical submerged culture vessel
has the features shown in the following figure
:

SUBMERGED CULTURE
FERMENTER

DESIGN
:
(1)Reactor Vessel (2)Jacket
(3)Insulation (4)Protective Shroud (5)Inoculum Connection (6)Ports of sensors of pH,
temperature and dissolved O2 (7)Agitator (8)Gas Sparger
(9)Mechanical Seal (10)Reducing
Gearbox (11)Motor (12)Harvest Nozzle (13)Jacket Connection (14)Sample valve with steam
connection (15)Sight Glass (16)Connections of acids, alkalis and antifoam agents (17)Air
inlet (18)Removable Top (19)Medium Feed nozzle (
20)Air exhaust Nozzle
-
connect to
condenser, not shown (21)Instrumentation ports for foam sensors pressure gauge and other
devices (22)Centrifugal foam beaker (23)Sight glass with light
-
not shown and steam
connection (24)Rupture disc nozzle

DIFFERENT TYPES

OF SUBMERGED CULTURE
FERMENTER
S

The major types of submerged cultures
fermenter

systems are

as follows:

1.

Stirred tank
fermenter

2.

Air lift
fermenter

3.

Bubble column
fermenter


12

4.

Flu
i
dized
-
bed
fermenter

5.

Trickle
-
bed
fermenter

1.

STIRRED TANK
FERMENTER

A

stirred tank

fermenter

is the simplest type of
fermenter

system
.


It is composed of a reactor and
a mixer such as a stirrer, a turbine wing or a propeller.

This is a cylindrical vessel with working
height to
-
diameter ratio (aspect ratio)of 3
-
4. A central shaft supports

three to four impellers ,
placed about 1 impellers diameter that direct the flow axially(parallel to shaft) or
radially(outwards from the shaft). Sometimes axial
-

and radial flow impellers are used on the
same shaft. The vessel is provided by four equall
y spaced vertical baffles, that extend from near
the walls in to vessels. Typically, the baffle width is 8
-
10% of the vessel diameter.


This reactor is useful for substrate solutions of high viscosity and for immobilized enzymes with
relatively low activ
ity. However, a problem that arises is that an immobilized enzyme tends to
decompose u
pon physical stirring. This

system is generally suitable for the production of rather
small amounts of chemicals.

2.

BUBBLE COLUMN
FERMENTER

A bubble column fermentation sys
tem

is an apparatus used for gas
-
liquid reactions first applied
by Helmut Gerstenberg.
This is a cylindrical vessel with a working aspect ratio

4
-
6.

The

13

introduction of gas takes place at the bottom of the column and causes a turbulent stream to
enable an optimum gas exchange.
In this way, the
compressed gas provides agitation
.
It is built
in numerous forms of construction. The mixing is done by the gas sp
arging and it requires less
energy than mechanical stirring. The liquid can be in parallel flow or counter
-
current.

Bubble column reactors are used in various types of chemical reactions like wet oxidation, or as
Algae bioreactor.
Although simple, it is n
ot widely used because of its
poor performance relative
to other

system
s
. It is
not
suitable for very vicious broths or those containing large amount of
solids.

3.

AIR LIFT
FERMENTER

Air
-
lift bioreactors are similar to bubble column reactors, but differ by
the fact that they contain
a draft tube.

The draft tube may be
an inner tube (called "air
-
lift bioreactor with an internal
loop

) or an external tube (called "air
-
lift bioreactor with an external loop

) which improves
circulation and oxygen transfer and eq
ualizes shear forces in the reactor.
In the internal
-
loop
designs the aerated riser and unaerated down corner are contained in small shell. In the external
-

14

loop configuration, the riser and the down comer are separate tubes that are linked near the top
and

the bottom. Liquid circulates between the riser (upward flow) and the down comer
(downward flow). The working aspect
ratio
of airlift
fermenter
s is 6 or greater.

Generally, these are very capable
fermenter
s, except for handling vicious broths. The abilit
y to
suspend solids and transfer O2 and heat is good. The hydronamic shear is low. The external loop
design is relatively little
-
used in industry.

4.

FLUIDIZED
-
BED
FERMENTER

These are similar to bubble columns with an expanded cross section near the top. Fresh or
recirculated liquid is continuously pumped into the bottom of the vessel, at a velocity that is
sufficient to fluidize the solids or maintain them in a suspension.The
se
fermenter
s need an
external pump. The expanded section slows down the local velocity of the upward flow, such
that the solids are not washed out of the bioreactors.


