1 - FST-607. Food Biotechnology (L # 1-10) - Republished

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02/25/2020 FST-607 by Dr Shahid Mahmood

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FST-607. FOOD BIOTECHNOLOGY 3 (2-1)

B. Sc. (Hons). Food Science and Technology
Semester-IIIV (Ex-PPP)
Spring-2020
Session: 2016-2020
Dr. Shahid Mahmood Rana

Associate Professor
INSTITUTE OF FOOD SCIENCE AND NUTRITION (IFSN)

UNIVERSITY OF SARGODHA, SARGODHA-PAKISTAN

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L # 1. BASICS OF FOOD BIOTECHNOLOGY
Outline
• Introduction
• Course Details
• Lecture Diary
• Exams and Evaluation

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ACADEMIC CALENDAR SPRING-2020

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Session 2016 - 2020

Academic Year Spring-2020
Course Teacher Dr. Shahid Mahmood
Program / Class B. Sc. (Hons). Food Science and Technology
Semester 8th
Section Ex-PPP
Corse Code FST - 607
Course Title Food Biotechnology
Credit Hours 3 (2-1)
Paper Theory + Practical
Commencement Monday: 27-01-2020
Exams Mid Term: 18-03-2020 (Wed) Final Term: 13/05/2020 (Wed)
Results Declaration 29-05-2020 (Friday)
02/25/2020
FST-607 by Dr Shahid Mahmood
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FST- 607. EXAMS & EVALUATION
EXAM DAY DATED MARKS
Quiz 1 Tuesday 28/02/2020 02

Assignment 1 Tuesday 04/03/2020 02
Pre Mid Tuesday 11/03/2020 02
Mid Wednesday 18/03/2020 30

Quiz 2 Tuesday 07/04/2020 02
Assignment 2 Tuesday 21/04/2020 02
Pre Final Tuesday 05/05/2020 02

Final (T+P) Wednesday 13/05/2020 50 (40+10)
Attendance ≤ 75 ≤ 80 ≤ 85 ≤ 90 ≤ 95
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(%) shortage 2 2 2 2
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FST-607. COURSE CONTENTS - T
Mid Term
1. Biotechnology: introduction, history.
2. Microbial metabolism.
3. Developments in metabolic and biochemical engineering: metabolites, range of
fermentation processes, components of fermentation processes.
4. Isolation and preservation of industrially important microorganisms.
5. Industrial fermentations: media, design and types of fermenters, process variables in
fermentation, recovery, purification of fermentation products.
Final Term
6. Production of organic acids, enzymes, amino acids, single cell proteins, carotenoids and
fermented food products.
7. Microbial genetics: conjugation, transduction, transformation.
8. GMO in food biotechnology.
9. Legal and social aspects of food
02/25/2020 biotechnology.
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FST-607. COURSE CONTENTS - P

1. Isolation, purification and maintenance of yeast and bacterial cultures.
2. Aerobic and anaerobic fermentation and production of various fermented
food products.

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RECOMMENDED BOOKS
1. El-Mansi, F. M. T., Bryee, C. F. A., Demain, A. L., and Allman, R. 2007.
Fermentation Microbiology and Biotechnology. CRC Press, Taylor and Francis
Group Boca Raton Florida, USA.
2. Shetty, K., Paliyath, G., Pometto, A., and Levin, R. E. 2005. Food Biotechnology.
Marcel Dekker Inc. New York, USA.
3. Borem, A., Santos, F. R. and Bowen, D. E. 2004. Understanding Biotechnology.

Pearson Education Inc. New Jersey, USA.
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STUDENTS’ AVERAGE RETENTION RATES

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FST-607. L # 1. Introduction of Food Biotechnology
Outline
• Definitions
• Oldest form of Biotechnology
• Examples of Applications
• Principles of Biotechnology
• Recombinant DNA Technology
• Steps to Genetically Modify Organism
• Cloning
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INTRODUCTION TO BIOTECHNOLOGY
“Biotechnology is the application of biological organisms, system or
process to manufacturing and service industries."
“Biotechnology is the integrated use of biochemistry, microbiology, and

engineering sciences in order to achieve technological (industrial)
application of the capabilities of micro-organisms or cultured tissue cells.”
(European Federation of Biotechnology).
“Biotechnology is the controlled use of biological agents, such as

microorganisms or cellular components.”
02/25/2020 FST-607 by Dr Shahid Mahmood (US National Science Foundation)
INTRODUCTION TO BIOTECHNOLOGY… 14
Oldest Forms of Biotechnology

• Making breads and curds with the help of microorganisms
• Application of fermentation in production of wine and other alcoholic beverages

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BIOTECHNOLOGY: MODERN APPLICATIONS

• Development of disease resistant plants
• Food crops that produce greater yields
• “Golden Rice" engineered to be more nutritious
• Genetically engineered bacteria that can degrade environmental pollutants
• Manufacture of biotechnological products like vaccine, enzymes, beverages, drugs
etc.
PRINCIPLES OF BIOTECHNOLOGY 16
Two important technique which enable development of modern biotechnology:
Genetic Engineering
Alteration of chemistry of DNA & RNA to introduce into host organism to
change phenotype of the host (e.g. Recombinant DNA Technology, Cloning)
Chemical Engineering
Maintenance of sterile ambience to enable growth of desired microbe /
bacterium in large quantities for manufacture of biotechnological products
like vaccine, enzymes, beverages, drugs etc.
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RECOMBINANT DNA TECHNOLOGY
• In Genetic Engineering - isolate & introduce only one or set of desirable genes
without introducing undesirable genes in target organisms
• First recombinant DNA was constructed - Stanley Cohen & Herbert Boyer
(1972) by linking gene encoding for antibiotic resistance with plasmid of
Salmonella typhimurium
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RECOMBINANT DNA TECHNOLOGY..
Recombinant DNA technology is accomplished with tools:
Restriction Enzymes: Enzymes which is used to make cut in DNA in recombinant DNA
technology. (has two types)
1. Restriction exonuclease – removes nucleotides from the end of DNA

