1.

Introduction
Fermentation:

The term fermentation

fermentation is derived from the Latin word  fevere,to
 fevere,to boil thus
describing
describing the appearance of the action
action of yeast on fruit extract or malted
malted grain
the boiling appearance is due to the production of CO 2 bubbles caused by the
anaerobic
anaerobic catabolism
catabolism of the sugar present in the extract. However fermentation
fermentation
has
has come
come to have
have diff
diffeerent
rent mean
meaniing to bioc
bioche
hem
mist
ists and
and to indus
ndustr
tria
iall
microbiologist. Its biochemical meaning relates to the generation of energy by
cata
catabo
bollism
ism of organ
rganiic comp
compou
ound
nd,, wher
wherea
eass its
its mean
meaniing in indusndusttrial
rial
microbiology tends to be much broader.

Antibiotics:

In common usage, an antibiotic (from the Ancient Greek: anti-"against",

and bios- "life") is a substance or compound that kills bacteria or inhibit their 
growth.
growth. Antibioti
Antibiotics
cs belong to the broader group of antimicrobi
antimicrobial
al compounds,
compounds,
used
used to trea
treatt infe
infect
ctio
ions
ns caus
caused
ed by micr
microo
oorg
rgani
anism
sms,
s, incl
includ
udin
ing
g fung
fungii and
and
 protozoa.

The
The term
term "ant
"antib
ibio
ioti
tic"
c" was
was coin
coined
ed by Selm
Selman
an Wa
Waksksma
man n in 1942
1942 to
describe any substance produced by a microorganism that is antagonistic to the
growt
growthh of othe
otherr micr
microooorga
rgani
nism
smss in high
high dilu
diluti
tion
on.. This
This orig
origin
inal
al defi
defini
niti
tion
on
excluded naturally occurring substances that kill bacteria but are not produced
 by microorganisms (such as gastric juice and hydrogen peroxide) and also
excluded synthetic antibacterial compounds such as the sulfonamides. Many
antibiotics are relatively small molecules with a molecular weight less than
2000 Da.

Antibiotics are commonly classified based on their mechanism of action,

chemical structure, or spectrum of activity. Most antibiotics target bacterial
functions or growth processes. Antibiotics that target the bacterial cell wall
(penicillin, cephalosporin), or cell membrane (polymixins), or interfere with
essential bacterial enzymes (quinolones, sulfonamides) are usually bactericidal
in nature. Those that target protein synthesis, such as the aminoglycosides,
macrolides,
macrolides, and tetracycli
tetracyclines,
nes, are usually
usually bacteriostat
bacteriostatic.
ic. Further
Further categorizat
categorization
ion
is base
basedd on thei
theirr targ
target
et spec
specif
ific
icit
ity:
y: "Narr
"Narrow
ow-sp
-spec
ectr
trum
um"" anti
antibi
biot
otic
icss targe
targett
 particular types of bacteria, such as Gram-negative or Gram-positive bacteria,
whereas broad-spectrum antibiotics affect a wide range of bacteria. In the last
few years, three new classes of antibiotics have been brought into clinical use.

1
Beta-lactam antibiotics:

The
The beta
beta-l
-lac
acta
tam
m grou
groupp of anti
antibi
biot
otic
icss is by far
far the
the larg
larges
estt grou
groupp of 
antibacterial agents used in clinical medicine. It includes the first true antibiotic
to be discovered and brought into clinical practice, and all the natural and semi-
synt
synthe
heti
ticc deri
deriva
vati
tive
vess with
with thei
theirr wide
wide-r
-ran
ange
ge of prop
proper
erti
ties
es and
and clin
clinic
ical
al
applications.

With a few notable exceptions, they remain a cornerstone of antibiotic

therap
therapy
y for the spectr
spectrum
um of seriou
seriouss system
systemic
ic infect
infection
ion.. Struct
Structural
urally,
ly, all are
 based upon the four member nitrogen containing beta-lactam ring that gives
these agents their antibacterial activity.

They
They can be divide
divided
d into
into four
four groups
groups - penici
penicilli
llins,
ns, cephal
cephalosp
ospori
orins,
ns,
carba
carbape
pene
nems
ms and
and mono
monobabact
ctam
amss - on the
the basi
basiss of the
the mole
molecucula
larr struc
structu
tures
res
surrounding and supporting this active site, A fifth group in clinical use are the
 beta-lactamase inhibitors that do not have intrinsic antibacterial activity.
activity.

Penicillin:

The penicillin may be divided into four main groups on the basis of their 
spectra of antibacterial activity. All the developments and manipulations of the
 penicillin nucleus have had the aim of extending the spectrum of the agents
against gram-negative bacteria or increasing their resistance to beta-lactamase
enzymes, or both.

All of this "improved" penicillin retains activity against bacteria such as

hemo
hemolylyti
ticc stre
strept
ptoc
ococ
occi
ci and
and clos
clostr
trid
idia
ia,, whic
which
h rema
remain
in sens
sensit
itiv
ivee to benz
benzyl
yl
 penicillin. However, it must be remembered that all the alterations to the basic
 penicillin structure necessary to achieve these particular advantages have been
at the expense of intrinsic activity against sensitive organisms.

Early penicillin, Benzyl penicillin (penicillin G), the original member of 
the group, remains the most active antibacterial agent against sensitive bacteria.
It is the drug of choice for serious infections with beta-hemolytic streptococci,
alpha-hemolytic streptococci (combined with an aminoglycoside in sub-acute
 bacterial endocarditis), the pneumococcus, the meningococcus and clostridia
other than Clostridium. Phenoxymethyl penicillin (penicillin V) remains useful
when oral treatment of streptococcal infections is adequate.

2
Beta-lactam antibiotics:

The
The beta
beta-l
-lac
acta
tam
m grou
groupp of anti
antibi
biot
otic
icss is by far
far the
the larg
larges
estt grou
groupp of 
antibacterial agents used in clinical medicine. It includes the first true antibiotic
to be discovered and brought into clinical practice, and all the natural and semi-
synt
synthe
heti
ticc deri
deriva
vati
tive
vess with
with thei
theirr wide
wide-r
-ran
ange
ge of prop
proper
erti
ties
es and
and clin
clinic
ical
al
applications.

With a few notable exceptions, they remain a cornerstone of antibiotic

therap
therapy
y for the spectr
spectrum
um of seriou
seriouss system
systemic
ic infect
infection
ion.. Struct
Structural
urally,
ly, all are
 based upon the four member nitrogen containing beta-lactam ring that gives
these agents their antibacterial activity.

They
They can be divide
divided
d into
into four
four groups
groups - penici
penicilli
llins,
ns, cephal
cephalosp
ospori
orins,
ns,
carba
carbape
pene
nems
ms and
and mono
monobabact
ctam
amss - on the
the basi
basiss of the
the mole
molecucula
larr struc
structu
tures
res
surrounding and supporting this active site, A fifth group in clinical use are the
 beta-lactamase inhibitors that do not have intrinsic antibacterial activity.
activity.