In this type of
fermenter
, a fluid (gas or liquid) is passed through a granular solid
material at high
enough velocities to suspend the solid and cause it to behave as though it were a fluid. This
process, known as fluidization, imparts many important advantages to the FBR. As a result, the
fluidized bed reactor is now used in many industri
al applications.

5.

TRICKLE
-
BED
FERMENTER


These consist of a cylindrical vessel packed with support material (e.g wood chips, rocks, plastic
structure). The support has large open spaces, for the flow of liquid and gas and the growth of

15

microoraganisms on t
he solid support. A liquid nutrient broth is sprayed onto the top of support
material, and trickles down the bed. Air may flow up the bed, countercurrent to the liquid flow.

These
fermenter
s are used in vinegar production, as well in other process.
These are suitable for
liquids with low viscosity and few suspended solids.


SOLID STATE
FERMENTER

SYSTEMS

SSF involves the growth of micro organisms on moist solid substrate where there is little water
in the spaces between the substrate molecules and a c
ontinuous gas phase. In the beginning it
was thought that liquid state fermentation or submerged fermentation (SLF) has more advantage
over SSF. But recent studies in the West
have shown

SSF to be
the
cheapest and more
environmentally friendly relative to
SLF in the production of value added industrial based
products such as enzymes, bio fuels and the likes. However in the East it is still
in the back
burners of the fermentation popularity just due to poor understanding and control of SSF.

Traditional uses
of SSF systems include the production of fermented foods, pigments, and koji in
the Far East. Within the past decade, the production of other, higher
-
value microbial metabolites
such as antibiotics ,biopesticides , aromas, gibberellic acid and bacterial am
ylase to name a few,
have been evaluated with highly promising results. Bread, sausages, and soy sauce are also some
familiar products of SSF.

Some of the advantages of SSF are listed below:


16



The use of little moisture may facilitate the production of some
specific compounds
which can’t be produced in SLF.



The products obtained in SSF are more thermo tolerant relative to those produced in SLF.



The low availability of water reduces the possibilities of contamination by bacteria and
yeast. This allows working
in aseptic conditions in some cases.



Simply designed reactors with few spatial requirements can be used due to the
concentrated substrates.

DIFFERENT TYPES OF SS
FERMENTER
S

The design of the fermenter is very important for a fermentation process.

Solid sub
strate
fermentation
fermenter
s vary in technical sophistication from the very primitive banana leaf
wrappings to highly automated machines used mainly in Japan.

There are many different types
of fermenters used for SSF. A few are explained below:

1.

TRAY
FERMENTER

This
fermenter

is one of the simplest and widely used
fermenter
s.

Its basic part is a wooden,
metal, or plastic tray, often with a perforated or wire mesh bottom to improve air circulation. A
shallow layer of less than 0.15 m deep, pretreated (e.
g., steamed) substrate is placed on the tray
for fermentation. Temperature and humidity
-
controlled chambers are used for keeping the
individual trays or stacks. A spacing of at least one tray height is usually allowed between
stacked trays. Cheesecloth may

be used to cover the trays to reduce contamination, but strict
monosepticity is not
attempted.
Inoculation and
occasional
mixing are done
manually, often
by hand.

Small
-

and medium
-
scale koji
operations in Asia
mostly use this
technology.







17



Disadvan
tages:

Despite some automation, tray
fermenter
s are



labor intensive



require a large area



Difficulties with processing hundreds of trays limit their scalability

2.

STATIC BED AND TUNNEL
FERMENTER
S

These are the modification of tray
fermenter

employing a single, larger and deeper, static bed of
substrate with forced aeration through the bed. The substrate is located in an insulated chamber.

Tunnel
Fermenter
:

In the tunnel
fermenter
, the bed of solids may be quite long but is usually no deeper than 0.5 m.
Tunnel
fermenter
s may be highly automated with mechanisms for continuous feeding, mixing
inoculation and harvest of substrate.


3.

ROTARY
-
DISK
FERMENTER
S

Rotary disk
fermenter
s are
used in large scale koji fermentations in Japan.

