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Fundament Fundame Fundamen ntals of Bioche tals of Biochem als of Biochem mical Enginee ical Enginee ical Engineering ring ring
Subject code Subject code-PCCH 4402
Chemical Engineering Department Chemical Engineering Department Chemical Engineering Department
th
7 Semester
Mrs. Ipsita D. Behera Mrs. Ipsita D. Behera
Asst. Professor Asst. Professor
I.G.I.T Sarang I.G.I.T Sarang
Email: - ipsitadbeheragmail.com eragmail.comDisclaimer:
This document does not claim any originality and cannot be used as a substitute
for prescribed textbooks. The information presented here is merely a collection
by the committee faculty members for their respective teaching assignments as
an additional tool for the teaching-learning process. Various sources as
mentioned at the reference of the document as well as freely available material
from internet were consulted for preparing this document. The ownership of the
information lies with the respective authors or institutions. Further, this
document is not intended to be used for commercial purpose and the committee
faculty members are not accountable for any issues, legal or otherwise, arising
out of use of this document. The committee faculty members make no
representations or warranties with respect to the accuracy or completeness of
the contents of this document and specifically disclaim any implied warranties
of merchantability or fitness for a particular purpose.SYLLABUS
Module I
Overview of microbiology, Aerobic & Anaerobic fermentation
processes, fermenter design, sterilization of microbial medium, kinetics
of microbial growth, enzymes and its kinetics, immobilization of
enzymes, chemostats.
Module II
Transport phenomena in Biochemical Engineering, Heat and Mass
transfer in Bioprocessing, oxygen transfer in fermenter, monitoring and
control of fementation process.
Module III
Downstream processing: - Recovery and Purification of products,
allied unit operation for product recovery, production of biogas and
ethanol, Effluent treatment by biological method
Text book
1. Bailey JB and oillis OR, Biochemical Engineering Fundamentals
2. Aiba S, Biochemical Engineering, Academic press
3. Rao D G, Introduction to Biochemical Engineering, Tata Mc Grow Hill
4.Michael L. Shuler/ Fikret Kargi, Bio Process Engineering , Pearson EducationLESSON PLAN
Class No.
Brief description of the Topic
1. Introduction of FBE
2. Overview of microbiology
3. Application of Microbiology
4. Aerobic & Anaerobic fermentation processes
5. SSF and SmF and applications
6. Fermentation Design
7. Microbial sterilization
8. kinetics of microbial growth
9.
Kinetics of microbial growth
10.
Problem discussion
11.
Problem discussion
12.
Enzyme and its characteristics
13.
Enzyme kinetics
14.
Problem discussion
15. Immobilization of enzymes & applications
16.
Chemostats
17. Question Discussion
18.
Heat transfer in bioprocessing
19. Mass transfer in bioprocessing
20. Oxygen transfer in fermenter
21. Monitoring and control of fementation process.22 Question discussion
23 Unit operation for product recovery
24 Effluent Treatment
Physical methods
25 Effluent Treatment
Chemical methods
26 Effluent Treatment Biological methods
27 Production of biogas
28 Production of ethanol
29 Question discussions
30 Semester question discussionFundamentals of Biochemical Engineering 1
Module-I
INTRODUCTION
Biotechnology is the art and science of converting reactants (substrate) into
useful products by the action of microorganisms or enzymes. Microorganisms
have been honestly serving the mankind. Thus any process in which microbes
or living organisms play a vital role in getting transformation of the feed into
useful products is termed as BIOPROCESSING. For example the way of
converting milk into curds, or fruit juice into wines, or sugar into alcohol.
BIOCHEMICAL ENGINEERING is of more recent origin, since the
biological industries did not recognize the importance of engineering inputs
until the experience of penicillin manufacture.
Microbiology
(Micro-small, bios-life) is the study of microscopic organism, which are
defined as any living organism i.e. either a single cell(unicellular), a cell
cluster or has no cell at all(a cellular). This includes eukaryotes, such as
fungi and protista and prokaryotes.