Penicillin:

The penicillin may be divided into four main groups on the basis of their 
spectra of antibacterial activity. All the developments and manipulations of the
 penicillin nucleus have had the aim of extending the spectrum of the agents
against gram-negative bacteria or increasing their resistance to beta-lactamase
enzymes, or both.

All of this "improved" penicillin retains activity against bacteria such as

hemo
hemolylyti
ticc stre
strept
ptoc
ococ
occi
ci and
and clos
clostr
trid
idia
ia,, whic
which
h rema
remain
in sens
sensit
itiv
ivee to benz
benzyl
yl
 penicillin. However, it must be remembered that all the alterations to the basic
 penicillin structure necessary to achieve these particular advantages have been
at the expense of intrinsic activity against sensitive organisms.

Early penicillin, Benzyl penicillin (penicillin G), the original member of 
the group, remains the most active antibacterial agent against sensitive bacteria.
It is the drug of choice for serious infections with beta-hemolytic streptococci,
alpha-hemolytic streptococci (combined with an aminoglycoside in sub-acute
 bacterial endocarditis), the pneumococcus, the meningococcus and clostridia
other than Clostridium. Phenoxymethyl penicillin (penicillin V) remains useful
when oral treatment of streptococcal infections is adequate.

2
 Fig: 1 Basic 3D structure of Penicillin G

Fig: 2 Penicillin G structure

As shown in the diagram, penicillin is not a single compound but a group

of closely related compounds, all with the same basic ring-like structure (a β-
lactam) derived from two amino acids (valine and cysteine) via a tripeptide
intermediate. The third amino acid of this tripeptide is replaced by an acyl group
(R in the diagram below) and the nature of this acyl group produces specific
 properties on different types of penicillin.

3
History of penicillin:

Penicillin was the first naturally occurring antibiotic discovered. There

are now more than 60 antibiotics, which are substances that are produced by
microbes and that fight bacteria, fungi and other microbes harmful to humans -
the word means against (anti) life (bio). Penicillin is obtained in a number of 
forms from Penicillium moulds.

In 1928, while working in St. Mary’s Hospital in London, bacteriologist

Alexander
Alexander Fleming was conducting research on the flu. He had been searching
searching
for antibacterial agents, influenced by his wartime experience. He had witnessed
the deaths of many soldiers that died, not from the wounds they received during
combat, but from secondary infections of those wounds. While he was on
holidays, a bit of blue-green mould had fallen into a discarded culture plate
containing Staphylococcus aureus,
aureus, forming a clear patch in the surrounding
area. From this he could conclude that the mould was producing an antibiotic
substance. He named the antibiotic penicillin, after the  Penicillium notatum
mould that produced it and 1929; he published the results of his investigations,
noting that his discovery might have therapeutic value if it could be produced in
quantity. Unfortunately it couldn’t, and it would be 10 years before another 
significant leap forward for penicillin would occur.

In 1938 Dr. Howard W. Florey came across Fleming’s paper on penicillin

(which oddly had languished in obscurity). While Fleming’s lab was poorly
equipped with no staff support, Florey’s lab was well equipped and staffed with
a team of scientists at Oxford University that included Dr. Ernst B. Chain. It
was
was Chai
Chain n who
who bega
begann extr
extrac
acti
ting
ng peni
penici
cill
llin
in into
into a puri
purifi
fied
ed and
and powe
powerf
rful
ul
antibiotic.

At first penicillin was made using old dairy equipment and after a great
deal of effort, enough was extracted for experimentation to begin. Eight white
mice were inoculated with deadly Streptococcus germs, followed by injection of 
 penicillin in half the mice. All of the untreated mice died the next day while the
treated mice all recovered. Now it was time for the first human test. Albert
Alexander, a 48-year-old London policeman had developed septicaemia as a
result of a small cut on his face. When treated with penicillin

Alexander began to recover within the day. However Florey’s team didn’t
have enough of the drug to see the patient through to a full recovery and he later 
re-lapsed
re-lapsed and died. However
However by 1941, it was acknowledged
acknowledged that penicillin
penicillin was
indeed a worthwhile drug and could save thousands of lives. In the same year 
Florey travelled to the United States (which at the time was still neutral) to
continue his work with penicillin.
4
 Because the United States intended to enter into World War II in another 
few months the penicillin project, which became declared a war project, was
given top priority (and funding). Florey and his team were able to use beer-
 brewing technology to produce the huge amounts of the mouldy liquor needed
for penicillin production. This underwent a slow purification process to produce
the large amounts of clinically usable penicillin that became available for 
military use in early 1940’s.

In Peoria, Illinois a blue-green mould was found growing on a mouldy

cantaloupe in a market. This mould was identified as  Penicillium chrysogenum
and produced approximately 200times as much penicillin than what Florey’s
team was working with ( Penicillium notatum).

Scientists began to try to increase the amount of penicillin produced by

 P. chrysogenum, by irradiating it with X-rays and UV rays in order to induce
mutations of this species. They succeeded and developed a mutant that
 produced 1000 times the amount of penicillin than Fleming’s original culture.
At the same time scientists began to grow the mould in the first deep tank 
fermenters.

By late 1943, mass production of the drug had commenced and by the
end of the war, many companies were manufacturing the drug, including the
Merck, Squibb and Pfizer. In 1945 Fleming was awarded the Nobel Prize in
Physiology and Medicine along with Florey and Chain.

In the coming years, strains and techniques were improved upon and
Penicillin saved tens of thousands of lives. However, it all began with a bit of 
 blue-green mould falling onto a discarded culture plate.

5
2. Penicillin production and Recovery
Penicillin is produced in large scale by fermentation. The success of the
 production depends upon proper selection of strain that we are going to use, raw
material selection, optimizing process parameters, monitoring and maintaining
the parameters, product recovery, recovery of solvents and also effective
effluent treatment.

Sequential steps in Penicillin G production in terms of department:

• Microbiology department
• Quality assurance
• Pilot scale plant
• Fermentation plant (Production)
• Quality control
• Product recovery plant

2.1 Microbiology department:

In microbiology department two main processes are carried over:

• Strain/colony selection
• Shake flask analysis

2.1.1 Strain/colony selection:

In strain/colony selection the colony of organism which has a high

 productivity and genetic stability is selected mainly by analyzing zone of 
inhibition assays.

The next step after isolation of microorganisms is their screening. A set

of highly selective procedures, which allows the detection and isolation of 
microorganisms producing the desired metabolite, constitutes primary
screening. Ideally, primary screening should be rapid, inexpensive, predictive,
specific but effective for a broad range of compounds and applicable on a large
scale.

6
Criteria for the choice of organism:
1. Nutritional characteristics of the organism.
2. Optimum temperature of the organism.
3. Suitability of the organism to the type of process to be used.
4. Stability of the microorganism.
5. Productivity of the organism.
6. Ease of product recovery.