They consist of a
upper and lower chambers, each with a circular perforated disk to support the substrate. A
common central shaft
rotates the disks.
Inoculated substrate
is introduced in the
upper chamber an
d
slowly moved to
the transfer screw.
The upper screw
transfers the partly
fermented solids
through a mixer to
the lower chamber
where further
fermentation
occurs. The mixer
breaks up the partly
fermented substrate

mycelium aggregates halfway through the f
ermentation process. Fermented
substrate is eventually harvested

using the lower transfer screw
.

Both chambers are aerated with
humidified, temperature
-
controlled air. Rotating
-
disc contactors have been used in effluent
treatment. They utilize a growing mi
crobial film on slow rotating discs to oxidize the effluent.



18





AUTOMATIC ROTARY
KOJI
FERMENTER

4.

TOWER
FERMENTOR

Tower fermenter is
simple in design and
easy to construct. It is
similar in concept to
rotary koji fermentor,

consisting
of a long
cylindrical

vessel with
an inlet at the bottom,
an exhaust at the top,
and a jacket to control
temperature.

A stack of several tray chambers form the tower. It does not require agitation hence
there are no shafts, impellers or blades. Tower fermentors are used for co
ntinuous fermentation
of beer, yeast and SCP.

In
1955 these were used in brewing industry.

TOWER FERMENTOR



Disadvantages
:

Despite of being simple and agitation free (to keep yeast cells in suspension) the tower
fermentors have following drawbacks
:

1.

Long
start up

2.

Technical complexity

3.

Skilled personnel Required

4.

No product consistency


5.

AGITATED TANK FERMENTOR

Helical ribbon
-
stirred tank fermentors have been employed for solid
-
state culture of fungi such
as
Chaetomium

cellulolyticum
on wheat straw.

Other simi
lar designs have utilized multiple
helical screws for agitation of large rectangular tanks.



19

6.

CONTINUOUS SCREW FERMENTOR

A screw fermentor is used for continuous fermentation process. Sterilized, cooled, and
inoculated substrate is fed at the inlet. The
screw moves the fermenting solids toward the harvest
port. The fermentation time depends on the length of the screw and the rotational speed.

As the device is not aerated, therefore onl
y anaerobic or microaerophilic
fermentations may be
done.

SCREW FERMENT
OR

7.

AUTOCLAVE FERMENTOR

Most fermentors are sterilized by autoclaving, or hot steam under pressure. For small laboratory
fermentors they are sterilized in autoclaves. In the case of large fermentors, most if not all are
equipped with in situ sterilization
facilities built into the fermentor sys
tem.

For most autoclaving sterilizations in both small and large fermentors, the accepted autoclaving
conditions is at 121 degrees Centigrade at pressure of 15 to 20 psi. The autoclaving holding time
is about 15 to 20

minutes.

There are certains point which should be kept in mind while autoclaving,they are as follows:
-

i.

To be efficient in autoclaving it is very important to drive off any air pockets that might
be present in the autoclave. Air is a poor heat conductor. I
f the air is not driven out it will
be difficult to bring the right temperature in all the autoclave. Let the autoclave heat and
steam up and release the hot steam through an escape valve before closing the valve and
starting the sterilization process
.

ii.

Ens
ure that the temperature recorded in the autoclave chamber is uniform throughout the
whole chamber. Make sure the temperature stated on the panel outside the autoclave is
the real temperature inside the autoclave chamber. We do not want under heating and
o
verheating to occur. Place thermo probes to measure the real temperature of the
autoclave and repair if needed
.

iii.

Do not overload the autoclaving chamber. This might lead to poor degree of sterilization
being achieved.

iv.

Ensure that the autoclave is not leakin
g or suffering from leak in pressure as it will affect
the sterilization process.


20

APPLICATIONS OF FERMENTORS IN INDUSTRY

Industrial fermentation

is the intentional use of

fermentation

by

microorganisms

such as

bacteria

and

fungi

to make products useful to
humans. Fermented products have applications as

food

as well as in general

industry.

FOOD FERMENTATION

Ancient fermented food processes, such as making

bread, wine, cheese
, curd, dosa
etc., can be
dated to more than

6000 yr ago
. They were developed long
before man had any knowledge of the
existence of the

microorganisms

involved. Fermentation is also a powerful economic incentive
for semi
-
industrialized countries, in their willingness to produce bio
-
ethanol.

PHARMACEUTICALS AND THE BIOTECHNOLOGY INDUSTRY

There are 5 major groups of commercially important fermentation:

1.