Microbiology is a broad term which includes virology, mycology,
parasitology etc.
It is the study of microorganisms which are not only microscopic and
exist as single cells, but also that ultramicroscopic organism which are
not cellular and hence cannot exist independently. E.g. viruses
Microbiology deals with study and functioning of cells, their interaction
with environment, other living organism and man.
It is studied with respect to two major aspects.
a) As basic biological science
b) As applied biological science
Basic biological science:
It provides a system to understand the nature of life processes, the principle
behind it and the genetics which is involved in the heritance of traits to next
generation.it has several sub streams such as
i) Medical microbiology: Study of pathogenic microorganism, the causes of
diseases and way to eliminate them.
Fundamentals of Biochemical Engineering 2
ii) Agricultural microbiology: Study of plant diseases, understanding various
beneficial interactions with plant system like soil fertility, crop-protection and
increasing field.
iii) Environmental microbiology: Study of relationship of microorganisms
with its habitat, pollution effect and its impact on environment from the stand
point of ecology balance and health.
iv) Food and dairy microbiology: Study of microorganisms that produce
various food and dairy products.
Applied biological science:
It deals with study of useful microorganisms as well as that of pathogenic
organism.
i) In dairy and food industry: Food microbiology not only includes the study
of those microbes which provides food to their high protein content but also
includes other those microbes which use our food supply as a source of
nutrients for their growth and result in deterioration of the food by increasing
their number, utilizing nutrients, contributing of flavours by means of
breakdown of food.
ii) Medical microbiology: Microbes causes infections resulting in diseases
among human and animals. On other side they help in creating a “disease free
world”, where people are from pain by this disease.
Application of microbiology
Microbes in food and dairy industries
Microbes in production of industrial products
Microbes in genetic engineering and biotechnology
Microbes in environmental microbiology
Microbes in medical microbiology
Microbes in agriculture
Microbes in bioterrorism
a. Food and dairy industry:
Provides food due to high protein content.
Food nutrient for their growth (deterioration of food), enzymatic changes,
contributing flavour.
Certain moulds used for manufacturing of food and ingredients of food.
Some moulds used in production of oriental food, soya sauce etc.
Fundamentals of Biochemical Engineering 3
Used for enzyme making like amylase.
Yeast: These are used in manufacturing of foods such as bread, beer,
wines, vinegar, surface ripened, and cheese. Some yeasts are grown for
enzymes and food.
Bacteria: Some pigmented bacteria cause changes in colour on the
surfaces of liquid food. Acetobacteria oxidises ethyl alcohol to acetic
acid. Some bacteria causes ropiness in milk and slimy growth cottage
cheese.
b. Microbes in production of industrial products:
Enzymes amino acid, vitamins, antibiotics, organic acid and alcohol are
commercially produced by microorganism.
Primary microbial product: These products are used by microorganism
for their growth. E.g. amino acids, enzymes, vitamins
Secondary microbial products: Not used by cell for their growth. E.g.
alcohol, antibiotics, organics acids.
Microbes produce some important amino acids such as glomatic acid,
lysine, and methionine.
c. Microbes in Genetic Engineering and Biotechnology:
Microbes used for mammalian proteins such as insulin and human growth
factor.
Making vaccine for microbial and viral genes and induce a new strain of
microbes.
Vaccine and diagnostic kits also depend on the improved strains of
microorganism.
Lactic acid as food preservative.
Acetic acid plays a major role in tanning and textile industry.
Interferons are produced in animal cell if included by viral infection.
These are used in testing interleukins (which stimulate T-lymphocytes).
Production of viral, bacterial or protozoan, antigen for protecting against
dysentery, typhoid, bacteria etc.
N fixing bacteria reduce nitrogen gas to ammonia required plant growth.
2
Fundamentals of Biochemical Engineering 4
d. Environmental Microbiology:
Plays an important role in recycling of biological elements such as
oxygen, carbon, N sulphur, and phosphorous.