Fig: 3 Zone of inhibition assay

2.1.2 Shake flask analysis:

Culture flasks (usually Erlenmeyer) of 250 or 500 ml or larger are used

for growing microorganisms; these are shaken, generally, by a gyratory shaker 
at 200-250 R.P.M. Shake cultures are usually applied to aerobic processes. At
this stage, the spores will begin to revive and form vegetative cells.
Temperature is normally maintained at 23-28°C and pH at ~6.5, although there
may be some changes made to facilitate optimum growth. In general,
filamentous microorganisms are grown for the production of secondary
metabolites, which begins 1-3 days after inoculation and continues 3-4 days
thereafter.

In all such cases, the shake cultures are used for:

• Strain improvement
• Determination of the optimum conditions for the fermentation process.
• In many industrial processes, they are also used for the initial stages of 
inoculum development.

7
 Shake cultures are a convenient method of growing microorganisms in
submerged cultures under aerobic conditions created by shaking; it is a small
scale equivalent of stirred tank bioreactor.

Both the devices are extensively used with filamentous micro-organisms

and, often, with other types of micro-organisms as well.

Usually, complex media are used for shake flask cultures, but the
objective is to devise a synthetic medium for the fermentation process. Studies
on inoculum size, temperature, agitation, nutrition are initially done using these
cultures to monitor their influences on growth and product formation.

2.2 Quality assurance:

Quality assurance, or QA for short, refers to a program for the systematic

monitoring and evaluation of the various aspects of a project, service, or facility
to ensure that standards of quality are being met. Quality assurance, in its
 broadest sense, is any action taken to prevent quality problems from occurring.
In practice, this means devising systems for carrying out tasks which directly
affect product quality.

Two key principles characterize QA: "fit for purpose" (the product should
 be suitable for the intended purpose) and "right first time" (mistakes should be
eliminated). QA includes regulation of the quality of raw materials, assemblies,
 products and components; services related to production; and management,
 production and inspection processes.

Even goods with low prices can be considered quality items if they meet
a market need. QA is more than just testing the quality of aspects of a product,
service or facility, it analyzes the quality to make sure it conforms to specific
requirements and comply with established plans. SOP’s are formulated and
maintained for maintaining the quality.

Steps for Quality Assurance Process

• Test previous article
• Plan to improve
• Design to include improvements and requirements
• Manufacture with improvements
• Review new item and improvements
• Test new item

8
Raw materials :

Quality of the raw materials used in the fermentation is the deciding

factor of the productivity; hence the quality analysis of each and every raw
material should be carried over.

When performing any kind of fermentation, the selection of media is of 

critical importance to the overall performance of the fermentation. The aim of 
the media is to provide all the elements required for the synthesis of cell
materials and the formation of the desired product. At the same time, the media
must provide a favourable environment for the culture in question (an example
of this would be control of pH by addition of calcium carbonate or inorganic
 phosphates). As well as this it must remain cost effective.

Typically, all microorganisms require Carbon, Hydrogen, Oxygen,

Sulphur and Nitrogen for cell growth and cell maintenance. In many cases,
microorganisms require small amounts of trace elements such as Cu, Mn and
Co (this will frequently depend on the water source as most water sources
contain small amounts of the elements) or growth factors such as vitamins or 
amino acids as well.

Each and every raw material is given a reference number which is called
M.I.N (Material inward number). Raw materials used in the fermentation of 
Penicillin-G are: Acetic acid, Activated carbon, Ammonium sulphate, n-butyl
acetate, n-butyl alcohol, Calcium carbonate, Corn steep liquor, Citric acid
monohydrate, Copper sulphate pentahydrate, Corrugated box, Demulsifier,
Dextrose monohydrate, Dextrose syrup, Ferrous sulphate heptahydrate, Filter 
aid, Formaldehyde, Maize oil, Magnesium sulphate heptahydrate,
Monopotassium phosphate, Pen G first crystal label, Maganese sulphate,
Phenyl acetic acid, Polypropylene glycol, Polythene bag, Potassium carbonate,
Sodium phenyl acetate solution, Sodium hydroxide solution, Sodium sulphate,
Sucrose, Sulphuric acid, Zinc sulphate, Ferrous sulphate and High maltose corn
syrup.

Documentation :

Every analysis result should be recorded and a clear documentation

should be prepared. All analysis should be performed as per the S.O.P prepared
and they should be recorded having a reference number for future reference.

9
2.3 Pilot scale plant:
Pilot scale reactor will be similar in design to the bench-top reactor 
except it will have a size of about 100-1000 litres. The aim here is to examine
the effect of scale-up on the culture. At this stage, hopefully growth will
continue as before, however, there are often sudden changes or loss in
 performance. This can be due to changes in the morphology of the culture
(remember   Penicillium chrysogenum is a filamentous fungi and hence
 pseudoplastic) that may or may not be correctable.

If the pilot-plant stage is successful then work can begin on an industrial

scale operation. This is now very much an engineering problem. The reactor 
must be capable of running aseptically and the design must reflect safety and
contamination requirements. At this stage the medium being added to the
reactor will change. Now we wish to emphasis penicillin production over 
growth while maintaining a constant volume. Carbon and nitrogen will be added
sparingly alongside precursor molecules for penicillin fed-batch style. Another 
note is that the presence of penicillin in the reactor is itself inhibitory to the
 production of penicillin. Therefore, we must have an efficient method for the
removal of this product and to maintain constant volume in the reactor. Other 
systems, such as cooling water supply, must also be considered.

In spic pharmaceuticals there are two 70litre fermenters, two1000 and

one 1500litre fermenters for pilot scale. Here pilot scale studies are carried over.
Optimization of media constituents, their flow rates, optimum temperature, pH
and various other additions are estimated. Pilot plant is equipped with latest
control system SCADA (Supervisory control and data acquisition, Eurotherm
suite).

Pilot plants are used to reduce the risk associated with construction of 
large process plants. They provide valuable data for design of the full-scale
 plant. Scientific data about reactions, material properties, corrosiveness, for 
instance, may be available, but it is difficult to predict the behavior of a process
of any complexity. Engineering data from other process may be available, but
this data cannot always be clearly applied to the process of interest. Designers
use data from the pilot plant to refine their design of the production scale
facility.

If a system is well defined and the engineering parameters are known,

 pilot plants are not used. For instance, a business that wants to expand
 production capacity by building a new plant that does the same thing as an
existing plant may choose to not use a pilot plant.

10
Fig: 3 Pilot scale fermeter (70 liter)
11
2.4 Fermentation plant:
Penicillin G production is done by two stage fed batch
fermentation. In the first stage, primary metabolism will be emphasized. Media
for this stage will typically be focused on achieving maximum growth and
 biomass production. In next stage (production stage) we desire to produce PenG
a secondary metabolite, hence the flow rates of the feed is adjusted to serve for 
only maintenance energy and not for growth, biomass production in this stage is
undesired. In SPIC pharmaceutical there about eleven 100m3 fermenters and six
60m3 seed fermenters are there for production.