Microbial cells or

biomass

as the product, e.g.

single cell protein,

bakers
yeast,

lactobacillus,

E. coli, etc.

2.

Microbial

enzymes:

catalase,

amylase,

protease,

pectinase,

glucose
isomerase,

c
ellulase,

hemicellulase,

lipase,

lactase,streptokinase, etc.

3.

Microbial

metabolites

:



Primary metabolites


ethanol,

citric acid
,

glutamic
acid,

lysine,

vitamins,

polysaccharides

etc.



Secondary metabolites
--

all antibiotics

fermentation

4.

Recombinant products:

insulin,

hepatitis B vaccine,

interferon,

granulocyte colony
-
stimulating factor,

streptokinase

5.

Biotransformations:

phenylacetylcarbinol,

steroid

biotransformation, etc.


NUTRIENT SOURCES FOR INDUSTRIAL FERMENTATION

Growth media are required for industrial fermentation, since any microbe requires water,
(oxygen), an energy source, a carbon source, a nitrogen source and

micronutrients for growth.


21

Carbon & energy source + nitrogen source + O2

+ other requirements → Biomass + Product +
byproducts + CO2

+ H2O + heat

PRODUCTION OF INDUSTRIAL ENZYME USING DIFFERENT
FERMENTORS

NEUTRAL PROTEASE:

Neutral protease is produced at
indrustial level using agro
-
industrial residues as substrate e.g.
wheat bran, rice husk, rice bran, spent brewing grain, coconut oil cake, palm kernel cake, sesame
oil cake, jackfruit seed powder and olive oil cake etc. while developing a production medium

it is

very important to monitor the cost
-
effectiveness of the medium so these agro
-
industrial residues
mentioned above are are very cheap and easily available. Among all substrates wheat bran is the
best. Seven fungal cultures, i.e. three strains of
Aspe
rgillus oryzae

and four strains of
Penicillium

species e.g.
P
.
funiculosum
,
P
.
funiculosum, P. pinophilum
,
P
.
aculeatum

were
evaluated using a plate assay for enzymeproduction, which showed a strain of
A. oryzae

NRRL
1808 as the most useful culture. Protea
se enzyme is produced in two fermentor systems, in solid
-
state fermentors (SSF) and sub
-
merged fermentors (SmF).

PRODUCTION OF NEUTRAL PROTEASE IN SSF AND IN SMF AND
THEIR COMPARISON:


In SSF a medium having an initial moisture content of 43.6%, when inoc
ulated with 1

ml of
spore suspension (8

×

108 spores) and incubated at 30

°C for 72

h (31.2

U enzyme per gram of
fermented substrate


U/gds) is used while in SmF medium (pH 7.5) containing 2% (w/v) wheat
bran, when inoculated with 3

ml of spore suspension

and incubated at 30

°C and 180

rpm for
72

h gave maximum enzyme yield of 8.7

U/gds is used. SSF gives best result comparative to
SmF because of 3.5
-
fold more enzymeproduction in SSF and it clearly demonstrating the
superiority of SSF over SmF.

Biesebeke e
t al. compared the molecular and physiological aspects of the fungus in submerged
and solid
-
statefermentation. He observed a number of differences correlated with the different
growth conditions. SmF has advantages in process control and easy recovery of e
xtracellular
enzymes, mycelia or spores. However, the products are dilute and enzymic extracts might be less

22

stable than those from SSF. SSF has been developed and described for fungal enzymeproduction
and its advantages include simplicity, lower productio
n costs, high enzyme yields and low
wastewater output. SSF has the added advantage since it is a static process without mechanical
energy expenditures, although problems such as temperature and pH control are encountered.

GLUCOAMYLASE

PRODUCTION OF
GLUCOAMYLASE IN SSF:


Aspergillus

sp. A3 is used for the production of glucoamylase under solid state fermentation.
Different substrates like wheat bran, green gram bran, black gram bran, corn flour, barley flour,
jowar flour, maize bran, rice bran and wh
eat rawa are the best substrate and give best results
among all these wheat bran showed the highest enzyme activity. The maximum enzyme activity
under optimum conditions was 247 U/g of wheat bran. The optimum conditions are fructose as
additive 1% w/w, ure
a as additive 1% w/w, incubation time of 120 h, incubation temperature at
30

°C, 2:10 (v/w) ratio of salt solution to weight of wheat bran, inoculum level 10% v/v,moisture
content of solid substrate 80%, 1:50 ratio of substrate weight to flask volume and p
H 5.0.

