INTRODUCTION BASIC PRINCIPLES OF FERMENTATION …

Introduction to IBT B.Sc 6th SEM,KLESNC

INTRODUCTIONBASIC PRINCIPLES OF FERMENTATION TECHNOLOGY ,SCREENING ANDISOLATION OF MICROORGANISMS ,MAINTENANCE OF STRAINS ,STRAINIMPROVEMENT (MUTANT SELECTION,RECOMBINANT DNA METHDS )

Biotechnology is the culmination of more than 8000 years of human experience usingliving organisms and the process of fermentation to make products such as bread, cheese, beerand wine. Today biotechnology is applied to manufacturing processes used in health care, foodand agriculture, industrial and environmental cleanup, among other applications. A widelyaccepted definition of Biotechnology is "Application of scientific and engineering principles toprocessing of materials by biological agents to provide goods and service". Some otherdefinitions replace rather ambiguous word ‘biological agents’ with more specific words such asmicroorganisms, cells, plant and animal cells and enzymes. When a biotechnological process isimplemented on a commercial scale there is every reason to believe that it will the in somebioreactor or fermenter. The entire process can be divided in three stages.

Stage I : Upstream processing which involves preparation of liquid medium, separation ofparticulate and inhibitory chemicals from the medium, sterilization, air purificationetc.,

Stage II: Fermentation which involves the conversion of substrates to desired product with thehelp of biological agents such as microorganisms; and

Stage III: Downstream processing which involves separation of cells from the fermentationbroth, purification and concentration of desired product and waste disposal or recycle.

A fermentation process requires a fermenter for successful production because it providesthe following facilities for the process such as contamination free environment, specifictemperature maintenance, maintenance of agitation and aeration, pH control, monitoringDissolved Oxygen (DO), ports for nutrient and reagent feeding, ports for inoculation andsampling, fittings and geometry for scale up, minimize liquid loss and growth facility for widerange of organisms.

Aseptic environment or contamination is defined as protection against entry of unwantedorganisms. Containment is defined as prevention of escape of viable cells from the process. Boththese environment is provided by a fermenter where ever required. Contamination is applicablein all process whereas containment is necessary when pathogenic organism is used for thefermentation process. The containment level varies based on the pathogenicity of the organismused. Some organism are termed GRAS ie. Generally Recognized As Safe. Criteria forassessment of hazardous organism are known pathogenicity of organism, virulence level, numberof organisms required to initiate infection, routes of infection, known incidence of infection,local existence of vectors and reserves of micro organisms, volume of organisms used in process,techniques used for cultivation and harvesting and prophylaxis and treatment facility. Based onall the criteria if an organism is termed pathogenic the containment of the fermentation process ismaintained. Good industrial large scale practice (GILSP) involves safe and highly productiveorganism for the process.

Head,Department Of Biotechnology KLE SNC Page 1

IBT deals with study of various techniques and methodologies, which are involved in large scale

production of products by using micro organisms.

Industrial biotechnology is the set of practices that use living cells such as algae,fungi,bacteria

yeast or cell components or enzymes to generate industrial products. IBT is the commercial

exploitation of m.o to produce valuable economic ,environmental and important chemical

products for chemical transformation

M.O are Beneficial also and harmful too.Following are the major steps in IBT • Isolation• Screening & strain improvement• fermenter design• Optimal growth• Product recovery• Recovered product microorganisms are useful in Agriculture/food &

beverages/Pharmaceuticals Several thousands of years(Babylonians & Sumerians- used yeast,during indian vedicperiod-used fermented milk product,fungal products-somaderived from mushrooms)• Scientific basis of fermentation (metabolic process that convrts sugar into

alcohol ,gases & acids )first examined 150 years ago• 1857 Louis Pasteur showed role of mo in alcoholic fermentation & also aerobic

&anaerobic microbes• Pasteur showed spoilage of wine & other liquid products are due to the growth of

unwanted microbes that leads to the formation of sour wine/beer • Pasteurization-It is the process of heating the liquid food or beverages to kill pathogenic

bacteria to make the food safe to eat.The use of pasteurization to kill pathogenic bacteriahas helped to reduce the transmission of diseases like typhoid fever,tuberculosis scarletfever polio and dysentry OR A process of heating a food (milk)which slows spoilage dueto microbial growth .In 1897,Louis pasteur suggested mild heating at a temp of 62.8 c forabout half hour (30 minutes) was enough to destroy the undesirable organisms withoutspoiling the taste.It is the process of killing microbes (sterilization).named after LouisPasteur

Flow chart of Downstream ‘Processing’