2. Restriction endonuclease – cut at specific positions within DNA

RECOMBINANT DNA TECHNOLOGY.. 19
DNA Ligase
• Restriction enzymes and DNA ligase make possible the combination of DNA
from different organisms into one DNA molecule called recombinant DNA
Vectors
• Gene transfer to host require Vector
• Commonly used vector - Plasmid (small, circular, double stranded, self
replicating extra chromosomal material of bacteria)
Host
• The organism DNA which has to change / alter

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RECOMBINANT DNA TECHNOLOGY..

Steps to Genetically Modify Organism

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CLONING
• Using bacteria to make multiple identical copies of a single strand of DNA
• Cloning Vector: Any vehicle that inserts a fragment of foreign DNA into the
genome of a host cell
• Used in gene therapy

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L # 2. MICROBIAL METABOLISM

Associate Professor


MICROBIAL METABOLISM
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Metabolism
• The sum of all chemical reactions in the body
• Metabolism is divided into two types
• Catabolism
• Anabolism

MICROBIAL METABOLISM 24

MICROBIAL METABOLISM… 25
Metabolism
• Catabolism is the chemical reactions that break down large compounds and
release energy.
• Anabolism is the chemical reactions that require energy to build large

compound
• Catabolic reactions furnish the energy needed to drive anabolic reactions.
• Energy harvested from catabolic reactions are stored in ATP molecules.
• ATP molecules are used to drive many anabolic reactions

BASIC CONCEPTS.. 26
ATP: A “Currency of Energy” for Cellular Reactions
• ATP stands for adenosine triphosphate.
• It is a nucleotide with three phosphate groups linked in a small chain.
• The last phosphate in the chain can be removed by hydrolysis (the ATP becomes
ADP, or adenosine diphosphate).
• ATP hydrolysis is used as an energy source in many biological reactions that

require energy

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GLYCOLYTIC PATHWAYS
• ATP as the cellular energy storage unit, can be formed during Respiration (R) or
Fermentation (F)
• Both contain the Glycolysis Pathway; which produces ATP, the electron carrier
molecule NADH (Nicotinamide adenine dinucleotide), and Pyruvate from
Glucose
• Aerobic Respiration will proceed via Krebs Cycle and an ETC if there is oxygen
to react as a terminal electron acceptor
• O2 is not the only possible terminal electron acceptor in some Bacteria (e.g.
NO02/25/2020
3 or SO4 can be used); called Anaerobic
FST-607 by Respiration
Dr Shahid Mahmood
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GLYCOLYSIS ENERGY YIELDS
• Maximum yield per Glucose = 38 ATP (30-32)
• Only achieved by aerobic respiration of mitochondria in eukaryote cells
• Anaerobic respiration is even less efficient
• Fermentation least efficient (2 ATP)
• FAD (flavin adenine dinucleotide)

GLYCOLYSIS: ENERGY YIELDS 29

FINALLY ATP PER MOLECULE OF GLUCOSE 30
Substrate-level Phosphorylation
• 2 ATP from Glycolysis + 2 ATP (directly GTP) from Krebs Cycle
Oxidative Phosphorylation
• 2 NADH+H+ from Glycolysis = 2 × 1.5 ATP (glycerol phosphate shuttle) or
= 2 × 2.5 ATP (malate-aspartate shuttle)
• 2 NADH+H+ from Oxidative Decarboxylation of Pyruvate and 6 from Krebs
Cycle = 8 × 2.5 ATP
• 2 FADH2 from Krebs Cycle = 2 × 1.5 ATP
• Altogether this gives 4 + 3 (or 5) + 20 + 3 = 30 (or 32) ATP per molecule of
Glucose

HYDROLYSIS OF MAJOR BIOMOLECULES 31
Enzymes of Hydrolysis
• Proteins Proteases
• Polysaccharide and other Carbohydrates Glycosidase
• Nucleic acids (DNA or RNA) Nucleases
• Lipids Lipases

HYDROLYSIS OF MAJOR BIOMOLECULES 32

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L # 3. AN INTRODUCTION TO FERMENTATION PROCESS

Associate Professor


WHAT IS FERMENTATION? 34
“Conversion of sugar to alcohol / acids using yeast or other microbes”.

“The science of fermentation is known as Zymology”.
• Yeasts are eukaryotic single celled microorganisms classified as members of
the fungus kingdom
• The first yeast originated hundreds of millions of years ago, and 1,500
species are currently identified
• They are estimated to constitute 1 % of all described fungal species
• Chemical conversion of carbohydrates into alcohols or acids
• The process is often used to produce wine and beer
• Also employed in preservation to create lactic acid in sour foods such as
pickled cucumbers, kimchi and yogurt

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BIOCHEMISTRY PERSPECTIVE
• Process that is important in anaerobic conditions when there is no oxidative
phosphorylation to maintain the production of ATP (Adenosine triphosphate) by
glycolysis
• During fermentation pyruvate is metabolised to various different compounds
• Homolactic fermentation is the production of lactic acid from pyruvate;
alcoholic fermentation is the conversion of pyruvate into ethanol and carbon
dioxide
• Heterolactic fermentation is the production of lactic acid as well as other acids
and alcohols