SSF holds tremendous potential for the production of enzymes. In case of crude fermented
product SSF is of special interest, may be used directly as enzyme source. For the SSF processes
Agro
-
industrial residues are generally considered the best sub
strates.

PRODUCTION OF GLUCOAMYLASE IN SMF:

Currently, glucoamulase enzyme is also produce in submerged fermentation (SmF), generally
employing genetically modified strains. Comparative to SSF in SmF the cost of production is
high and is uneconomical.

So as a result the SSF should be considered as an attractive method and it has has many
advantages over submerged fermentation for the production of enzyme.



23

ALGAE BIOFUELS PHOTOBIOREACTOR


Ability of microalgae to mitigate CO
2

emission and produce oil wit
h a high productivity show
that it has the potential for applications of producing the third
-
generation of biofuels. For
microalgae biofuel production there is a need of identification of preferable culture conditions
for high oil productivity, developmen
t of effective and economical microalgae cultivation
systems, as well as separation and harvesting of microalgal biomass and oil.

Chisti in 2007 proposed that under suitable culture conditions, some microalgal species are able
to accumulate up to 50

70% o
f oil/lipid per dry weight. And Gouveia and Oliveira in 2009
proposed tha the fatty acid profile of microalgal oil is suitable for the synthesis of biodiesel.
Chisti also proposed the major reason of using microalgal oil for biodiesel production which is
t
he tremendous oil production capacity by microalgae, as per hectare, they could produce up to
58,700

L oil, which is one or two magnitudes higher than that of any other energy crop.
However, it also faces a number of technical hurdles that render the curre
nt development of the
algal industry economically unfit. In addition, it is also necessary, but very difficult, to develop
cost
-
effective technologies that would permit efficient biomass harvesting and oil extraction.
Nevertheless, since microalgae product
ion is regarded a feasible approach to mitigate global
warming, it is clear that producing oil from microalgal biomass would provide significant
benefits, in addition to the fuel. Photobioreactor could be effective to grow microalgae by using
favorable lig
ht source and reactor configuration. Collection and concentration of microalgal
biomass from cultivation systems contribute heavily to the operation cost of the overall process.

METHANE FERMENTATION SYSTEM FOR FOOD RECYCLING

Keeping in mind the applicabil
ity of food waste leachate (FWL) in bioreactor landfills or
anaerobic digesters to produce methane as a sustainable solution to the persisting leachate
management problem and this research was made in Korea. Taking into account the climatic
conditions in K
orea and FWL characteristics, the effect of key parameters, i.e. temperature,
alkalinity and salinity on methane yield was investigated. The monthly average moisture content
and the ratio of volatile solids to total solids of the FWL were found to be 84% a
nd 91%,
respectively
.

The biochemical methane potential experiment under standard digestion conditions

24

showed the methane yield of FWL to be 358 and 478

ml/g

VS after 10 and 28 days of digestion,
respectively, with an average methane content of 70%. Elemen
tal analysis showed the chemical
composition of FWL to be C
13.02
H
23.01
O
5.93
N
1
. The highest methane yield of 403

ml/g

VS was
obtained at 35

°C due to the adaptation of seed microorganisms to mesophilic atmosphere, while
methane yields at 25, 45 and 55

°C we
re 370, 351 and 275

ml/g

VS, respectively, at the end of
20 days. Addition of alkalinity had a favorable effect on the methane yield. Dilution of FWL
with salinity of 2

g/l NaCl resulted in 561

ml CH
4
/g

VS at the end of 30 days. Considering its
high biodeg
radability (82.6%) and methane production potential, anaerobic digestion of FWL in
bioreactor landfills or anaerobic digesters with a preferred control of alkalinity and salinity can
be considered as a sustainable solution to the present emergent problem.