Harvest fermentation broth for desired product in culture broth

Recovery from Intracellular product/ extracellular product

Removal of microbial cells, debris and other solids

Cell disruption / Cell disintegration(Physical,Chemical,Enzymatic Methods )

Separation(broth with solids &liquids)

Extraction/ Concentration-evaporatio,liqid –liqid extraction,membrane filtration,ppt,adsorption


Purification(gel filtration,ion-exchange,affinit ,hydrophobic interaction,HPLC)

Formulation(drying,freeze-drying,crystallization)

FINAL PRODUCT

• Microbial activity Exploited for making wide range of industrial products • Real boost to IBT-discovery of antibiotics which developed a close growth between

pharmaceutical and microbial industry.• 1929-Alexander Fleming -Penicillin (gram positive ba)1941(Industrial productionof

penicillin)W.Florey ,Ernest Chain -Oxford university.USA

•

• Possible optimization of cultural conditions to grow the fungus in large culture vesselscalled –fermenter .

1945-3 microbiologist shared Nobel prize for the production of penicillin at larger scale• Streptomycin (gram -ve)–Selman Waksman 1944 isolated from Actinomycetes-

Streptomyces griseus.• Search for newer antibiotics led to the discovery of several other chemical substances of

industrial imp from m.o


• M.o can be utilized for the conversion of inexpensive substrate (nutrients) into desirableend products such as alcohol/drugs and other industrial solvents

• Application of Microbial activity in mining to leach out metals from low grade ores indealing with pollution and in control of pathogens/pests/oil spills............

M.O Products

Alconol &solvent

Saccharomyces cervisiae Ethanol

Cloustridium acetobutyricum Acetone,isopropanol/butanol

Acetobacter sp Sorbitol

Bacillus sp Propyleneglyol

Saccharomyces glycerol

Organic acids

Aspergillus niger Citric acid

Lactobacillus sp &Rhizobium Oryzae

Lctic acid

Bacillus sp Acrylic acid

Acetobacter sp Acetic acid

Rhizopus sp Fumeric acid

Vitamins

Pseudomonas dentrificans Vit-B12

Gluconobacter sp Vit-C

M.O Product

Enzymes

Aspergillus niger/Oryzae Glucoamylase

Bacillus subtilis Amylase /Protease

Tricoderma sp Cellulase

Saccharomyces cervisiae Invertase

Rhizopus oryzae/Bacillus sp Protease

Saccharomyces cervisiae Lactase

Polysaccharides

Xanthomonas campestris Xanthumgum Industrial uses ,common food additive ,effective thickening


agent & stabilizer to prevent ingredients fromseparating ,produced by simple sugars using fermentationprocess

• All m.o –fermentative in nature• Key areas-production of industrial enzymes/antibiotics/vitamins/biofuels • Lipases/proteases/xylanase • Potential application in leather ,detergents,dairy ,pharmaceuticals& paper industry • Recent devel includes-monoclonal abs for clinical diagnosis and therapeutic purposes

(major development in BT)• Applications of immobilized cells and enzymes in industrial fermentation process is

another significant development in recent years .

(Immobilized biocatalyst can work in a column reactor, the substate being fed continuously atone end and product being recovered from the other end )

• Conversion of raw materials in to end product • Basic requirements for industrial fermentation • Devel.of molecular biology like GE have - tremendous influence on various fermentation

process – new valuable products • One of the most routinely used organism E.coli used as host for the production of large

no of human proteins of medicinal importance Proteins/pharmaceutical =GM.E.Coli Insulin-,interleukins,calcitonin ,erythropoietin,serum albumin .Epidermal growthfactor,urokinase,DNA vaccines-immunizatn aganist viral infections• Applications of immobilized cells and enzymes in industrial fermentation process is

another significant development in recent years .

Conversion of raw materials in to end product • Basic requirements for industrial fermentation

Major requirement - Industrial fermentation • The organism

• Medium(substrate)

• Process for product recovery (fermented product)

The organism (Producer)• A suitable organism is a critical requisite for any fermentation process• Selected organisms-strain(particular organisms isolated from population of sub species)• Most suitable organism isolated from nature/by creating specific genetically modified

strains of m.o that yields high levels of the product through GET. • Strain selected should hv genetically stable characteristics • ability to grow rapidly and vigoursly


• Strain should be essentially be non pathogenic and non producer of any unwantedbyproducts such as toxins

• Best producer of desired products & should produce product in sufficient quantitiies tomake it commercially viable