Seed stage:

At this stage, sugar will usually be the primary source of carbohydrate for 
the culture. The seed media contains sugar, MSP, AMS, CSM, CaCO3, CSL,
and PPG. Before inoculation Steam is used to keep the reactor running
aseptically. This is achieved because the reactor is designed as a pressure vessel
and steam is sent through at a minimum temperature/pressure of 121°C/15 psi
for 15-30min.

The media thus prepared is maintained under positive pressure by giving

sterile air to avoid contamination. About 10% of volume of the reactor is
inoculated in the vessel, the Penicillium chrysogenum starts to adapt itself in the
environment.

Sugar is given as carbohydrate source because it is easily used for energy

metabolism and thus gives the best yields in terms of growth. At this stage
substrate corn steep liquor is frequently added that will give a readily usable
source of Nitrogen.

Both these substrates allow maximal growth of the culture at the expense
of product (antibiotic) formation. This is because growth and antibiotic
 production are mutually interrelated and inversely proportionate. Readily
available carbon and nitrogen sources tend to inhibit antibiotic production (this
is known as Catabolite Repression, where a secondary metabolite is inhibited
 by the presence of a more readily usable substrate).

The parameters to be maintained in seed stage are:

Temperature : 25°C
Pressure : 0.5Kg/Cm2
Airflow : 1.0vvm
Agitation : 2.3m/s
12
The seed maturation criteria:
Age : 60-64 hrs
PMV : above 20%
pH : increasing trend

Production stage:

In this stage the Penicillin G is produced. The constituents used for this
 production are sugar (45%), MSP, AMS, CSM, CSL, CaCO3, CSL, and PPG.

Once the broth is seeded with the seed broth from a matured seed
fermeter the time is noted as log0. At the log0 the fermenter is aerated and
agitated to maintain DO2, temperature is set at 25°C; pH 6.5 is maintained with
AMH.

After 5hrs of feeding, precursor PAA is added as NaPA and care should
 be taken that its concentration should be in the range of 0.3-0.8g/l if 
concentration exceeds it is toxic to organism.

Once feeding is started Pen-G production will start. The concentration of 
PenG or PAA, Nitrogen, and PMV are monitored at every 8 hr intervals.
Depending on residual PAA value PAA addition is given.

Initially the growth of the microorganism will go on up to 50hrs and the

 production will be increasing slowly (log phase).

From 50-120hrs the production will be maximum (production phase),

after 170 hrs the production will decrease and come to an end due to lysis of 
organism.

The parameters to be maintained in seed stage are:

Temperature : 25°C
Pressure : 0.6Kg/Cm2
Airflow : 1.0vvm
DO : 60%
PAA : 0.3-0.8g/l

13
 Fig: 4 100m3 Fermenter used for production

The parameters for the production are monitored by a centralized control

system called DCS (Distributed Control System). By this system all the
 parameters can be monitored and can be controlled by using various control
valves.

Harvesting of fermenter:

Once the production is stopped, 0.5% of formaldehyde is added to the

 broth to kill the microorganism. This is sent to recovery plant to recover Pen-G.

2.5 Quality control:

Quality control is a process by which entities review the quality of all

factors involved in production. This approach places an emphasis on three
aspects: Total Quality Control is the most important inspection control of all in
cases where, despite statistical quality control techniques or quality
improvements implemented, sales decrease.

If the original specification does not reflect the correct quality

requirements, quality cannot be inspected or manufactured into the product.

14
 For instance, the parameters for a pressure vessel should include not only
the material and dimensions, but also operating, environmental, safety,
reliability and maintainability requirements.

Elements such as controls, job management, defined and well managed

 processes, performance and integrity criteria, and identification of records
competence, such as knowledge, skills, experience, and qualifications soft
elements, such as personnel integrity, confidence, organizational culture,
motivation, team spirit, and quality relationships.

The quality of the outputs is at risk if any of these three aspects is

deficient in any way.

Instruments used:
1. KF titrator (Karl Fischer)

Principle:

The determination of total moisture by Karl Fisher titration is a

calculation based on the concentration of iodine in the KF titrating reagent (i.e.
titer) and the amount of KF re-agent consumed in the titration. The end-point of 
the titration is determined by the dead-stop end-point method.

Reagents
 Karl Fisher reagent
 Methanol (moisture free)
 Sodium sulphate (Na2SO4), moisture free
 Sodium tartrate dihydrate (Na 2C4H4O6 • 2 H2O)

Procedure
Standardization:

 New bottles containing ‘Composite 5’ or ‘Titrant 5’ must be standardized

against sodium tartrate dihydrate. 230.10g sodium tartrate dehydrate
corresponds to 36.4g H2O.

Using approx 0.1g of sodium tartrate dihydrate as sample. Fresh solvent

is used between each standardization. The standardization is accepted when two
determinations agree within 0.5% relative.

15
 Fig 5: KF Titrator 

2. Kjeldahl Method for Determining Nitrogen

The Kjeldahl method was developed over 100 years ago for determining
the nitrogen contents in organic and inorganic substances. Although the
technique and apparatus have been modified over the years, the basic principles
introduced by Johan Kjeldahl still endure today.

Kjeldahl nitrogen determinations are performed on a variety of 

substances such as meat, feed, grain, waste water, soil, and many other samples.
Various scientific associations approve and have refined the Kjeldahl method,
including the AOAC International (formerly the Association of Official
Analytical Chemists), Association of American Cereal Chemists, American Oil
Chemists Society, Environmental Protection Agency, International Standards
Organization, and United States Department of Agriculture.The Kjeldahl
method has three main steps: digestion, distillation, and titration.

Digestion

Digestion is accomplished by boiling a homogeneous sample in

concentrated sulfuric acid. The end result is an ammonium sulfate solution.
The general equation for the digestion of an organic sample is shown below:

Organic N + H2SO4 → (NH4)SO4 + H2O + CO4 + other sample matrix

 byproducts
16
Distillation:

Excess base is added to the digestion product to convert NH4 to NH3 as

indicated in the following equation. The NH3 is recovered by distilling the
reaction product.

(NH4)2SO4 + 2NaOH → 2NH3 + Na2SO4 + 2H2O

Titration:

Titration quantifies the amount of ammonia in the receiving solution. The

amount of nitrogen in a sample can be calculated from the quantified amount of 
ammonia ion in the receiving solution.

There are two types of titration—back titration and direct titration. Both
methods indicate the ammonia present in the distillate with a color change.

In back titration (commonly used in macro Kjeldahl), the ammonia is

captured by a carefully measured excess of a standardized acid solution in the
receiving flask. The excess of acid in the receiving solution keeps the pH low,
and the indicator does not change until the solution is "back titrated" with base.