2,
Microbes in biochemical cycle: Photosynthesis and chemosynthesis
microorganism convert CO into organic carbon. Methane is generated
2
anaerobically from CO and H by metheogenic archaea. The organic
2 2
forms of N are interconverted by metabolic activities of microorganism
2
which maintain the natural N balance.
2
Microbes in pollution microbiology: Biological sewage treatment and
self-purification. Both results in mineralisation of organic, pollutants and
in utilisation of dissolved oxygen.
e. Medical Microbiology:
Vaccination: Small pox, diphtheria, whooping cough.
f. Computer application:
Optimisation via computer: Computers are used on scale of, to store,
evaluate effects of individual parameters on metabolic behaviour of
culture.
Control via computer: Control fermentation process.
g. Microbes in agriculture:
During compost formation by the crop residue like wheat straw, rice
straw, sugar cane bagasse are very difficult to degrade due to presence of
highly resistant lignified tissues. So, breakdown of these complex organic
materials can be done by microbes by a short span.
Biogas production through anaerobic fermentation is must reverent to
fulfil their energy demand in rural population.
The productivity of leguminous crop largely depend on efficient and
suitable management of the ecosystem by specific rhizobial association.
Some bacterial helps in killing a wide range of insects like beetles,
mosquitoes, flies, aunts, termites which is very useful for agricultural
industries
h. Microbes in Bioterrorism: It has been defined as deliberate release of
disease causing germs, microorganism with the intent of killing large
number of people. Accordingly microorganisms are used as weapons of
mass destruction of people and causes small pox, plague, cholera and also
anthrax.
Fundamentals of Biochemical Engineering 5
Mode of transmission:
Air droplets and dust
Food fruits and vegetables
Drinking water
Fundamentals of Biochemical Engineering 6
FERMENTATION
Fermentation is the word derived from the Latin verb FERVERE (to boil),
which describe the evolution of carbon dioxide bubble in anaerobic conditions
by the action of yeast on fruit juice.
General Requirements of Fermentation Process:-
Since fermentation is a biochemical process brought about by the
intervention of living organisms, it is essential that any fermentation process
should have:
A microorganism for carrying out the bioconversion.
A substrate to get converted into useful products.
Maintenance of fermentation conditions.
Effluent treatment section.
A provision for recovery and purification of the products.
Facilities for packaging and marketing.
Fermentation carried out in the presence of air / O is known as aerobic
2
fermentation, where as in absence of air is called anaerobic fermentation.
Anaerobic Fermentation:-
Yeast fermentation process to produce alcohol requires a small amount of
aeration for the cells to multiply. After word no air is required.
On the other hand air is detrimental for the process which will otherwise
oxidise the substrate.
Most of the anaerobic fermentation produces carbon dioxide gas.
Many times gas covers the surface and acts as a blanket to prevent the
effect of O
2.
The evolved CO will also help in better mixing conditions, which is more
2
evident in large industrial tanks because of longer pathways for the gas
bubbles to go before they leave the fermenter.
Aerobic Fermentation:-
Sparaging air /O is very common phenomenon in fermentation process to
2
supply O for cells to meet their specific O demand .Such fermentation process
2 2
which are associated with the bubbling of O are termed as aerobic
2
fermentation.
Fundamentals of Biochemical Engineering 7
Solid state and submerged fermentation and their Applications
Solid state fermentation:-
SSF is a method of growing microorganisms in an environment of limited
moisture without having free flowing water. The microorganisms grow on a
solid surface which is moistened and which has also got free access to air.
It is also known as “ Koji” fermentation , for the production of soya
products such as tempeh, soya sauce etc. certain metabolites. It has some
advantages that lower manufacturing cost because of the use of crude solid agro
wastes like wheat bran. The solid surface directly comes in contact with the air
and hence the aeration costs are avoided. The other economic advantages are
Low capita investment and recurring expenditures.
Low water utilization and hence negligible outflow of water.