FWL is a mechanically pretreated, easily soluble substrate, which can be handled by
environmentally friendly biological practices such as landfilling and anaerobic digesters to
obtain both economic and environmental co
-
benefits. The lab
-
scale BMP test show
ed the
methane yield of FWL to be 478

ml/g

VS at 35

±

2

°C after 28 days of digestion. Methane gas in
the digestion process accounted for over 70% (v/v) of the total biogas produced. Methane yield
was highest (403

ml/g

VS) at a mesophilic temperature of 35

°C in a period of 20 days.
Alkalinity addition had positive effect on the methane yield. Dilution of FWL with salinity of
2

g/l NaCl resulted in 561

ml

CH
4
/g

VS at the end of 30 days. Taking into account its elemental
composition C
13.02
H
23.01
O
5.93
N
1

and
high biodegradability (82.6%), FWL can be used as a highly
desirable feedstock for methane production in bioreactor landfills or anaerobic digesters and can
be treated as a sustainable solution to the present emergent problem for a clean and renewable
ener
gy resource.

The Bioenergy Co. of Japan has decided to construct a power plant in Tokyo Bay to recycle food
waste as part of its methane fermentation power generation project, which is part of the "Super
Eco
-
Town Project" promulgated by the Tokyo Metropoli
tan Government. The plant will come
on stream in fiscal 2005.

Under the Food Recycling Law enacted in 2001, all business entities in the food industry are
obliged to reduce or recycle food waste by more than 20 percent by 2006. Methane fermentation
power g
eneration is recommended in the law as one way to recycle food waste.




25

BUBBLE COLUMN FOR CITRIC ACID PRODUCTION

Citric acid is produced from the cells of the yeast

Candida guilliermondii in a bubble column,
that

have been immobilized by adsorption onto
sawdust. At a dilution rate of 0.21 h−1

in a
nitrinogen
-
limited medium containing glucose, a reactor productivity of 0.24 g l−1

h−1

has been
achieved which is twice that observed in a batch fermenter culture using freely suspended cells.
The corresponding
specific production rate was 0.024 g citrate g−1

biomass h−1

while the yield
was 0.1 g citrate g−1

glucose utilized. These latter values were lower than those observed using
freely suspended cells, indicating that further improvements can be made to the op
eration of the
reactor. In comparison with literature reports describing other cell immobilization techniques,
adsorption onto sawdust allows similar reactor productivities while being cheap and permitting
simple immobilization and reactor operation.

KOJI
FERMENTATION SOLID STATE

Aspergillus oryzae

has two glucoamylase
-
encoding genes,

glaA

and

glaB
, their patterns of
expression are different. Expression of the

glaB

gene is marked in solid
-
state culture (koji), but
low in submerged culture. To elucidate the
induction mechanism of the

glaB

promoter in solid
-
state culture (koji), a fusion gene system using the

glaA

or

glaB

promoter and the

Escherichia
coli uidA

gene encoding β
-
glucuronidase (GUS) is employed. The expression of

glaB
-
GUS was
induced by starch or
maltooligosaccharides in a similar manner to that

glaA
-
GUS, but other
physical factors were found to be required for the maximal expression of the

glaB

gene in solid
-
state culture (koji). The time
-
course of

glaB
-
GUS expression in solid
-
state culture (rice
-
koji
making) suggested that its expression is induced by low water activity (
Aw
) of the medium and
high temperature. When mycelia grown on a membrane under standard conditions were
transferred to low
-
Aw

and high
-
temperature conditions (membrane
-
transfer cu
lture,
MTC),

glaB

expression was markedly induced, but that of

glaA

was not. Additionally,

glaB
-
GUS production was induced in MTC using a membrane with smaller pore size, suggesting that
a physical barrier against hyphal extension could regulate

glaB

expre
ssion. Under conditions
found to induce

glaB

expression, namely, starch, low
-
Aw
, high
-
temperature and physical

26

barriers, approximately 6400 U/mg
-
protein was obtained, equivalent to that in solid
-
state culture
(koji). In conclusion, glucoamylase production
under these induction conditions achieved in
MTC reached 274 U/ml
-
broth, which was equivalent to the level observed in solid
-
state culture
(koji). Northern blot analysis indicated that

glaB

expression was induced at the level of
transcription 4 h after the

transfer to the inducible conditions described above.