2NH3 + 2H2SO4 → (NH4)2SO4 + H2SO4

(NH4)2SO4 + H2SO4 + 2NaOH → Na2SO4 + (NH4)2SO4 + 2H2O

In direct titration, if boric acid is used as the receiving solution instead of 
a standardized mineral acid, the chemical reaction is:

 NH3 + H3BO3 → NH4 + H2BO-3 + H3BO3

The boric acid captures the ammonia gas, forming an ammonium-borate

complex. As the ammonia is collected the color of the receiving solution
changes.

2NH4 + H2BO3 → H2SO4 (NH4)2SO4 + 2H3BO3

The boric acid method has the advantages that only one standard solution
is necessary for the determination and that the solution has a long shelf life.

17
Applications

The Kjeldahl method's precision and reproducibility have made it the

internationally-recognized method for estimating the protein content in foods
and it is the standard method against which all other methods are judged.

Fig 6: Kjeldahl Method for Determining Nitrogen

3. HPLC

High-performance liquid chromatography (or high-pressure liquid

chromatography, HPLC) is a form of column chromatography used frequently
in biochemistry and analytical chemistry to separate, identify, and quantify
compounds based on their idiosyncratic polarities and interactions with the
column's stationary phase.

HPLC utilizes different types of stationary phase (typically, hydrophobic

saturated carbon chains), a pump that moves the mobile phase(s) and analyte
through the column, and a detector that provides a characteristic retention time
for the analyte. The detector may also provide other characteristic information
(i.e. UV/Vis spectroscopic data for analyte if so equipped). Analyte retention
time varies depending on the strength of its interactions with the stationary
 phase, the ratio/composition of solvent(s) used, and the flow rate of the mobile
 phase.

18
 In QC we use HPLC mainly for determining PAA and PenicillinG
concentration and activity. We use reverse phase chromatography, where the
stationary phase is non-polar and mobile phase is polar. The mobile phase we
commonly used for PenG activity estimation is Acetonitryl in 0.1%PBS and the
stationary phase is ODS (Octo Dodecyl Silane).

Fig 7: HPLC

With HPLC, a pump (rather than gravity) provides the higher pressure
required to propel the mobile phase and analyte through the densely packed
column. The increased density arises from smaller particle sizes. This allows for 
a better separation on columns of shorter length when compared to ordinary
column chromatography.

4. Gas chromatography (GC)

It is a common type of chromatography used in analytic chemistry for 

separating and analyzing compounds that can be vaporized without
decomposition. Typical uses of GC include testing the purity of a particular 
substance, or separating the different components of a mixture (the relative
amounts of such components can also be determined). In some situations, GC
may help in identifying a compound. In preparative chromatography, GC can be
used to prepare pure compounds from a mixture.

In gas chromatography, the moving phase (or "mobile phase") is a carrier 

gas, usually an inert gas such as helium or an unreactive gas such as nitrogen.
The stationary phase is a microscopic layer of liquid or polymer on an inert

19
solid support, inside a piece of glass or metal tubing called a column. The
instrument used to perform gas chromatography is called a gas chromatograph.

The gaseous compounds being analyzed interact with the walls of the
column, which is coated with different stationary phases. This causes each
compound to elute at a different time, known as the retention time of the
compound. The comparison of retention times is what gives GC its analytical
usefulness.

Gas chromatography is in principle similar to column chromatography (as

well as other forms of chromatography, such as HPLC, TLC), but has several
notable difference. Firstly, the process of separating the compounds in a mixture
is carried out between a liquid stationary phase and a gas moving phase,
whereas in column chromatography the stationary phase is a solid and the
moving phase is a liquid. (Hence the full name of the procedure is "Gas-liquid
chromatography", referring to the mobile and stationary phases, respectively.).

Secondly, the column through which the gas phase passes is located in
an oven where the temperature of the gas can be controlled, whereas column
chromatography (typically) has no such temperature control. Thirdly, the
concentration of a compound in the gas phase is solely a function of the vapor 
 pressure of the gas. Gas chromatography is also similar to fractional distillation,
since both processes separate the components of a mixture primarily based on
 boiling point (or vapor pressure) differences.

Fig 8: Gas chromatography

20
5. Polari-meter :
A polarimeter is a scientific instrument used to measure the angle of 
rotation caused by passing polarized light through an optically active substance.
Some chemical substances are optically active, and polarized light will rotate
either to the left (counter-clockwise) or right (clockwise) when passed through
these substances. The amount by which the light is rotated is known as the angle
of rotation.

Polarimeters measure this by passing monochromatic light through the

first of two polarizing plates, creating a polarized beam. This first plate is
known as the polarizer. This beam is then rotated as it passes through the
sample. The sample is usually prepared as a tube where the optically active
substance is dissolved in an optically inactive chemical such as distilled water,
ethanol and methanol. Some polarimeters can be fitted with tubes that allow for 
sample to flow through continuously.

After passing through the sample, a second polarizer, known as the

analyzer, rotates either via manual rotation or automatic detection of the angle.
When the analyzer is rotated to the proper angle, the maximum amount of light
will pass through and shine onto a detector.Semi-automatic polarimeter requires
visual detection but use push-buttons to rotate the analyzer and offer digital
displays. The most modern polarimeters are fully automatic, and simply require
the user to press a button and wait for a digital readout.

The angle of rotation of an optically active substance can be affected by:

 Concentration of the sample Wavelength of light passing through

the sample (generally, angle of rotation and wavelength tend to be
inversely proportional)
 Temperature of the sample (generally the two are directly
 proportional)
 Length of the sample cell (input by the user into most automatic
 polarimeters to ensure better accuracy)

Polarimeters can be calibrated – or at least verified – by measuring a

quartz plate, which is constructed to always read at a certain angle of rotation
(usually +34°, but +17° and +8.5° are also popular depending on the sample).
Quartz plates are preferred by many users because a solid sample are much less
affected by variations in temperature, and does not need to be mixed on-demand
like sucrose solutions. Applications Because many optically active chemicals
are stereoisomers, a polarimeter can be used to identify which isomer is present

21
in a sample – if it rotates polarized light to the left, it it a levo-isomer, and to the
right, a dextro-isomer.

If the specific rotation of a sample is already known, then the

concentration and/or purity of a solution containing it can be calculated. Most
automatic polari-meters make this calculation automatically, given input on
variables from the user.

Concentration and purity measurements are especially important to

determine product or ingredient quality in the food & beverage and
 pharmaceutical industries. Samples that display specific rotations that can be
calculated for purity with a polarimeter include:

 Antibiotics
 Vitamins
 Amino Acids
 Starches
 Sugars

Polarimeters are used for determining quality of sugar. Often it is used a

modified polarimeter with a flow cell called a saccharimeter. These instruments
use the International Sugar Scale (as defined by ICUMSA).

Fig: 9 Polarimeter 

22
6. LOD analyzer:

The classic laboratory method of measuring high level moisture in solid

or semi-solid materials is loss on drying (LOD). In this technique a sample of 
material is weighed, heated in an oven for an appropriate period, cooled in the
dry atmosphere of a desiccators, and then reweighed.