Low energy requirements for the fermentation process because of
absence of agitation.
Absence of foam formation because of absence of excess water.
High reproducibility of the result.
Simple fermentation media.
Less fermentation space, and les complex plant and machinery.
Absence of rigorous control techniques.
Any level of scale of operation.
Absence of elaborate aeration requirements.
Ease in controlling bacterial contamination.
Facilities of using wet and dry fermented solids directly.
Ease in induction and suppression of spores.
Lower costs of downstream processing.
Submerged Fermentation:-
In case of submerged fermentation (SmF) the microorganisms and the
substrate are present in the submerged state in the liquid medium, where a large
quantity of solvent is present. This has many advantages over SSF.
Since the contents are in submerged state in the liquid medium, the
transfer of heat and mass is more efficient, and is amenable for
modelling the process.
The scaling-up the process is very easy.
Fundamentals of Biochemical Engineering 8
Differences between SSF and SmF
Characteristics feature SSF SmF
Condition of microorganisms and static Agitated
substrate
Status of the substrate Crude Refined
Nature of the microorganism Fungal system ______
Availability of water Limited High
Supply of oxygen By diffusion By bubbling/
sparging
Contact with oxygen Direct Dissolved O
2
Requirement of fermentation medium Small Huge
Energy requirement Low High
Study of kinetics Complex Easy
Temp and concentration gradient Steep Smooth
Controlling of reaction Difficult Easy
Chances of bacterial contamination Negligible High
Quantity of liquids to be disposed Low High
Pollution problems Low high
Applications:-
Citric acid can be produced by both SSF and SmF. Generally, the later
technique is used in industrially. SSF method has not yet become
commercial success because of its labour intensity.
Soya-based oriental foods like tempeh and soya sauce are produced by
SSF alone.
Production of Roquefort cheese from sheep milk is a classical example of
SSF.
Mushroom cultivation is another example of the growth of fungus on
solid medium like paddy straw.
Fish and meat production are preserved in the form of sausages as
fermented foods.
Fundamentals of Biochemical Engineering 9
FERMENTER DESIGN
A fermenter is a type of bioreactor for containing and controlling
microorganisms during a fermentation process.
BASIC FUNCTIONS OF FERMENTERS
The main function of a fermenter is to provide a controlled environment
for growth of a microorganism, or a defined mixture of microorganism, to
obtain a desired product.
(Bioreactors refer to production units of mammalian and plant cell culture)
CRITERIA USED IN DESGINING AND CONSTRUCTING A
FERMENTER-
Vessel should be capable of being operated aseptically and should be
reliable for long term operation
Interplay of the transport parameters
Supply of adequate quantity of oxygen so that cells do not suffer from
inadequacy of oxygen supply
Adequate aeration and agitation to meet the metabolic requirements of
the microbes
Adequate amount of mixing should be ensured without causing damage
to the cells
Vessel geometry should be such that it should facilitate scale-up
Flexibility in operation of the fermenter for various purposes, so that the
vessel should be suitable for a range of processes
Low power consumption
Temperature and pH control
Low evaporation losses
Minimal use of labour in operation, harvesting, cleansing and
maintenance
Proper sampling facility
Cheapest and best materials should be used
Adequate service provisions must be available for individual plants.
Fundamentals of Biochemical Engineering 10
TYPES OF FERMENTER-
Based on shape it can be classified as-
(i) Tabular &
(ii) Stirred tank
(Cooling coils are provided to maintain constant temperature inside the
bioreactor. It can be operated aseptically for many days and simple in
construction.
Disadvantages-
high power requirement
shearing on the organisms caused by vigorous agitation and inhibition
exercised by the product)
(i) Fluidized Bed Bioreactor: - It is more popular in chemical industry
rather new to biochemical industry. These are mostly used in
conjuction with immobilized cells or enzyme system and are operated
continuously.