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BIOCHEMISTRY PERSPECTIVE …
• Industrial microbiologists have extended the term - fermentation - to describe:
“Any process for the production of product by the mass culture of

microorganism”
“Brewing and the production of organic solvents may be describes as

fermentation in both sense of the word”

PRODUCTS OF FERMENTATION 37

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COMMERCIALLY IMPORTANT FERMENTATIONS

There are five major groups of commercially important fermentations:
• Those that produce microbial cells (or biomass) as the product
• Those that produce microbial enzymes
• Those that produce microbial metabolites
• Those that produce recombinant products
• Those that modify a compound which is added to the fermentation - the transformation
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THE COMPONENT PARTS OF A FERMENTATION PROCESS

• The extraction of the product and its purification
• The disposal of effluents produced by the process

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THE COMPONENT PARTS OF A FERMENTATION PROCESS

• The formulation of media to be used in culturing the process organism during
the development of the inoculums and in the production fermenter
• The sterilization of the medium, fermenters and ancillary equipment
• The production of an active, pure culture in sufficient quantity to inoculate the

production vessel
• The growth of the organism in the production fermenter under optimum

conditions for product formation
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L # 4. TYPE OF ENERGY / NUTRIENTS’ SOURCES AND

MEDIA OF FERMENTATION

Associate Professor


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MEDIA OF FERMENTATION
• All micro-organisms require water, sources of energy, carbon, nitrogen, mineral element
and vitamin plus oxygen in their growth medium
• On a small scale, it is simple to device a medium containing pure compounds, but the
resulting medium although satisfy the growth, may be unsuitable for use in a large scale
process
• Carbon Sources : Molasses, Cereal grains, Glucose, Sucrose , Lactose
• Nitrogen Sources : Ammonium salts, Urea, Nitrates, Soya bean meal, Slaughter-
house waste , Fermentation residues

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MEDIA OF FERMENTATIO - CRITERIA

On a large scale one must use sources of cheap nutrients to create a medium
which will meet as many as possible of the following criteria
1. It will produce a maximum yield of product or biomass per gram of substrate
used
2. It will produce a maximum concentration of product or biomass
3. It will permit the maximum rate of product or biomass
4. It will be the minimum yield of undesired product
5. It will be cheap and of a consistent quality and is readily available throughout
the year
6. It will cause minimal problem in other aspects of production and agitation,
extraction, purification and waste treatment

MEDIA OF FERMENTATION: FERMENTOR DESIGN 45
The medium selected will affect the design of the fermenter to be used
• A laboratory medium may not ideal in a large fermenter with a low
gas-transfer pattern
• Media with a high viscosity will also need a higher power input for
effective stirring
• Besides the requirement for growth and product formation,
medium may also influence pH variation, foam formation,
oxidation-reduction potential and the morphological form of the
organisms
• It may also be necessary to provide precursors or metabolic
inhibitors

MEDIUM FORMULATION 46
Medium formulation is an essential stage in the design of

• Successful laboratory experiments
• Pilot-scale development
• Manufacturing processes
The constituents of a medium must satisfy
• The Elemental requirements for Cell Biomass and Metabolite production
• Must be an adequate supply of Energy for Biosynthesis and Cell

Maintenance

WATER
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• Major component of all fermentation media

• Factors need to be considered
 pH
 Dissolve salt
 Effluent contamination
• Mineral content of water is very important in Brewing and most critical in

the Mashing Process and historically influenced the Siting of Breweries and
the Type of Beer produced

BREWING 48
“Brewing is the production of beer by steeping a starch source (commonly

cereal grains, the most popular of which is barley) in water and fermenting
the resulting sweet liquid with yeast”.
• A brewery or brewing company is a business that makes and sells beer.
• The place at which beer is commercially made is either called a brewery or a
beerhouse
“Mashing is the brewer's term for the hot water steeping process which
hydrates the barley, activates the malt enzymes, and converts the grain
starches into fermentable sugars”.
• There are several key enzyme groups that take part in the conversion of the
grain starches to sugars.

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MAJOR ENZYME GROUPS AND FUNCTIONS IN BREWING

Enzymes Temperature (°F) pH Functions
Lowers the mash pH
Phytase 86-126 5.0-5.5 No longer used
Debranching enzymes 95-113 5.0-5.8 Solubilization of Starches
β-Glucanase 95-113 4.5-5.5 Best gum breaking rest
Peptidase 113-131 4.6-5.3 Produces Free Amino Nitrogen (FAN)
Protease 113-131 4.6-5.3 Breaks up large proteins that form haze
β-Amylase 131-150 5.0-5.5 Produces maltose

Produces a variety of sugars,
α-Amylase 154-162 5.3-5.7 including maltose
WATER
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• Nowadays, the water may be treated by deionization or other techniques

and salts added, or pH adjusted, to favour different beers so that breweries
are not so depend on the local water sources
• In large continuous culture plants it is common practice to recycle the water

streams
• It may even be possible to reuse all the water in the fermenter fed with
appropriate adjustment of nutrient levels

ENERGY SOURCES 51
• Energy for growth comes from either the oxidation of medium components
or from light
• Most industrial micro-organisms are chemo-organotrophs, therefore the

commonest sources of energy will be the Carbon source such as
Carbohydrates, Lipids and Proteins
• Some micro-organisms can use Methane or Methanol as Carbon and energy

sources

NUTRIENTS’ SOURCES
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Carbon requirement for the medium is normally provided by:

Nutrients Raw material
Sucrose Sugar, Sugarcane / Sugar Beet Molasses
Glucose Corn, Sugar, Starch, Cellulose
Lactose Milk Whey
Fats Vegetable oil
Starch Maize Grains, Cereals, Potatoes and Casava
Hydrocarbons Petroleum fractions
MALT 53
• Malt is germinated cereal grain that has been dried in a process known as
"malting".
• The grain is made to germinate by soaking in water and is then halted from
germinating further by drying with hot air.
• Malt is the product that is left over after a cereal grain has been dried, allowed to
sprout, air dried again, then heated in an oven.
• Any of a variety of cereal grains, including rice, wheat, oats and rye can be used
to make malt.
NUTRIENTS’ SOURCES 54
Barley Grains
• It may be partially germinated and heat treated to give material known as
malt
• Malt:
• Contains a variety of sugar besides starch
• The main substrate for brewing in many countries
• Malt extract may also be prepared from malted grain
Sucrose
• Obtained from sugar cane and sugar beet
• Commonly used in fermentation media in very impure form as beet or cane
molasses

NUTRIENTS’ SOURCES 55
Lactose and Crude Lactose(milk Whey)
• Now is extremely limited in media formulation since the introduction of

continuous-feeding process
• Whey is used as a substrate for biomass production
Vegetable oil
• Olive, Maize, Cotton seed, Linseed, Soya bean, etc
• Source of Carbon : oleic, linoleic and linolenic acid
• As an antifoam in association with a surface active agent

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L # 5-6-7. FERMENTERS: DESIGN AND TYPES

Associate Professor


FERMENTATION: DEFINITIONS 58
• Preservation methods for food via microorganisms (general use)
• Any process that produces alcoholic beverages or acidic dairy products (general use)
• Any large scale microbial process occurring with or without air (common definition used in industry)
• Any energy releasing metabolic process that takes place only under anaerobic conditions (becoming
more scientific)
• “Any metabolic process that releases energy from a sugar or other organic molecule, does not require
oxygen or an electron transport system, and uses an organic molecule as the final electron acceptor”.
(most scientific)
• The word "ferment" is derived from the Latin verb fervere, which means to boil.

FERMENTER / FERMENTOR 59
• “A fermentor (bioreactor) is a closed vessel with adequate arrangement for

aeration, agitation, temperature and pH control, and drain or overflow vent to
remove the waste biomass of cultured microorganisms along-with their products”.
• Fermentor: An organism that causes fermentation
• Fermentor: An apparatus that maintains optimal conditions for the growth of

microorganisms, used in large-scale fermentation and in the commercial production
of antibiotics and hormones, enzymes
• The heart of the fermentation process is the FERMENTER

Fermenters are containers used to grow bacteria and fungi in large amounts
• Transgenic bacteria (genetically modified bacteria that carry the gene from
other sources and are used for the production of desired gene product at large
scale.)
• Penicillium mould for producing penicillin (an antibiotic)
• Fusarium mould for producing mycoprotein (a fungus-based meat substitute)
• Fermenters are usually made from a metal that will not corrode, such as
stainless steel.
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ENZYME USAGE IN FOOD APPLICATION
SYNTHESIZED
from
MICROBES

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ENZYME SOURCE ACTION IN FOOD APPLICATION IN FOOD
Aspergillus spp. Amylase Dough Softening

Bacillus spp. Wheat Starch Increased Bread Volume
α-Amylase Microbacterium Hydrolysis Production of Sugars for Yeast
imperiale Fermentation
Reduction of Wine Maturation Time

α-Acetolactate Bacillus subtilis Converts Acetolactate by circumventing need of
to Acetoin Decarboxylase for secondary
Fermentation of Diacetyle to Acetoin
Hydrolyzes Starch One stage of High Fructose Corn syrup

Amyloglucosida Aspergillus niger Dextrin to Glucose production.
se Rhizopus spp. (Saccharification) Production of Lite Beers
Release free Amino

Aminopeptidas Lactococcus lactis acids from N-terminus Debittering Protein Hydrolyzates
e Aspergillus spp. of Proteins and accelerating Cheese Maturation
Rhizopus oryzae Peptides
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Aspergillus niger Break down H2O2 to O2 Removal Technology combined

Catalase Micrococcus luteus H2O & O2 with Glucose oxidase
Aspergillus niger Fruit Liquefaction in Juice

Cellulase Hydrolyze Cellulose
Trichoderma spp. Production
Aspergillus awamori Hydrolyzes K- Casein Coagulation of Milk for Cheese

Chymosin Kluyveromyces lactis making
Cyclodextrin Synthesize Cyclodextrins are Food grade

glucanotransfera Bacillus spp. Cyclodextrins from microencapsulant for Color
se liquefied Starch Flavors and Vitamins
Sweetening Milk and Whey

Hydrolyzes Milk products for Lactose Intolerant
β-Galactosidase Aspergillus spp. Lactose to Glucose individuals
(lactase) Kluyveromyces spp. and Galactose Reduction of crystallization in Ice
Cream containing whey
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Hydrolyzes β-glucans Filtration aids
Aspergillus ssp.
β-Glucanase in Haze prevention in Beer
Bacillus subtilis
Beer mashes production
Actinoplanes missouriensis Production of High Fructose

Bacillus coagulans Converts Corn Syrup
Glucose isomerase Streptomyces lividans Glucose to Fructose (Beverage sweetener)
Streptomyces rubiginosus
O2removal from packaging

Aspergillus niger Oxidizes
Glucose oxidase Removal of Glucose from Egg
Penicillum chrysogenum Glucose to Gluconic acid
White to prevent Browning
Hemicellulase Cyclodextrins are Food grade