If the volatile content of the solid is primarily water, the LOD technique
gives a good measure of moisture content. Because the manual laboratory
method is relatively slow, automated moisture analyzers have been developed
that can reduce the time necessary for a test from a couple hours to just a few
minutes. These analyzers incorporate an electronic balance with a sample tray
and surrounding heating element. Under microprocessor control the sample can
 be heated rapidly and a result computed prior to the completion of the process,
 based on the moisture loss rate, known as a drying curve.

Fig: 10 LOD analyzer 

7.Bulk density apparatus:

The bulk and tapped density of pharmaceutical powders are often

measured for processability. The tapped density is measured for two primary
 purposes: (i) the tapped value is more reproducibly measured than the bulk 
value, and (ii) the "flowability" of a powder is inferred from the ratio of these
two measured densities.

The density ratio method is compared to flowability measurements on a

Johanson Flow Indicizer. These methods are evaluated for monodisperse glass
 beads as well as for common pharmaceutical excipients and blends. The
"tapped" density of a pharmaceutical powder is determined using a tapped
23
density tester, which is set to tap the powder at a fixed impact force and
frequency. The methods for measurement in the U.S. pharmaceutical industry
are specified in the U.S. Pharmacopeia (USP). Tapped density by the USP
method is determined by a linear progression of the number of taps. In light of 
experiments performed over the past decade, exhibiting a slow relaxation
approach to the final packing state (logarithmic with the number of taps), the
tapped density measured with the USP method is further explored. Additionally,
the dependence of the tapped density on the conditions specified in the USP
method, e.g., sample size and time separation between taps, is examined.

Fig:11 Bulk density analyzer 

2.6 Product recovery plant:

Penicillin G recovery is carried over by 8 steps:

1. Extraction with n-Butyl acetate

2. Removal of color(carbon treatment)

3. Extraction of PenG in aqueous phase

4. Crystallization using butanol

5. Crystal washing

6. Drying

24
 1st Extraction unit 2nd Extraction unit 3rd Extraction unit

Dryer Crystal washing Crystallization

Fig: 12 Schematic representation recovery processes

1 st Extraction unit:

In 1st stage the fermentation broth is collected in a two 70m 3 reactor and
one more reactor is kept for receiving partial discharge. The broth is first treated
with H2SO4 and the pH is brought down to 2. This is done because in this pH
Pen G soluble in organic phase which is provided by adding n-Butyl acetate.
The broth can form emulsion and the extraction will become difficult, to avoid
this situation we are adding a demulsifying agent (Quaternary ammonium salt).

With these additions Pen G is extracted in n-Butyl acetate as Rich BAC

and this is then sent to color removal unit. The color removal is done by using
Activated carbon. Our penicillin rich solution is then treated with 0.25-5%
activated carbon to remove pigments and impurities.

Activated carbon is an amorphous solid, and absorbs molecules from the

liquid phase through is highly developed internal pore structure. It is obtained in
 powered, pelleted or granular form and is produced from coal, wood and
coconut shells.

2nd Extraction unit:

In this unit PenG is converted into Potassium salt so that it can be

extracted in aqueous phase. This is achieved by adding 13-15% K 2CO3 solution.
In this unit PenG salt is recovered in aqueous phase, then this phase along with
some BAC. Then the rich aqueous phase is sent to 3rd extraction unit.
25
 Fig: 13 Scheme of the extraction process

3rd Extraction unit:

In third extraction unit the residual BAC is completely removed and rich
aqueous phase pH is adjusted to 6.2-6.5 with KOH. Then the RAS is transferred
to crystallization unit. The exhaust BAC is sent to SRP for recovery.

Crystallization unit:

Crystals are highly organized inert matters. If grown without external

interference, they grow in polyhedral shapes and exhibit many degrees of 
symmetry. Penicillin G is an odorless, colorless or white crystal, or crystalline
 powder. Crystallization is essentially a polishing step that yields a highly pure
 product. It is done through phase separation from a liquid to a solid.

In crystallization unit we use a Butanol as solvent. Batch crystallization is

the most the most used method for polishing antibiotics, including penicillin G.
They are slowly cooled to produce supersaturation. Seeding causes nucleation
and growth is encouraged by further cooling until the desired crystals are
obtained. While the crystallization procedures product of very high purity,
improves appearance and has a low energy input, the process can be time
consuming due to the high concentration of the solutions during crystallization.
It can also be profoundly affected by trace impurities and batch crystallization
can often give poor quality, nonuniform product.

After crystallization gets over the slurry is sent to further 

 purification and the exhaust Butanol is sent to SRP for recovery.

26
Crystal washing:

While the penicillin G crystals we have formed are essentially pure in

nature but adsorption and capillary attraction cause impurities from its mother 
liquor on their surfaces and within the voids of the particulate mass. Because of 
this the crystals must be washed and pre-dried in a liquid in which they are
relatively insoluble. This solvent should be miscible with the mother solvent.
For this purpose we use anhydrous l-Propanol, n-Butanol or another volatile
solvent.

Dryer:

Drying is done by fluid bed dryer. Fluid bed processing involves drying,
cooling, agglomeration, granulation, and coating of particulate materials. It is
ideal for a wide range of both heat sensitive and non-heat sensitive products.
Uniform processing conditions are achieved by passing a gas (usually air)
through a product layer under controlled velocity conditions to create a
fluidized state.

In fluid bed drying, heat is supplied by the fluidization gas, but the gas
flow need not be the only source. Heat may be effectively introduced by heating
surfaces (panels or tubes) immersed in the fluidized layer.

In fluid bed cooling, cold gas (usually ambient or conditioned air) is used.
Conditioning of the gas may be required to achieve sufficient product cooling in
an economically sized plant and to prevent pick up of volatiles (usually
moisture). Heat may also be removed by cooling surfaces immersed in the
fluidized layer.

Agglomeration and granulation may be performed in a number of ways

depending upon the feed to be processed and the product properties to be
achieved. Fluid bed coating of powders, granules, or tablets involves the
spraying of a liquid on the fluidized powder under strictly controlled conditions.
The dry PenG powder is sent to pulverizing unit.

27
3. Butyl
acetate

3-5% BAC Exhaust BAC

Exhaust
with spent
BAC with H2O
 broth

Solvent recovery plant

Solvents used in recovery plant are costly hence these should be recycled
inorder to reduce production cost, so a solvent recovery plant is been installed.
The two main solvents which are used in recovery of PenG are BAC and
Butanol. These solvents after extraction are sent to solvent recovery plant at
different stages, these solvents are treated by different techniques and they are
recycled.