• Able to convert raw materials into products under given conditions • Grow well in pure culture especially in liquid culture• Genetically stable when maintained in the laboratory for long periods through serial

subculture without loosing genetic ability and viability• Should adopted to grow well in large culture vessels (fermenter) without undergoing

stress • Should sporulate ,so that standardizatuion of inoculum ll be easy High growth rate when an organism is fast growing• product production –quick• Contamination –less • Control of different parameter –easy • Total cost – less Non pathogenic and could not be in any way harmful to plants ,animals or humans Free from toxin production ,toxins even in small quantitiies are not acceptable in food

products The separation of cells from the culture medium & at the end of fermentation process

should not be difficult (some cells stick together and produce slime ) Organism must undergo genetic manipulatios such as mutant selection,recombinant DNA

protocols which are aimed at strain improvement The medium (nutritional and hormonal)Optimisation of nutrient media is vital thing to

ensure maximum yield. This production process include substrate mixture containing least expensive compounds

,that are readily available and produce highest yield of the desired product .

Industrial strategy for isolation of m.o • ISOLATION OF MICROBES FROM NATURAL SOURCE

• IDENTIFICATION OF DESIRED MICROBES

• CHARECTERIZATION OF MICROBES

• SCREENING OF DESIRED MICROBES

• INOCULUM PREPARATION

• STRAIN IMPROVEMENT

• FERMENTATION

Culture Medium


Carbon source –corn sugar,starch cellulose ,sugar cane,sugarbeet,mollases,milkwhey ,vegetable oils .

Nitrogen –soyabean meal,cornsteep liquor,pureamonia,urea,nitrate salts and ammonium salts

Phosphate –phosphate saltsVitamins and growth factors –thiamin,biotin,beef extract,yeast

extract ,wheat ,cotton seed meal,corn steep liquor,corn seed mealIn many instances it has been practiceble to utilize nutrient

containing wastes from the dairy industry (whey) the paper industry (waste liquor) and other commercial operations ,sugar processing wastes can be basis of nutrient media.

Macro nutrients Micronutrients Additional factors

C,H,N,S,P.Mgsources,water,sugars,lipids,aa,salt minerals

Trace elements ,fe,Zn, Cu, Mn,Metals,Mo,Co,Vitamins

Growth factors,transportproteins,attatchment proteins

Dr.Prathibha.K.S HOD BIOTECHNOLOGY 22

Media -2 types

• Complex/crude/undefined/natural

• Synthetic /defined

Undefined media-made from naturally available vegetable/animal sources.

Synthetic media-made from chemicals,advantages are easy to monitor that can be

designed to get high yield

• Synthetic media -Chemical compositions available are in pure form throughout the

work ,they cause no foaming during fermentation /agitation of the broth. recovery of the

product is very simple.

• Disadvantage –they are expensive & can be used only for the production of speciaity

chemicals

From industrial point of view ,production of fermentation can be divided into

•

• 1.Commodity chemicals

• 2.Speciality chemicals

Commodity chemicals -Produced in bulk ,sold at low cost(raw materials used should

necessarily be inexpensive. eg.ethanol acetone, biofertilizers ,bioremediation

agents,organic acids SCP,SCO,enzymes(cellulase,pectinase,lipase,alkaline protease) for

leather/textile and food industry


Speciality chemical-as the name suggests,are those used for special purpouses such as

disease therapy/nutrition/catalysis of metabolic reaction

• used in minute quantities & require tremendous care in their production as they are

required in high purity following rigorous quality control

• Their production cost is high and are antibiotics,vitamins,hormones and

enzymes(RE,Ligases,polymerases)

• Produced in small capacity fermenters of not more than 10 thouands lts.( control of

contamination becomes very difficult in large fermenters of 50thousands litrs)

Type of products from microbial fermentations

Industrial microbes are cultivated under controlled conditions to optimize their growth and to

produce target product.(food & food additives/alcohols/organic acids /health care

products/industrial enzymes/beverages etc.

Final product is recovered

• Cell harvesting,dsruption of cells,product purification and extraction by using suitable

solvent medium(process development and DSP)

• During growth process in the fermenter ,the organism goes through TROPHOPHASE &

IDIOPHASE

TROPHOPHASE -Nutrient supply is abundant and the organism grows at a rapid rate

producing primary metabolites which are necessary for the growth process

(primary metabolites are the products of the main pathway of metabolism which is a part of

growth and essential for the functions of the organism,without these organism can not survive)

primary metabolites produced in the trophophase when nutrients are in plenty

Majority of the products obtained from microbial fermentations are secondary matabolites

IDIOPHASE-–growth stops due to exhaustion of nutrients and the organism starts

producing sec metabolites.sec. met very complex mol requiring large no of rx to produce

one product.25 rx productn of tetracyclin -72 rx tetracyclin

(products of sec metabolites are not required for growth of the organism products of

pri.metabolism can serve as source for sec metabolism)

The scale up process

• Begins after establishment of production of a new valuable useful products by m.o at the

laboratory level (shake culture flask-good producers but stop fermenting in large volume

of broth –capacities of 4,000 lts)


• Methodology of finding out optimal conditions for the best yield of a product under

industrial conditions called bioprocess optimization.