MUREE BREWERY : AN INDUSTRIAL VISIT TO STUDY FERMENTOR
SYSTEMS:

AN INDUSTRIAL OVERVIEW

“Murree Brewery” is
an ISO 14001 Certified Company

established in the year 1860 in the
British era is leading beer industry in Pakistan. It has two leading manufacturing units one
located in Rawalpindi and other in Hattar (KPK), Pakistan. It was established for the ever
increasing demand of the beer by t
he personal British Raj .it is the oldest venture in Pakistan for
beer market up till now. The Murree Brewery at Ghora Galli was among the first modern beer
breweries established in Asia. The virtues of beer brewed from barley malt & hops as a light
alcoho
lic beverage were not lost on the local population who rapidly became avid consumers. By
the turn of the 20 century, the name "Murree" was famous for its beer in keg and bottle in the
bars, beer halls and army messes of British India. Murree Beer was first

awarded a medal for
product excellence at the Philadelphia Exhibition in 1876, followed by numerous awards over
the past 150 years, Murree brewery has potential distributors with high quality beers throughout
globe. The industry has expanded its business
beyond Pakistan and is available to rest of the
world.

MUREE BREWERY PRODUCTS

Murree brewery has a wide range of products which are categorized into alcoholic and non
alcoholic products but the main product of export is the beer and its different types produced
there
Our Premium products include



27

NON ALCOHOLIC PRODUCTS
:



Apple malt



P
each malt



Lemon malt



Strawberry malt



Murree Sparklets (mineral water)



Cindy Malt



Malt 79

ALCOHOLIC PRODUCTS:



Whisky



Malt whisky



Beer



Millennium beer(7% alcohol)



Classic (alcohol 5
-
3%)



Murree beer(4.8
-
4.9%)



Vodka



Twelve years old Single Malt Whiskies



Vintage with a blend of a Scotch Grain Whisky,



Silver Top Gin,



Bolskaya Vodka



Doctor's Brandy.






28

INDUSTRIAL VISIT OF GROUP
THREE:










BEER PRODUCTION BY FERMENTATION


Group three visited Murree brewery on October 12 , 2012 to study the fermentor systems there
and studied the beer fermentation process and each and every step involved in detail. The visit
was organized by Mr.khalil who was very warm and welcoming briefed

us each and every step
of the beer production in detail and made us possible to study the fermentor system and its
aspects in detail. In order to understand the process we
must look in to account the concept of
brewing that is beer fermentation from barl
ey which can be defined as
Fermentation

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
.

The equation is as follows:

C
6
H
12
O
6

→ 2 C
2
H
5
OH + 2 CO
2




29

1.Malting

2.Steeping

3.Gemination

4.Mashing

BEER FERMENTATION THE PROCESS

The basic ingredients of beer are water, barley(starch source) able to be fermented (converted
into alcohol),
brewer's

yeast

to produce the fermentation and flavoring agents such as hops.
Barley seeds of particular
quality are imported from Australia and are graded for the production
of a particular product they are categorized as grade A, grade B, grade C.

The first pla
ce in the brewery which we visited was the brew house to observe the malting which
is broken into three steps germination of barley and its mashing and germination. The process is
categorized into four steps:






FIG 1 and 2 depict the germinating barley in the brew house of Murree brewery in the first step of beer
production (malting)



30


MALTING:

The process of making barley grains ready for the process of brewing is kn
own as malting.

S
TEEPING:


The seed is soaked in water in a vat and aerated for 1 to 1.5 days for sprouting to activate the
enzyme at a low temperature from 10
-
12`C. barley seeds are graded as A, B and C depending
upon the type of moisture(40
-
45% moisture
) grade A(beer) and grade B seeds (malt)
production.

GERMINATION:


The germination of seeds is carried out in brew house for five days where barley seeds are
aerated and maintained at particular temperat
ures
during day 1 and 2(18
-
19`C). After
germination
is done the seeds converted to malt by the process of chitting .In chitting the seends
are germinated with sprouts on them .

KILINING:

The germinated seeds are taken to the kiln and the sprouted seeds are roasted at 180`C in a kiln.
The kiln at Murree brew
ery was really of old and antique style .kilning basically modifies the
germinating seeds by enhancing flavors and modification of barley seeds for malt and other
products production.

MASHING:


Mashing is the process of combining a mix of milled grain
(
typically
malted

barley

with
supplementary grains

such as
corn
,
sorghum
,
rye

or wheat), known as the "
grain bill
", and water,
known as "liquor" which is used to crush malt in a vessel called a "mash tun" with the help of
heating. Mashing allows the
enzy
mes

in the malt to break down the
starch

in the grain into
sugars, typically
maltose

to create a malty liquid called
wort
. Mashing of barley produces wort
.hard water is maintained

at 180`C and the crushed malt is transferred to a lautering unit.