3.1 Recycling of BAC:

n-Butyl acetate solvent used in L-L extraction of Penicillin is costly, so

recycling of BAC is necessary to minimize the cost of production. BAC is sent
to SRP plant in three stages:

1. BAC after 1st extraction process, it contains about 3-5% of BAC

with spent broth

2. Exhaust BAC after 2nd extraction process, it contains only BAC

3. Exhaust BAC with H2O, this comes after L-L extraction in 3 rd stage

28
 Fig:14 Scheme of the BAC sent to SRP

BAC received at different stages are collected separately and treated in

different methodologies.

3.1.1 BAC with spent broth:

BAC with spent broth is treated by (KNIK) distillation column.

29
 Fig: 15 Counter-current Distillation column

The liquid inlet is given at the top of the column. The pressure at the top
of the column will be around 100mm.Hg and at the bottom it will be around
280mm.Hg. Pressure drop is used as key in this distillation process. After 
vapourisation the gas phase is passed to condenser and then to a phase
separator. BAC is collected in a tank and recycled back to PenG recovery plant.

Feed in
K  Condenser  Vent condenser 

 N Vacuum pump
Phase separator 
I

K 
BAC
To waste treatment plant

Fig: 16 Recycling of BAC

3.1.2 Rectification of exhaust BAC:

In this stage of BAC the water will be present in addition; this can be
rectified by using same strategy. Here a process heater is attached in order to
vaporize the heat. Then it is passed to distillation column then to condenser and
then to a phase separator. BAC and water can be collected separately from the
 phase separator.

Distillation Condenser 
column

Phase separator 
30
 Process
heater  Feed inlet BAC

Water 

Fig: 17 Recycling of BAC and water 

3.1.3 Stripping of exhaust BAC:

When the feed contains low amount of BAC and high amount of water 
this kind of procedure is used. This kind of mixture is received after the third
extraction unit. In this BAC concentration will be lower when compared to feed
received after second extraction. Usually BAC concentration will be less
than1%.

First the exhaust BAC is sent to the process heater and then it is sent the
distillation column then to the condenser and then to the phase separator. The
water from the phase separator is recycled back to the distillation column. Until
the clear separation is achieved recycle is continued.

Distillation Condenser 
column

Phase separator 

BAC

Water 
Process
heater 
Feed inlet

Fig: 18 Stripping of BAC

31
3.2 Recovery of Butanol:

Butanol used in crystallization and crystal wash procedures should be

recovered and recycled to PenG recovery plant. Like BAC Butanol is also
received in different conditions. This can be categorized by the quantity of the
water present with the Butanol and also with the presence of BAC.

Butanol present in the effluent of PenG recovery process can be

categorized into 4 classes of feed and they are:

1. Low exhaust Butanol:

In this butanol-water concentration ratio will be around 20:80. This

can be recycled by stripping unit. This feed is got during drying
 process.

2. High exhaust Butanol:

In this feed the composition of Butanol and water will be around

80:20. This can be recycled by using rectification unit. Feed
filtration after crystallization has this property.

3. Mother liquor:

Water will be present in butanol in trace amounts (93:7); the

complete recycling can be achieved by using a rectification unit.
This feed is got after crystal wash.

4. Ternary phase:

In this feed BAC is present along Butanol and water (10:75:15).

This forms a tertiary phase and these can be separated by
hydrolysis.

3.2.1 Stripping of low exhaust Butanol:

32
 Stripping of Butanol is done by distillation and phase separation
techniques as used in the recovery BAC. The difference in boiling points is
exploited for this stripping process.

The pressure in the column is reduced so that the boiling point difference
can be used as a tool for removing Butanol from the water. As the water 
concentration is more we have to recycle the water to distillation column until
complete stripping of Butanol takes place.

This can be schematically represented as:

Distillation Condenser 
column

Phase separator 

High
exhaust
Butanol
Water 
Process
heater 
Low exhaust butanol

Fig: 19 Stripping of Low exhaust Butanol

3.2.2 Rectification High exhaust Butanol:

In exhaust butanol water amount will be low, so the after phase

separation we have to recycle the feed so that the water is removed completely.
Initially after phase separation we will get another high exhaust butanol on
continuous recycling we will get butanol alone. We also get some low exhaust
 butanol which has to be sent to stripping column for further treatment.

Distillation Condenser 
column
33
 Phase separator 

Process heater 

Butanol High exhaust Low exhaust

High exhaust butanol Butanol Butanol

Fig: 20 Rectification unit for high exhaust Butanol

3.2.3 Rectification of mother liquor:

In mother liquor the water concentration is low hence the recovery can be
achieved by simple distillation column setup. Recycle of condensate is not
necessary but when water concentration is high we can recycle and butanol can
 be recovered.

Distillation Condenser 
column

Process Butanol
heater 

Mother liquor 

Fig: 21 Schematic representation of rectification column

3.2.4 Ternary phase:

In this feed BAC is present along water and Butanol. Dissocitive

extraction is used. Dissociation extraction is the process of using chemical
reaction to force a solute to transfer from one liquid phase to another. One
example is the use of a sodium hydroxide solution to extract phenolics, acids, or 
mercaptans from a hydrocarbon stream.

34
 The opposite transfer can be forced by adding an acid to a sodium
 phenate stream to spring the phenolic back to a free phenol that can be extracted
into an organic solvent. Similarly, primary, secondary, and tertiary amines can
 be protonated with a strong acid to transfer the amine into a water solution, for 
example, as an amine hydrochloride salt. Conversely, a strong base can be
added to convert the amine salt back to free base, which can be extracted into a
solvent. This procedure is quite common in pharmaceutical production.

Distillation Condenser 
column

Phase separation

Process heater 

Butanol + Water  High exhaust

BAC + water  Butanol
+ NaOH

Fig: 22Butanol, BAC recovery by water hydrolysis

The NaOH is first mixed with the feed, here phase dissociation takes
 place. Then it is passed to process heater and then to distillation column then to
condenser then to phase separation. Till separation is finished the butanol phase
is recycled. Butanol is recovered as high exhaust butanol and this is sent as feed
to the high exhaust butanol rectification unit.

35
4. Effluent treatment plant
Effluent Treatment Plant or ETP are used in the pharmaceutical and
chemical industry to purify water and remove any toxic and non toxic materials
or chemicals from it. These plants are used by all companies for environment
 protection.

Importance of ETP Plants

The treatment of effluents in pharmaceutical industry is essential to

 prevent pollution of the receiving water. The effluent water treatment plants are
installed to reduce the possibility of pollution; biodegradable organics if left
unsolved, the levels of contamination in the process of purification could
damage bacterial treatment beds and lead to pollution of controlled waters. The
COD reduction is mainly carried over in ETP; due to addition of formaldehyde
organisms are dead and hence the BOD value will be appropriate so there is no
need for the methods to reduce BOD.

COD - Chemical Oxygen Demand

The chemical oxygen demand (COD) is commonly used to indirectly

measure the amount of organic compounds in water. Most applications of COD
determine the amount of organic pollutants found in surface water (e.g. lakes
and rivers), making COD a useful measure of water quality. It is expressed in
milligrams per liter (mg/L), which indicates the mass of oxygen consumed per 
liter of solution.