• 1.challenging task & requires experts

• 2.medium sized vessel-pilot plant of 5000 to 10000 lits

Problems in fermetrs

• Volume is more for given surface area

• Gas transfer becomes difficult in big tanks, as industrial products are the result of aerobic

fermentations, proper oxygen transfer in the broth is essential for good yield

• Fluid dynamics studies hv to be conducted with the help of bioengineers

• The product and its recovery

Fermenters with huge capacities should be designed with optimal environmental conditions to

produce maximal yields of the products.

The final product will be mixed with numerous other chemicals like live and dead microbial

cells.

The process of recovery of the product in the pure form is the final important step and is called

DSP Which includes number of separation techniques such as centrifugation, filtration,

ultracentrifugation and chromatography

Quality Assurance and packaging

• Quality control

• Quality assurance

• ISO

Packaging

Quality-meeting the requirements

In Pharmaceutical – follow of specifications for developing system for certain materials

to be tested or evaluated with reference to speccifications.

Chemical/drug –specifications for each quality is required like specific

gravity,colour,odour,taste,solubility,antimicrobial spectrum,toxicity, PH and specified

values /standards have to be met

Quality control-techniques and activities /methodology used to ful fill the requirement.

ISO-deals with all fields ,sets standards(except electricals and electronics)

Q Assurance –planned and systematic action taken by the industry to provide adequate

confidance to the user that product meets the standards and requirements.


Q A and development attached to each industry which oversees all aspects of Research

and Development ,manufacture and quality control

Chemical/drug manufactured by a particular process has generally 5 variables

Labels

Packaging materials vary depending on inputs

Raw materials

In- process items

Finished products

Labels/packaging/raw materials purchased by manufacturer-less likely to be tested for

chemical and microbiological properties,type of material used for labels,nature of

ink/type of prinng ,type of glue used shd be decided with care.

The packaging for tablets,powders,liquids,bulk drugs and bulk enzymes require different

assembly lines and different specifications ,(robotics)

Bulk products are usually lyophillized and packaed in glass jars which are adequtely

sealed.the final packed item shd be tested for sterility,fill volume(quantiy),quality and

purity

Finally it is also imp for the industry to maintain all the scientific documents for each

chemicals and drug of that is manufactured

ISOLATION, SCREENING AND STRAIN IMPROVEMENT

Industrial strategy for isolation of m.o

• ISOLATION OF MICROBES FROM NATURAL SOURCE

• IDENTIFICATION OF DESIRED MICROBES

• CHARECTERIZATION OF MICROBES

• SCREENING OF DESIRED MICROBES

• INOCULUM PREPARATION

• STRAIN IMPROVEMENT•

• FERMENTATION Dr.Prathibha.K.S HOD BIOTECHNOLOGY 40


• After isolation of bacteria ,it is imp to identify the specific bacteria.

• Identification is done with the help of following characteristics

Morphology

Selective and diagnostic media

Cultural characteristics

Additional recognised biochemical tests

Profile of microbial strains

Rapid identification methods

STEPS

Isolation of industrially important microbes

• Screening

• Strain improvement

• Inoculum Development

• Fermentation, (comes from the Latin verb fevere, which means to boil.

It originated from the fact that early at the start of wine fermentation gas bubbles are released

continuously to the surface giving the impression of boiling)

Downstream processing

• First step in industrial biotechnology is obtaining right organism that can yield the

maximum product

• Isolation from diverse environmental sources

• Subjected to screening for specific products

• Newly found organisms subjected to strain improvement trails to increase product yield

• Improved m.o subjected to bioprocess optimization –large scale production

(Methodology of finding out optimal conditions for the best yield of a product under industrial

conditions called bioprocess optimization)

• Preservation of cultures –part of IBT

• Isolation of micro organisms

• Serial dilution method/technique

• Pour plate

• Spread plate

• Streak plate

• Enrichment culture

• Special isolation methods


• Serial dilution method/technique

• A pure culture may be obtained from a sample containing a mixture of bacteria by

serially diluting the sample with sterile broth in culture tubes

• Serial dilution decreases the cell concentration in the tubes finely diluted tube is cultured

by transferring to medium and well isolated individual colonies can be obtained.

• Pour plate method

• This method involves plating of diluted sample mixed melted agar medium .