Synthesize Cyclodextrins
& Bacillus spp. microencapsulate for Color
from liquefied Starch
Xylanase Flavors and Vitamins
Aspergillus spp. Hydrolyzes Hemicellulose

Β-Galactosidase Bread improvement through
Bacillus subtilis (insoluble non-starch
(lactase) improved crumb structure
Trichoderma reesei polysaccharide in flour)
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Lipase / Aspergillus spp. Hydrolyzes Triglycerides to Flavor enhancement in Cheese

Estrase Candida spp. Fatty acids and Glycerol products
Aspergillus spp.
Clarification of Fruit Juice by De-
Pectinase Penicillum Hydrolyze Pectin Pectinization
funiculosum
Removal Methyl groups from With Pectinase in De-Pectinization

Pectinesterase Aspergillus spp.
Galactose units in Pectin technology
Hydrolyzes 1-6 bonds that

Bacillus spp. Starch Saccharification (improves
Puliulanase form branches in Starch
Klebsiella spp. efficiency)
structure
Aspergillus spp. Milk Coagulation for Cheese making

Protease / Bacillus spp. Hydrolyzes of K-Casein Hydrolyzates production for Soups
Proteinase Penicillium Hydrolysis of Wheat Glutens and Savory foods
citrinum Bread Dough improvement

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• Aspergillus is a genus consisting of a few hundred mold species found in

various climates worldwide
• Bacillus is a genus of Gram-positive, Rod-shaped (bacillus) bacteria; can be
Obligate aerobes (O2 reliant), or Facultative anaerobes (O2 reliant or NOT)
• Microbacterium is a genus of bacteria in the family Microbacteriaceae;
consists of 63 species
• Rhizopus is a genus of common Saprophytic Fungi on plants and specialized
parasites on animals
• Saprophytes are plants, fungi, or micro-organisms more accurately called
myco-heterotrophs because they actually parasitize fungi, rather than dead
organic matter directly. They live on dead or decomposing matter.
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• Lactococcus lactis is a Gram-positive bacterium used extensively in the

production of Buttermilk and Cheese
• Buttermilk (LASSI) refers to a number of dairy drinks (liquid left behind
after churning butter)
• Kluyveromyces is a genus of Yeasts in the Ascomycetes family of
Saccharomycetacea
• Trichoderma is a genus of Fungi that is present in all soils, where they are
the most prevalent culturable fungi
• Candida is a genus of yeasts and is the most common cause of fungal
infections worldwide
• Actinoplanes species are Gram-positive, soil-inhabiting, filamentous bacteria
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• Butyric acid fermentation, also known as late blowing defect (LBD), is a

major cause of spoilage in semi-hard and hard cheeses. It results in the
appearance of texture and flavor defects that generate severe economic
losses at the cheese industry.
• Wine aging refers to a group of reactions that tend to improve the taste
and flavor of a wine over time. The term wine 'maturation' refers to
changes in wine after fermentation and before bottling.
• Malt is germinated cereal grain that has been dried in a process known
as "malting". The grain is made to germinate by soaking in water and is
then halted from germinating further by drying with hot air.
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In general:
 Stirred Vessel H/D  3
 Volume 1-1000 m3 (80 % filled)
 Biomass up to 100 Kg dry weight/m3
 Product 10 mg/L –200 g/L

FERMENTOR DIAGRAM 70

BASIC DESIGN OF A FERMENTER 71

VARIOUS COMPONENTS OF AN IDEAL FERMENTER 72

MONITORING AND CONTROLLING PARTS OF FERMENTER 73

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Feature Function Reason

An aseptic precaution to prevent
Hot steam sterilises the inside of the
Steam Inlet contamination by unwanted
fermenter
microorganisms
Allows sterile nutrients to enter the Microorganisms need nutrients so that
Nutrient Inlet
fermenter they can grow and reproduce
Water Jacket with Keeps the temperature inside Microorganisms grow best at an
Cooling Water constant optimum temperature
Microorganisms need oxygen for
Air Inlet Provides a source of oxygen
aerobic respiration
An aseptic precaution to prevent
Stops microorganisms getting inside
Filter on Air inlet contamination by unwanted
the fermenter
microorganisms
Mixes the microorganisms with the
Keeps the mixture inside the
Stirring Paddles nutrients and keeps the temperature
fermenter agitated (stirred)
even
BASIC FUNCTIONS OF A FERMENTER 75
• It should provide a controlled environment for optimum biomass/product yields

• It should permit aseptic fermentation for a number of days reliably and
dependably, and meet the requirements of containment regulations. Containment
involves prevention of escape of viable cells from a fermenter or downstream
processing equipment into the environment
• It should provide adequate mixing and aeration for optimum growth and
production, without damaging the microorganisms/cells
• The power consumption should be minimum
• It should provide easy and dependable temperature control

BASIC FUNCTIONS OF A FERMENTER… 76
• Facility for sampling should be provided
• It should have a system for monitoring and regulating pH of the fermentation

broth
• Evaporation losses should be as low as possible
• It should require a minimum of labour in maintenance, cleaning, operating and

harvesting operations
• It should be suitable for a range of fermentation processes. But this range may
often be restricted by the containment regulations
In General
 Stirred Vessel H/D  3
 Volume 1-1000 m3 (80 % filled)
 Biomass up to 100 Kg dry weight/m3
 Product 10 mg/L –200 g/L