Class A water:

COD level is above 7000

36
Class B water:

COD level is below 4000

Class C water:

COD level is above 400

 Normally the effluent coming from plant has following properties its pH
is around 2-2.5 and its temperature is around 50-60°C. Total solids in present in
the effluent will be in the range of 20-25%.

The effluent is first sent to decanter or evaporator and then effluent is

transferred to double drum dryer. In this step about 80% of moisture is reduced
to 20%. Then temperature of the effluent is then reduced by using suitable heat
exchangers.

There are about two UASB systems and an anaerobic sludge digestion
system is present. The intention is to reduce the COD of the effluent. In addition
to this a Multi effect evaporator is present.

4.1 UASB:

Upflow anaerobic sludge blanket (UASB) technology, normally referred

to as UASB reactor, is a form of anaerobic digester that is used in the treatment
of waste water.

UASB uses an anaerobic process whilst forming a blanket of granular 

sludge which suspends in the tank. Wastewater flows upwards through the
 blanket and is processed (degraded) by the anaerobic microorganisms. The
upward flow combined with the settling action of gravity suspends the blanket
with the aid of flocculants. The blanket begins to reach maturity at around 3
months.

Small sludge granules begin to form whose surface area is covered in

aggregations of bacteria. In the absence of any support matrix, the flow
conditions create a selective environment in which only those microorganisms,
capable of attaching to each other, survive and proliferate. Eventually the

37
 aggregates form into dense compact biofilms referred to as "granules". A
 picture of anaerobic sludge granules can be found here.

The blanketing of the sludge enables a dual solid and hydraulic (liquid)
retention time in the digesters. Solids requiring a high degree of digestion can
remain in the reactors for periods up to 90 days. Sugars dissolved in the liquid
waste stream can be converted into gas quickly in the liquid phase which can
exit the system in less than a day. A properly designed UASB can reduce upto
75-85% COD.

38
 Fig: 23 UASB plant

4.2 Anaerobic digestion:

39
 Anaerobic digestion is a series of processes in which microorganisms
 break down biodegradable material in the absence of oxygen, used for industrial
or domestic purposes to manage waste and/or to release energy. It is widely
used as part of the process to treat wastewater. As part of an integrated waste
management system, anaerobic digestion reduces the emission of landfill gas
into the atmosphere.

Anaerobic digestion is widely used as a renewable energy source because

the process produces methane and carbon dioxide rich biogas suitable for 
energy production, helping to replace fossil fuels. The nutrient-rich digestate
which is also produced can be used as fertilizer.

The digestion process begins with bacterial hydrolysis of the input

materials in order to break down insoluble organic polymers such as
carbohydrates and make them available for other bacteria.

Acidogenic bacteria then convert the sugars and amino acids into carbon
dioxide, hydrogen, ammonia, and organic acids. Acetogenic bacteria then
convert these resulting organic acids into acetic acid, along with additional
ammonia, hydrogen, and carbon dioxide. Finally, methanogens convert these
 products to methane and carbon dioxide.

5. Utility and Instrumentation

40
5.1 Utility:

Utility plant provides the steam for sterilization, De-mineralized water,

cold water for heat exchanging purposes and a nitrogen plant for production of 
nitrogen.

The accessories present in this plant are boilers, chillers, compressors and
nitrogen plant.

DM water plant:

Dm water is used to avoid corrosion and to have control over process

feed inputs. Entire plant uses demineralized water hence it should be produced
economically.

The water is got from SIPCOT and this is sent to pretreatment plant and
then to aeration tank. Inorder to convert ferrous iron to ferric iron we are
aerating it, such that it is converted to ferric iron and in the presence of 
coagulating agent like aluminium sulphate ferric is coagulated and this is
removed by filters and sent to reservoir.

Water from the reservoir is transferred to DM-Plant. In DM plant it is

first passed through filter then through Strong acid cation filter. SAC filter 
contains strong acid cation resin (Duolite C-20). This resin filters ions like Ca 2+,
Mg2+, Na2+ etc.

Then this water is transferred to weak base anion filter. WBA filter has
weak base anion resin (Duolite A-368). This can filter Cl -, SO4-, CO2 then this
water is passed to degasser where CO2 is reduced.

After water passed to degasser it is passed to Strong base anion filter to

ensure that SiO2 and CO2 are completely removed. This demineralized water is
then passed to process plants.

Boilers:

41
 Steam for sterilization and for various processes should be produced in
large volume but economically, since this is a most power consuming process
 power consumption should be taken care.

There 3 boilers in the plant:

• 8Tonne/hr coal boiler 

• 15tonne/hr coal boiler 

• 15tonne/hr fuel boiler 

Chiller:

Cold water for heat exchange purposes and for various processes is
 produced by chilling unit. The water should be cooled down to 5°C. This water 
after fulfilling its purpose and gaining heat it will be around 11-15°C. This
water is then processed in chilling unit and recycled back.

Brine chillers are used to chill down the brine (Methyl ethylene glycol).
This is done because the brine doesn’t lose chillness readily. There are two
 brine chilling units.

There are two kinds of chillers:

• Water chiller 

• Brine chiller 

There are two cooling towers one is utility cooling tower and other one is
for process cooling tower for cooling process water.

Nitrogen plant:

 Nitrogen is a cool, dry, inert, gas that can displace Oxygen in a situation
where Oxygen may provide the catalyst for combustion or where it may affect
the quality of a product. Two nitrogen plants are there in the plant:

• Capacity 40Nm3/hr: 6.5Kg/Cm2

• Capacity 60 Nm3/hr: 6Kg/Cm2

42
5.2 Instrumentation department:

Instrumentation is the branch of engineering that deals with measurement

and control. The department should check its accuracy, reliability and validation
of the instruments.

An instrument is a device that measures or manipulates variables such as

flow, temperature, level, or pressure. Instruments include many varied
contrivances which can be as simple as valves and transmitters, and as complex
as analyzers. Instruments often comprise control systems of varied processes.
The control of processes is one of the main branches of applied instrumentation.

Control instrumentation includes devices such as solenoids, valves,

circuit breakers, and relays. These devices are able to change a field parameter,
and provide remote or automated control capabilities.

Instrumentation plays a significant role in both gathering information

from the field and changing the field parameters, and as such are a key part of 
control loops.

Pressure measurement:

Pressure is an effect which occurs when a force is applied on a surface.

Pressure is the amount of force acting on a unit area.

Type:
 Pressure guage
 Draft guage
 Diaphragm seal transmitter 
 Differential pressure instrument

Flow measurement:

The volumetric flow rate in fluid dynamics and hydrometry, (also known
as volume flow rate or rate of fluid flow) is the volume of fluid which passes
through a given surface per unit time (for example cubic meters per second [m3
s-1] in SI units, or cubic feet per second [cu ft/s]). It is usually represented by
the symbol Q.

Type:
• Orifice plate
• Venturimeter 

43