(ii) Loop or Air Lift Bioreactor: - In the conventional bioreactor, oxygen
is supplied by vigorous agitation of the bioreactor content. The heat is
generated which is a problem in conventional type. In this cooling
becomes simpler due to the position of inner or outer loop.
(iii) Membrane Bioreactor: - These consist of a semipermeable
membrane made up of cellulose acetate or other polymeric materials.
The primary purpose of the membrane is to retain the cells within the
bioreactor, thus increasing their density, while at same time allowing
metabolic products to pass through the membrane.
(iv) Pulsed Column Bioreactor: - It has a column bioreactor generator
connected to the bottom of the column. It can be utilised as an aerobic
bioreactor, enzyme bioreactor or as separation unit.
(v) Bubble Column Bioreactor: - Multistage bubble column bioreactor
are suitable in the equivalent batch process. In this it is possible to
provide different environmental conditions in various stage. It is not
suitable for fungal fermentation due to oxygen demanding system.
(vi) Photo Bioreactor: - For the growth and production of photosynthetic
organisms, a light source is required. In this, there is an important
‘reactant’, the photons which must be absorbed in order to react and
produce products. The design of the light source is critical in the
Fundamentals of Biochemical Engineering 11
performance of this type of bioreactor. Example- Annular Reactor. In
this source of radiation is a cylinder with a annular section, which
enclose the lamp completely. The nutrient passing from the product is
removed from the top. It is used for Spirulina(SCP) and other algal
protein production.
(vii) Packed tower Bioreactor: - It consists of cylindrical column packed
with an inert material like wood shavings, twigs, cake, polythene or
sand. Initially, both medium and cells are fed into the top of the
packed bed. Once the cells adhered to the support and were growing
well as a thin film fresh medium is added at the top of the packed bed
and the fermented medium removed from the bottom of the column. It
is used for vinegar production, sewage effluent treatment and enzymes
conversion of penicillin to 6-amino penicillanic acid.
The design of fermenter involves the co-operation between experts in
microbiology, biochemistry, mechanical engineering and economics.
CONSTRUCTION OF FERMENTERS
The criteria considered before selecting materials for construction of a
fermenter are:
(a) The material that have no effect on sterilisation
(b) Its smooth internal finish-discouraging lodging of contamination.
(c) Internal surface should be corrosion resistant.
There are two types of such materials:
(i) Stainless Steel, and
(ii) Glass.
The construction of the fermenter depends upon the following-
(i) Control of Temperature. Since heat is produced by Microbial
Activity and the mechanical agitation, thus it is sometimes necessary
to remove it. On the other hand, in certain processes extra heat is
produced by using thermostatically controlled water bath or by using
internal heating coil or jacket meant for water circulation.
Fundamentals of Biochemical Engineering 12
(ii) Aeration and Agitation. The main purpose is to provide oxygen
require to the metabolism of microorganisms. The agitation should
ensure a uniform suspension of microbial cells suspended in nutrient
medium. There are following necessary requirements for this purpose:
(a) The agitator (impeller) for mixing: The size and position of the
impeller in the vessel depends upon the size of the fermenter.
More than one impeller is needed if adequate aeration agitation is
to be obtained. Ideally, the impeller should be 1/3 or 1/2 of the
vessel diameter (D) above the base of the vessel. The number of
impeller may vary from size to size to the vessel.
(b) Stirrer glands and bearings meant for aseptic sealing: Four
basic types of seals assembly have been used-
The packed gland seal
The simple bush seal
The mechanical seal and
The magnetic drive.
(c) Baffles for checking the vortex resulting into foaming: The
baffles are incorporated into the agitated into the agitated vessels
to prevent a vortex ant to improve aeration efficiency. They are
metal strips roughly one-tenth of the vessel diameter and attached
radially to the walls.
Fundamentals of Biochemical Engineering 13
(d) The sparger (aeration) meant for introducing air into liquid: A
sparger is a device for introducing air into the liquid into a
fermenter. It is important to know whether the sparger is to be
used on its own or with mechanical agitation as it can influence
equipment design to determine initial bubble size.