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L#6

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FERMENTER DESIGN
• A bioreactor is a device in which a substrate of low value is utilized by living
cells or enzymes to generate a product of higher value.
• On the basis of the agent used, bioreactors are grouped into the following two
broad classes:
• Those based on living cells
• Those employing enzymes
• But in terms of process requirements, they are of the following types:
• Aerobic
• Anaerobic
• Solid state
• Immobilized cell bioreactors
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BIOREACTOR
A bioreactor should provide for the following:
• Agitation
• Aeration
Agitation
The medium must be suitably stirred to keep the cells in suspension and to make
the culture homogeneous; it becomes increasingly difficult with the scaling up.
Various types of stirrers range from simple magnetic stirrers, flat blade turbine
impellers, to marine impellers, to those using pneumatic energy, e.g. airlift
fermenter, and those using hydraulic energy, e.g., medium perfusion

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BIOREACTOR
Aeration
Aeration may be achieved by medium perfusion, in which medium is continuously
taken from culture vessel, passed through an oxygenation chamber and returned
to the culture. The cells are removed from the medium taken for perfusion so
that the medium can be suitably altered, e.g. for pH control. Perfusion is used
with glass bead and more particularly with micro-carrier systems
The following components of the fermenter are required for aeration and
agitation:
• Agitator (impeller)
• Stirrer glands and bearings
• Baffles
• Sparger (the aeration
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system)
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AGITATOR (IMPELLER)
• Agitators achieve the following objectives:
• Bulk fluid and gas-phase mixing
• Air dispersion
• Oxygen transfer
• Heat transfer
• Suspension of solid particles
• Maintenance of a uniform environment throughout the vessel
• These objectives are achieved by a suitable combination of the most
appropriate agitator, air sparger and baffles and the best positions for nutrient
feeds, acid or alkali for pH control and antifoam addition
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TYPES OF AGITATOR (IMPELLER)
• Agitators are of several different types:
• Disc turbines
• Vaned discs
• Open turbines of variable pitch
• Propellers

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L#7

85
STIRRER GLANDS AND BEARINGS

• The satisfactory sealing of the stirrer shaft assembly has been one of the most
difficult problems; this is very important for maintaining aseptic conditions over
long periods.
• Four basic types of seal assembly have been used in fermenters:

• The stuffing box (packed- gland seal)
• The simple bush seal
• The mechanical seal
• The magnetic
02/25/2020 drive FST-607 by Dr Shahid Mahmood
86
BAFFLES
• Baffles are metal strips roughly one-tenth of the vessel diameter and attached
radially to the fermenter wall
• They are normally used in fermenters having agitators to prevent vortex
formation and to improve aeration efficiency
• Usually, four baffles are used, but larger fermenters may have 6 or 8 baffles
• Extra cooling coils may be attached to baffles to improve cooling
• Baffles may be installed in such a way that a gap exists between the baffles and
the fermenter wall
• This would lead to a scouring action around and behind the baffles, which
would minimise microbial growth on the baffles and the fermenter wall

87
AERATION SYSTEM (SPARGER)

• The device used to introduce air into the fermenter broth is called sparger
• Spargers are of the following three basic types:

• Porous spargers
• Orifice spargers
• Nozzle spargers
• Porous spargers may be made of sintered glass, ceramics or a metal

88
TEMPERATURE REGULATION
• The fermenter must have an adequate provision for temperature control
• Both microbial activity and agitation will generate heat
• If this heat generates a temperature that is optimum for the fermentation
process, then heat removal or addition may not be required
• But in most cases, this may not be the case; in all such cases, either additional
heating or removal of the excess heat would be required
• Temperature control may be considered at laboratory scale, and pilot and
production scales
• The heating/cooling requirements for a specific fermentation process can be
accurately estimated by taking into account the overall energy balance of the
process, which is described by the following formula:
• Qmet02/25/2020
+ Qag + Qgas = Qacc + Qexch + Qevap
FST-607 Qsen Mahmood
by Dr+Shahid
89
TEMPERATURE REGULATION…
• Qmet – the rate of heat generated by microbial metabolism
• Qag = the rate of heat produced by mechanical agitation
• Qgas = the rate of heat generated by aeration power input
• Qacc = the rate of heat accumulation in the system
• Qexch = the rate of heat transfer to the surroundings and/or heat exchanger, i.e.,
heating/cooling device
• Qevap = the rate of heat loss due to evaporation
• Qsen = the rate of sensible enthalpy gain by the flow streams (exit-inlet)

90
FOAM CONTROL
• Foam is produced during most microbial fermentations
• Foaming may occur either due to a medium component, e.g., protein present in
the medium, or due to some compound produced by the microorganism
• Proteins are present in corn-steep liquor, pharma media, peanut meal, soybean
meal
• These proteins may denature at the air-broth interface and form a protein film
that does not rupture readily
• Foaming can cause removal of cells from the medium; such cell wills undergo
autolysis and release more proteins into the medium
• This, in turn, will further stabilize the foam

91
PATTERNS OF FOAMING
• Foaming remains at a constant level throughout the fermentation. Initial
foaming is due to the medium, but later microbial activity contributes to it
• Foaming declines steadily in the initial stages, but remains constant thereafter,
this type of foaming is due to the medium
• The foaming increases after a slight initial fall’, in this case, microbial activity is
the major cause of foaming
• The foaming level increases with fermentation duration; such foaming pattern is
solely due to microbial activity
• A complex foaming pattern that combines features of two or more of the above
patterns

92
ANTIFOAM
• Ideal antifoam should have the following properties:
1. It should disperse rapidly and act fast on existing foam
2. It should be used at a low concentration
3. It should prevent new foam formation for a long time
4. It should not be used up or degraded by the microorganism
5. It should be nontoxic (to the microorganism as well as animals, including humans)
6. It should not interfere with downstream processing
7. It should not cause problems in effluent treatment
8. It should be safe to handle
9. It should be cheap
10. It 02/25/2020
should not affect oxygen transfer
93
L # 8. TYPES OF FERMENTERS