• The original sample should be suitably diluted with sterile water or broth to ensure

development of isolated colonies.

• The agar medium is maintained in liquid state by maintaing temp at 45 C and aliquot of

diluted inoculum added to it.

• The medium mixed with the inoculum is poured into sterile petri dishes and the plates

incubated

• Pour plates will exhibit both surface and subsurface colonies as some cells will be

trapped deep in the medium

• A combination of serial dilution of the original sample with pour plate method is suitable

for isolation of soil bacteria,fungi and actinomycetes.


•

Spread plate method

• Bacterial suspension is suitably diluted is not mixed with the molten medium before

pouring

• After agar medium is properly solidified ,a loopful of suspension is transferred to the

solidified surf

• A loopul of suspension is transferred to the surface of the medium .

• Susension is made to spread uniformly on the surface of the medium with the help of

sterilized glass spreader

• The plate is then incubated overnight to find individual colonies

Streak plate method

• A small amount of the mixed inoculum is taken with a sterile nichrome wire loop and

streaked over the surface of agar medium in a petri dish.

• Streak should be in such a way it should provide successive dilutions within a single plate

• Five dilutions can be effected in a single plate and each subsequent streak thinning out

the inoculum from the first streak

• On incubation of the plate,in the final ,individual colonies are likely to appear.


• Pure cultures may be obtained by transferring cells from the individual colonies to agar

slants

Enrichment culture

• Special culture method wherein an environment is created to favour a specific group of

microorganisms

• Introducing a specific nutrient and by modifying the Ph and temp

• The conditions will be unsuitable for majority of microorganisms which are to be

eliminated

• Ex- nitrobacteria can be obtained by inoculating soil with a slat solution containing

sodium nitrate at ph 8.5 and incubating it in air in a dark incubator at 25-30 c

• Nitrobacteria is capable of oxidizing nitrates and the presence of nitrate favours the

growth of this bacterium

• Ex-selective enrichment - isolation of vibrio cholerae from chloera patients.(Most

intestinal vibrios grow at the surface of culture broths bcz of the need of oxygen Ph 8-

9,this pH retards many of the ba associated with vibrios in faecal matter.

• V.cholera metabolises peptone & hydrolyses egg ,thus alkanine egg peptone sol.

Inoculated with faces of cholera patient gives rise to almost pure surface growth of

V.cholerae. Surface film gives pure culture of the bacteria

• Special isolation methods

• Selective media with chemical which can suppress unwanted m.o –isolation of specific

m.o

• Ex-Rosebengal added to media –fungi from soil/air(chemicals suppress soil /atmospheric

bacteria)

• C.V/Brilliant green inhibit gram + ba and help in the isolation of gram negative ba from

sewage

• Sodium azide inhibits cytochrome oxidase and therefore inhibits organisms cytochrome

system


And allows lactic acid bacteria which lack cytochrome system to grow

• On a differential medium ,eosin –methylene blue agar(EMB)Escherichia coli produces

colonies with brilliant green metallic sheen .

• on the same medium Aerobatic aerogenes produces pink colonies with dark centres

On Differential Medium

E.coli Aerobacter

Baiting techniques

• Isolation of Phytophthora sp from soil is effected through baits(trap/hook)(apples, green

water melon etc)buried in soil sample for 4-5 days .

• Pythium sp can be isolated from soil by buring mustard or corn seeds

• Burying rice culms (straw)in soil helps in the isolation of Drechslera oryzae from soil.

• Water moulds are often isolated on baits of killed insects floated on a sample of water

• Keratinophilic ba are isolated by burring hair in soil as bait.

Screening of industrially useful microorganisms

Right sp for an industrial process is initially obtained by screening large number

of mo isolated from diverse environment for a particular product(soil, litter water).

B/W 1940 and 1980 ,several thousands of mo were screened for production of antibiotics

mainly against bacteria. Large number of soil actinomycetes were laboriously screened for

antibiotics using inhibition zone method on plate cultures techniques containing sensitive test

microorganisms.

Different test organisms were used to determine the antimicrobial spectrum of the isolated

organisms.

Analytical methods were then used to identify the chemical nature of inhibitory compound and

toxicity were performed to assess the toxicity to plant and animal cells

1970s focus shifted to screening microbes for newer industrial products such as antiparasitic ,

antiifungal,anticancer and anti viral agents substances useful against hypercholesteromia


(leading to high BP) acting as immunomodulators were also obtained.(mediators used to

regulate /normalize the immune system/active agents of immune therapy),Semiautomatic

methods of screening are saving time and labour.