Three basic types of sparger are:
(i) Porous sparger
(ii) Orifice sparger
(iii) Nozzle sparger
(e) Microbial sensors: It consists of a microorganism immobilized on
a membrane and an electrode. The principle of working is the
change in respiration or the amount of metabolites produced as a
result of the assimilation of substrate by the microorganism. A
wide range of thermophilic microbes have been used for the
manufacturing of microbial sensors as mentioned in the table
below.
Immobilised yeast, Trichosporoncutaneumhas been used to develop
an oxygen probe for BOD estimation in sewage and other water
samples. The BOD sensor includes an oxygen electrode that consists
of a platinum cathode and an aluminium anode bathing in salt KCl
solution and a Teflon membrane. Immobilised yeast cells are crapped
between the pores of a porous membrane and the Teflon sensor can
measure BOD at 3-60/mg/litre. Methanotrohic bacteria is used in
measuring methane as well as oxygen. Similarly, ammonia and nitrate
biosensors consist of immobilised nitrifying bacteria. This is used to
determine ammonia in waste water based on the conversion of nitrate
to N O by an immobilised denitrifying Agrobacterium sp.The nitrate
2
biosensor has been used to measure nitrate profiles in biofilm.
APPLICATIONS: Microbial biosensors have several uses in:
clinical analysis,
general health care monitoring,
veterinary and agricultural applications,
industrial product processing,
monitoring and control of environment pollution and
in military and defence for detection of chemical and biological species
used in weapons.
Fundamentals of Biochemical Engineering 14
DESIGN AND OPERATION
The basic purpose of design of a fermenter or bioreactor is to visualise the size
of the unit to deliver the product both qualitatively and quantitatively. After the
size is designed, the next task is to achieve the transport properties i.e;
Fluid mechanics
Heat transfer
Mass transfer effects.
Fermenters are designed to provide support to best possible growth and
biosynthesis for industrially important cultures ant to allow ease of
manipulation for all operations associated with the use of fermenters.
These vessels must be strong enough to resist the pressure of large
volume of agitating medium.
The product should not corrode the material nor contribute toxicity to the
growth medium. This involves a meticulous design of every aspect of the
vessel parts and other openings, accessories in contact, etc.
In fermentations, provisions should be made for the control of
contaminating organisms, for rapid incorporation of sterile air into the
medium in such a way that the oxygen of air is dissolved in the medium
and therefore, readily available to the microorganisms and CO produced
2
from microbial metabolism is flushed from the medium.
Fundamentals of Biochemical Engineering 15
Some stirring devices should be available for mixing the organisms
through the medium so as to avail the nutrients and the oxygen.
The fermenter has a possibility for the intermittent addition of antifoam
agent.
Some form of temperature control efficient heat transfer system is also
there for maintaining a constant predetermined temperature in the
fermenter during the growth of organism.
The pH should be detected.
Other accessories in the fermenter consist of additional inoculum tank or
seed tank in which inoculum is produced and then added directly to the
fermenter.
Media Design
Any fermentation process proceeds through the action of microorganisms which
perform in the presence of a medium. Hence, proper design of the medium is an
essential component in the design of a fermentation process. Thus, detailed
investigations are needed to identify the most suitable medium for any
fermentation process to proceed.
Medium Requirements
Since the medium is desired to support the functioning of microorganisms, the
requirements of the medium are decided by those of the microorganisms. They
are:
• carbon
• nitrogen
• energy source
• minerals
• other nutrients like vitamins, etc. • oxygen/air for aerobic processes.
• water.
The medium used in a laboratory-scale process, or for that matter even in the
pilot plant-scale level, can be reasonably composed of pure components; but
such a luxury is not affordable in the case of commercial production, where the
cost of production rules the economic viability, and hence the commercial
viability of the process. Thus, for large-scale productions, we look for a
medium, which has the following attributes:
• It should be cheap, and easily available at a consistent cost and quality.
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