Associate Professor


94
TYPES OF FERMENTERS
Stirred Tank Fermenter
• These are glass (smaller vessels) or stainless steel (larger volumes)
• These are closed systems with fixed volumes and are usually agitated with
motor-driven stirrers with considerable variation in design details, e.g., water
jacket in place of heater type temperature control, curved bottom for better
mixing at low speeds, mirror internal finishes to reduce cell damage
• Many heteroploid cell lines can be grown in such vessels

95
TYPES OF FERMENTERS…
Airlift Fermenter
• An airlift fermenter consists of a gas light baffled riser tube or draught tube (broth rises
through this tube) connected to a down-comer tube (broth flows down through this
tube)
• The riser tube may be placed within the down-comer tube or it may be externally
located and connected to the latter
• Air/gas mixture is introduced into the base of the riser tube by a sparger
• The aerated medium/broth of the riser tube has a lower density, while that in the down-
flow tube it is relatively much less aerated and, as a consequence, has a higher density
• The lighter medium in the rise tube flows upward till it reaches the gas disengagement
space of the fermenter
• The O2 is continuously consumed by the cells and CO2 is generated by respiration
96

97
Tower Fermenter
• A tower fermenter has been defined by Green-shields and co-workers as an:
“elongated non-mechanically stirred fermenter that has an aspect ratio (height
to diameter ratio) of at least 6 : 1 for the tubular section and 10:1 overall, and
there is a unidirectional How of gases through the fermenter”
• There are several different types of tower fermenters, which are grouped as
follows on the basis of their design:
• Bubble columns
• Vertical-tower beer fermenter
• Multistage fermenter systems

98
TOWER FERMENTER
Bubble Column Tower Fermenters
These are the simplest type of tower fermenters; they consist of glass or metal
tubes into which air is introduced at the base. Fermenter volumes from 3 / to up to
950 / have been used, and the aspect ratio may be up to 16 : 1. These tower
fermenters have been used for citric acid and tetracycline production and for a
range of other fermentations based on mycelial fungi
Vertical-Tower Beer Fermenters
These fermenters were designed for beer production and to maximise yeast biomass
yields. A series of perforated plates are placed at intervals to maximise yeast yields.
It has a settling zone free of gas; in this zone, yeast cells settle down to the bottom
and return to the main body of the tower fermenter, and clear beer could be
removed from the fermenter. Tower of up to 20,000 / capacity and capable of
producing up to 90,000 I beer
02/25/2020 perbyday
FST-607 haveMahmood
Dr Shahid been installed
99
TOWER FERMENTER…
Multistage Tower Fermenters
• In these fermenters, a column forms the body of vessel, which is divided into
compartments by placing perforated plates across the fermenter
• About 10 % of the horizontal area of plates is perforated
• In a variant of this type of fermenter (down-flow tower fermenter), the
substrate is fed in at the top and overflowed through down spouts to the next
section and the air is supplied from the base
• These fermenters have been used for continuous culture of E. coli, S. cerevisiae
(baker’s yeast) and activated sludge

100
Bubble-up Fermenter
• It is a bubble column fermenter that is fitted with an internal cooling coil
• Air is introduced from the bottom of the column
• In this vessel, the cooling coil effectively separates the column into an inner
riser/draught tube and the outer down-flow tube
• The cooling coil assembly functions as a leaky draught tube
• The culture broth rises in the compartment enclosed by the cooling coils and it
moves down in the compartment outside the coil, although back- mixing also
occurs through the coils
• The region above the cooling coil shows good mixing, and there were no poorly
oxygenated zones in the vessel
101
L # 9-10. FERMENTATION VARIABLES

Associate Professor


102
TYPICAL VARIABLES
• pH
• Acidity or varying acid profile
• Initial sugar concentration
• Type of sugar (glucose, fructose, sucrose)
• Temperature
• Yeast strain
• Yeast preparation
• Usually investigated against a ‘control’

103
pH
• Yeast will ferment sugar to alcohol over a very large pH range
• Wine making pH range is typically 3.0 - 4.0
• Changing initial pH generally has little effect on fermentation kinetics or
products, or final alcohol levels
• Very low pH (<3) will impede yeast
• Higher pH >4 will favour bacteria and other competing organisms (Acetobacter)
• Very high pH >4.5 will favour other pathways of sugar catabolism (reduced
alcohol production)
• pH will affect role of any SO2 present as action of SO2 is pH dependent
• pH does not usually change much during normal fermentation
104
ACIDITY
• Acids in fruits are weak organic acids
• Acid profile varies with fruit
• Most acids do not take significant part in fermentation metabolism
• Tartaric acid may precipitate as tartrate salt (loss of acidity)
• Malic acid may be metabolised to lactic acid (loss of acidity) by yeast or MLF
bacteria
• Faulty ferment may produce excess acetic acid (increased acidity)
• Acidity and pH may change slightly due to production of alcohol (changes buffer
capacity)

105
CHANGES IN ACIDITY AND ACID PROFILE DURING FERMENTATION
• Acidity (TA) may increase or decrease overall
• Succinic acid, acetic acid produced via normal alternative pathways (increase)
• Some yeast strains may produce malic acid, more may convert some of malic
acid to lactic acid (increase or decrease)
• Tartaric acid is stable to microbial action but can precipitate with liberated
potassium ions (as potassium tartrate or potassium hydrogen tartrate)

106
L # 10

107
SUGAR
• Sugars in fruit are usually a combination of glucose, fructose and sucrose
• Grapes approx 1:1 glucose : fructose, trace sucrose (other fruits, see handout)
• Yeast may ferment glucose faster than fructose
• Sucrose is inverted by yeast enzymes to glucose + fructose