Antibiotic Spectrum of Activity

• No antibiotic is effective against all microbes

Dr.Prathibha.K.S HOD BIOTECHNOLOGY 62

Antibiotics - Screening of mo for antibiotic production is done routinely by Cross- streakiing

method for this processs , it is imp to hv cultures of as many as possible pathogenic mo .the

newly isolated mo tested for its ability to inhibit the growth of one or more of the pathogenic mo


in the collection. Dr.Prathibha.K.S HOD BIOTECHNOLOGY 64

On agar plate on a suitable medium, the candidate organism isolated from The environment is

streaked on the left side in a straight line , on the same plate, three different test organisms

(pathogens )are streaked at right angle to the candidate organism streak. the plate is incubated

overnight for 48 hrs the candidate streak will show full growth where as among the 3 test

organisms growth may be fully or partially inhibited by the metabolite , produced by the

candidate organism. Depending on the degree of metabolite production,the zone of inhibition

will be smaller or bigger.

• Strain Improvement-Once a sp having industrial applications found,a research

programme is undertaken to increase the capacity of the mo to produce the desired

products

• The classical example is penicillin .Penicillium notaum sp –Alexander Fleming –very

low quantities of penicillin in static culture ,and commercial production was nonviable.

• Extensive screening of soil samples led to the isolation of a strain of penicillium

chrysogenum from soil of Peoria ,USA,that could be cultured in shake flask cultures and

was thus more productive and commercially useful.

• Genetic improvement of this strain through induced mutation using X-rays,UV,and

mustard gas treatment and selection led to a much improved strain that could yield 55

times more penicillin than original strain.


• Since conditions in Britain at that time were not suitable for any funding for research,

Americans, attracted by the reports on penicillin invited Florey and Chain to work on

Penicillin. Working in USA, they were able to produce large quantities of Penicillin for

trial and subsequent use. In the year 1945, for the discovery of Penicillin, Flemming,

Florey and Chain shared the Nobel Prize in physiology and medicine (Flemming however

was not in volved in the work done in USA on Penicillin).

What is the Need?

• With the exception of the food industry, only a few commercial fermentation processes

use wild strains isolated directly from nature.

• Mutated and recombined mo’s are used in production of antibiotics, enzymes, amino

acids, and other substances.

What Should We Look for when We Plan a Strain Improvement Program?

• In general economic is the major motivation.

• Metabolite concentrations produced by the wild types are too low for economical

processes.

• For cost effective processes improved strain should be attained.

• Depending on the system, it may be desirable to isolate strains:

• · Which shows rapid growth

• · Which shows Genetic stability

• · Which are non-toxic to humans

• · Which has large cell size, for easy removal from the culture fluid.

• Having ability to metabolize inexpensive substrate.

• which require shorter fermentation times,

• which do not produce undesirable pigments,

• which have reduced oxygen needs, With lower viscosity of the culture so that

oxygenation is less of a problem,

• Which exhibit decreased foaming during fermentation,

• With tolerance to high concentrations of carbon or nitrogen sources

Methods of Strain Improvement

• The use of recombinant DNA techniques.

• Protoplast fusion,

• Site-directed mutagenesis,


• Recombinant DNA methods have been especially useful in the production of primary

metabolites such as amino acids, but are also finding increasing use in strain development

programs for antibiotics.

Culture Preservation

• Preservation of cultures by freezing, drying, or a combination of the two processes is

highly influenced

• Methods to protect against the negative effects of dehydration include adaptation of pre-

incubation in high osmotic pressure solutions.

Damage caused by thawing after freezing can be minimized by rapid melting and by the

composition of the medium used for growth after preservation.

• There are various preservation methods .

Serial Transfer

Preservation under Oi

Lyophilization

Storage over Silica Gel

Preservation on Paper

Preservation on Beads

Liquid Drying

Cryopreservation

Preservation in Liquid Nitrogen,(Preservation in liquid nitrogen is still the most

successful long-term method)

Serial Transfer-Based upon its ease of use, serial transfer is often the first “preservation”

technique used by microbiologists.

The disadvantages of relying upon this method for culture maintenance include

contamination, loss of genetic and phenotypic characteristics, high labor costs, and loss

of productivity.

Preservation under Oil-One of the earlier preservation methods was the use of mineral

oil to prolong the utility of stock cultures.

Mineral oil has been found to prevent evaporation from the culture

Decrease the metabolic rate of the culture by limiting the supply of oxygen.

This method is more suitable than lyophilization for the preservation of non-

sporulating strains.


Lyophilization-One of the best methods for long-term culture preservation of many

microorganisms is freeze-drying (lyophilization).