31


LAUTERING PROCESS:

Lautering is the separation of the
wort

(the liquid containing the sugar extracted during mashing)
from the grains which is done in a lauter turn at 75`C temperature .The seed is separated from the
extract and

un

dissolved extract is removed.

WORT BOILING:

Boiling of malt extracts is called as wort. It basically ensures its sterility, and thus prevents a lot
of infections. During the boil hops are added, which contribute bitterness, flavor, and aroma
compounds

to the beer. The wort is boiled at 100`C and unsuspended phenols and extract is
pumped to the boiler. The boil lasts between 15 and 120 minutes, depending on its intensity, the
hop addition schedule, and volume of water the brewer expects to evaporate. Th
e wort is
separated on the basis of specific gravity and it is clarified aerated and cooled then transferred to
the fermentor for beer production.



Fig1(a) Lautering turn fig1(b) wort sep
arator.

Fig1(C)Temperature And Pressure Control Unit Fig1(D) Wort Boiler




32








BEER FERMENTATION

BATCH FERMENTATION:


Murree brewery uses the open batch fermentation process to ferment beer from yeast. The yeast
strain of
saccharomyces

cerevisiae

imported from Germany which is grown in the form of stock
solutions to be added to the fermentor. The yeast stock solution is made in 100ml beakers and
their serial dilutions are made from 1to 10 liters the inoculums is then transferred to the
Fermentors
in a batch. Yeast require one day for activation which is maintained at the
temperature of (14
-
16`C). The process is known as yeast publication .fermentation produces beer
of different categories depending upon the wort density.

FERMENTOR DESIGN:

Murree br
ewery had 24 multiple open batch bubble column fermentor systems in their
fermentation unit for beer production. Fermentors installed at Murree brewery was a cylindro
-
conical vessel or CCVs, with a conical bottom and a cylindrical top lined inside with
the
rmocouple to maintain the temperature and temperature sensor at the bottom and a pressure
regulating unit as well. The cone's aperture of the batch fermentor was typically around 60°at
this angle it allows the yeast to flow towards the cone's apex, but is
not so steep as to take up too
much vertical space. This type of confirmation handles both the fermenting and conditioning in
the same tank. Fermentor was made up of stainless steel lined with a layer of concrete with a size

33

of one hectoliter (100liters).
At the end of fermentation, the yeast and other solids which have
fallen to the cone's apex can be simply flushed out of a port at the apex. Fermentation takes place
of the wort in the presence of yeast to beer. After fermentation beer is obtained dependin
g upon
the density of the wort. The beer with 12% alcohol content is used to make classic beer and
millennium beer, with (6
-
7%) and light or Murree beer has low content of alcohol.

BEER CONDITONING AND STORAGE

After initial or primary fermentation, the bee
r is now conditioned, matured or aged in one of
several ways, which can take from 2 to 4 weeks, several months, or several years, depending on
the type of beer. The beer is usually transferred into a second container, to a trub which is free
from dead yeas
t, secondary metabolites and other undesirable flavors that are a result of primary
fermentation .

PACKAGING:

Packaging is putting the beer into the containers in which it will leave the brewery. Typically,
this means putting the beer into bottles, alumini
um cans and kegs, but it may include putting the
beer into bulk tanks for high
-
volume customers.

RESEARCH PLANNING AND DEVLOPMENT LAB

We visited research planning and quality control lab of Murree brewery as well our team met
their quality control manager
Mr Muhammad Sohail he briefed us about the fermenting
problems., batch , new product ranges , future products and quality control problems. The
monitoring of the products at each step to avoid any contamination. The yeast culture is checked
at the end of e
very batch of beer and malt before packaging. Random sampling and OCU are
done to check the quality of their products. The whole batch is pasterurized at 20`c then 40 `C
-
70`C for thirty seconds and labeled in their specialized packaging unit.

FUTURE PROD
UCTS:



Big peach

pineapple malt



Cola whisky

leechi malt


34








35






Fig 1.

Batch fermentor systems 2. An open batch fermentor 3. Group 3 with Fermentors

4. Yeast publication unit



36

PACKAGING AND STORAGE:




37

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Fig1(C)Temperature And Pressure Control Unit Fig1(D) Wort Boiler