108
SUGAR CONCENTRATION
• Typically 20-25 % in wine making
• This is high enough to delay onset of fermentation (longer lag phase)
• High sugar >250g/L:
• Cell viability reduced
• Cell division retarded
• Possible increased sensitivity to alcohol toxicity
• Increased production of acetic acid
• Greater likelihood of stuck ferment

109
TEMPERATURE
• Along with sugar concentration, temperature is one of the most important
fermentation variables
• Growth rate of yeast strongly temperature dependent
• Cell division: every 12 hours at 10˚, every 5 hours at 20˚, every 3 hours at 30˚
• At temperatures over 20, yeast viability declines rapidly at the end of ferment
• For many reasons, the preferred temperature for wine making is below that
known to be optimal for ethanol production or yeast growth
110
LOW TEMPERATURE FERMENTS

• 15-20˚C typical for white wine styles
• Yeast growth retarded, but yeast viability enhanced (reduces toxicity effects of
alcohol)
• Slower ferment rate – longer to complete fermentation (note: too cold will
arrest fermentation)
• Higher production of alcohol
• Increased synthesis and retention of fruit esters and fatty acid ethyl esters
• Better flavour concentration for whites

111
HIGHER TEMPERATURE FERMENTS

• 24-27˚C for reds
• Higher temperatures favours extraction of anthocyanins (colour) and tannins
• Shorter lag phase = earlier alcohol production, which also favours colour and
tannin extraction
• Higher temps can favour undesirable consequences such as increased
production of acetic acid, aldehyde and acetoin, lower ester production
• will be less noticeable in reds due to their more complex composition

112
YEAST STRAIN
• Yeast strains vary considerably in many factors, such as:
• Alcohol production and toxicity tolerance
• Temperature range
• Acetic acid production
• SO2 production
• Sugar metabolism (glucophilic, fructophilic)
• Flavour production and metabolism
• Selection of yeast strain is a critical decision in commercial
winemaking

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FST-607. FOOD BIOTECHNOLOGY 3 (2-1)

Dr. Shahid Mahmood Rana

INSTITUTE OF FOOD SCIENCE AND NUTRITION (IFSN)

02/25/2020 FST-607 by Dr Shahid Mahmood

L # 1. BASICS OF FOOD BIOTECHNOLOGY

• Exams and Evaluation

02/25/2020 FST-607 by Dr Shahid Mahmood

ACADEMIC CALENDAR SPRING-2020

02/25/2020 FST-607 by Dr Shahid Mahmood

Session 2016 - 2020

Quiz 1 Tuesday 28/02/2020 02

Mid Wednesday 18/03/2020 30

Pre Final Tuesday 05/05/2020 02

FST-607. COURSE CONTENTS - P

02/25/2020 FST-607 by Dr Shahid Mahmood

3. Borem, A., Santos, F. R. and Bowen, D. E. 2004. Understanding Biotechnology.

STUDENTS’ AVERAGE RETENTION RATES

02/25/2020 FST-607 by Dr Shahid Mahmood

“Biotechnology is the integrated use of biochemistry, microbiology, and

“Biotechnology is the controlled use of biological agents, such as

Oldest Forms of Biotechnology

02/25/2020 FST-607 by Dr Shahid Mahmood

BIOTECHNOLOGY: MODERN APPLICATIONS

• Food crops that produce greater yields

• “Golden Rice" engineered to be more nutritious

• Genetically engineered bacteria that can degrade environmental pollutants

• Manufacture of biotechnological products like vaccine, enzymes, beverages, drugs

Two important technique which enable development of modern biotechnology:

RECOMBINANT DNA TECHNOLOGY

without introducing undesirable genes in target organisms

(1972) by linking gene encoding for antibiotic resistance with plasmid of

1. Restriction exonuclease – removes nucleotides from the end of DNA

02/25/2020 FST-607 by Dr Shahid Mahmood

02/25/2020 FST-607 by Dr Shahid Mahmood

RECOMBINANT DNA TECHNOLOGY..

02/25/2020 FST-607 by Dr Shahid Mahmood

• Using bacteria to make multiple identical copies of a single strand of DNA

genome of a host cell

• Used in gene therapy

02/25/2020 FST-607 by Dr Shahid Mahmood

FST-607. FOOD BIOTECHNOLOGY 3 (2-1)

Dr. Shahid Mahmood Rana

INSTITUTE OF FOOD SCIENCE AND NUTRITION (IFSN)

02/25/2020 FST-607 by Dr Shahid Mahmood

• The sum of all chemical reactions in the body

• Metabolism is divided into two types

02/25/2020 FST-607 by Dr Shahid Mahmood

02/25/2020 FST-607 by Dr Shahid Mahmood

• Anabolism is the chemical reactions that require energy to build large

• Catabolic reactions furnish the energy needed to drive anabolic reactions.

• Energy harvested from catabolic reactions are stored in ATP molecules.

• ATP molecules are used to drive many anabolic reactions

ATP: A “Currency of Energy” for Cellular Reactions

• ATP stands for adenosine triphosphate.

• It is a nucleotide with three phosphate groups linked in a small chain.

• ATP hydrolysis is used as an energy source in many biological reactions that

02/25/2020 FST-607 by Dr Shahid Mahmood

• Maximum yield per Glucose = 38 ATP (30-32)

• Only achieved by aerobic respiration of mitochondria in eukaryote cells

• Anaerobic respiration is even less efficient

• Fermentation least efficient (2 ATP)