The commonly used cryoprotective agents are skim milk (15% [wt/vol] for cultures

grown on agar slants and 20% for pelleted broth cultures) or sucrose (12% [wt/vol] final

concentration).

It should be noted that some plasmid--containing bacteria are successfully preserved by

this method.

Storage over Silica Gel

• Neurospora has successfully been preserved over silica gel.

Preservation on Paper

• Drying the spores on some inert substrates can preserve spore-forming fungi,

actinomycetes, and unicellular bacteria.

• Fruiting bodies of the myxobacteria, containing myxospores, may be preserved on pieces

of sterile filter paper and stored at room temperature for 5 to 15 years.

Preservation on Beads

The method involving preservation on beads (glass, porcelain) , developed by Lederberg,

is successful for many bacteria.

Liquid Drying To avoid the damage that freezing can cause, a liquid—drying preservation

process is applied.

• It has effectively preserved organisms such as anaerobes that are damaged by or fail to

survive freezing.

• This procedure was preferred over lyophilization for the maintenance of the

biodegradation capacity of gram-negative bacteria capable of degrading toluene.

• Malik’s liquid-drying method was also found to be markedly superior to lyophilization

for the preservation of unicellular algae.

Cryopreservation-Microorganisms may be preserved at - 5 to - 20°C for 1, to 2 years by

freezing broth cultures or cell suspensions in suitable vials.

• Deep freezing of microorganisms requires a cryoprotectant such as glycerol or dimethyl

sulfoxide (DMSO) when stored at -70°C or in the liquid nitrogen at -156 to -196°C.

• Broth cultures taken in the mid--logarithmic to late logarithmic growth phase are mixed

with an equal volume of 10 to 20% (vol/vol) glycerol or 5 to 10% (vol/vol) DMSO.

• Alternatively, a 10% glycerol-sterile broth suspension of growth from agar slants may be

prepared.


Preservation in Liquid Nitrogen

• Storage in liquid nitrogen is clearly the preferred method for preservation of culture

viability

INTRODUCTION to USP & DSP

Biotechnology is the culmination of more than 8000 years of human experience using

living organisms and the process of fermentation to make products such as bread, cheese, beer

and wine. Today biotechnology is applied to manufacturing processes used in health care, food

and agriculture, industrial and environmental cleanup, among other applications. A widely

accepted definition of Biotechnology is "Application of scientific and engineering principles to

processing of materials by biological agents to provide goods and service". Some other

definitions replace rather ambiguous word ‘biological agents’ with more specific words such as

microorganisms, cells, plant and animal cells and enzymes. When a biotechnological process is

implemented on a commercial scale there is every reason to believe that it will the in some

bioreactor or fermenter. The entire process can be divided in three stages.

Stage I : Upstream processing which involves preparation of liquid medium, separation of particulate and inhibitory chemicals from the medium, sterilization, air purification etc.,

Stage II: Fermentation which involves the conversion of substrates to desired product with the help of biological agents such as microorganisms; and

Stage III: Downstream processing which involves separation of cells from the fermentation broth, purification and concentration of desired product and waste disposal or recycle.

A fermentation process requires a fermenter for successful production because it providesthe following facilities for the process such as contamination free environment, specifictemperature maintenance, maintenance of agitation and aeration, pH control, monitoringDissolved Oxygen (DO), ports for nutrient and reagent feeding, ports for inoculation andsampling, fittings and geometry for scale up, minimize liquid loss and growth facility for widerange of organisms.

Aseptic environment or contamination is defined as protection against entry of unwantedorganisms. Containment is defined as prevention of escape of viable cells from the process. Boththese environment is provided by a fermenter where ever required. Contamination is applicablein all process whereas containment is necessary when pathogenic organism is used for the


fermentation process. The containment level varies based on the pathogenicity of the organismused. Some organism are termed GRAS ie. Generally Recognized As Safe. Criteria forassessment of hazardous organism are known pathogenicity of organism, virulence level, numberof organisms required to initiate infection, routes of infection, known incidence of infection,local existence of vectors and reserves of micro organisms, volume of organisms used in process,techniques used for cultivation and harvesting and prophylaxis and treatment facility. Based onall the criteria if an organism is termed pathogenic the containment of the fermentation process ismaintained. Good industrial large scale practice (GILSP) involves safe and highly productiveorganism for the process.

Depending on the type of product, the concentration levels it is produced and the puritydesired, the fermentation stage might constitute anywhere between 5-50% of the total fixed andoperating costs of the process. Therefore, optimal design and operation of bioreactor frequentlydominates the overall technological and economic performance of the process.

In any biological process, the following are unique features.

(a) The concentrations of starting materials (substrates) and products in the reaction mixture arefrequently low; both the substrates and the products may inhibit the process. Cell growth, thestructure of intracellular enzymes, and product formation depend on the nutritional needs ofthe cell (salts, oxygen) and on the maintenance of optimum biological conditions(temperature, concentration of reactants, and pH) with in narrow limits.

(b) Certain substances inhibitors effectors, precursors, metabolic products influence the rate andthe mechanism of the reactions and intracellular regulation.

(c) Microorganisms can metabolize unconventional or even contaminated raw materials(cellulose, molasses, mineral oil, starch, ores, wastewater, exhaust air, biogenic waste), aprocess which is frequently carried out in highly viscous, non-Newtonian media.

(d) In contrast to isolated enzymes or chemical catalysts, microorganisms adapt the structure andactivity of their enzymes to the process conditions, whereby selectivity and productivity canchange. Mutations of the microorganisms can occur under sub optimal biological conditions.

(e) Microorganisms are frequently sensitive to strong shear stress and to thermal and chemicalinfluences.

(f) Reactions generally occur in gas-liquid -solid systems, the liquid phase usually beingaqueous.

(g) The microbial mass can increase as biochemical conversion progresses. Effects such asgrowth on the walls, flocculation, or autolysis of microorganisms can occur during thereaction.


(h) Continuous bioreactors often exhibit complicated dynamic behavior.

Due to above mentioned demands made by biological systems on their environment,there is no universal bioreactor. However, the general requirements of the bioreactor are asfollows:

(a) The design and construction of biochemical reactors must preclude foreigncontamination (sterility). Furthermore, monoseptic conditions should be maintainedduring the fermentation and ensure containment.

(b) Optimal mixing with low, uniform shear achieved by proper designing of agitator andaerator

(c) Adequate mass transfer (oxygen) achieved by monitoring the speed of agitator andagitator

(d) Clearly defined flow conditions that can be maintained by proper opening valves andmonitoring devices

(e) Feeding of substrate with prevention of under or overdosing by proper feed ports andmonitoring

(f) Suspension of solids(g) Gentle heat transfer(h) Compliance with design requirements such as: ability to be sterilized; simple

construction; simple measuring, control, regulating techniques; scaleup; flexibility; longterm stability; compatibility with up- downstream processes; antifoaming measures.

DESIGN OF FERMENTER

Components of fermenter

1. Basic component includes drive motor, heaters, pump, etc.,

2. Vessels and accessories

3. Peripheral equipment (reagent bottles)

4. Instrumentation and sensors

Various components of an ideal fermenter for batch process are:

S.No. Part Purpose

1 Top plate cover (made of steel)

2 Clamp top plate compressed onto vessel using clamp

3 Seal separates top plate from vessel (glass) to prevent air leakage

4 Vessel glass, jacketed, steel with ports for various outputs, inputs, probes

etc


5 Drive motor used to drive mixing shaft

6 Drive shaft mixes the medium evenly with its impeller

7 Marine impeller for plant tissue culture

8 Baffles prevent sedimentation on sides and proper mixing

9 Sparger air supplier / after filtration via membranes – ensures efficient

dispersal – by attached to impeller

10 Exit gas cooler like condenser remove as much moisture as possible from exhaust

11 Inoculation port to add inoculum

needle

12 Feed pumps regulates the flow rates of additives (medium, nutrients) variable

speed

13 Peristaltic fixed speed pumps – used for continuous sampling

pumps

14 Syringe pump using a syringe – mostly used in batch

15 Exit gas analysis CO2 analyzer, O2 analyzer, mass spectrometer

16 Sample pipe through which samples are drawn

17 3 way inlet to insert different probes

Monitoring and controlling parts of fermenter are:

S.No Part Use

1 Pt100 temperature sensor (platinum resistance electrode)

2 Foam probe kept above the medium level to sense foam formation

3 pH electrode senses pH

4 O2 sensor Monitors dissolved oxygen level

5 Heater pad directly heats the medium

6 Cold finger after direct heating – used to cool the vessel contents (closed

coil/pipe to pass cool water)


7 Rotameter variable air flow meter – indicates rate of air flow into vessel

– attached to air sparger

8 Pressure valve attached to rotameter for safer operation

9 Air pump supply of air

10 Peristaltic pump to pump in medium, acids, bases, antifoam

Fig.1 Ideal fermenter

BODY CONSTRUCTION Construction materials differ with small scale, pilot and large scale. In small scale for vesselconstruction glass or stainless steel may be used. For pilot and large scale process, stainlesssteel (>4% chromium), mild steel (coated with glass or epoxy material), wood, plastic orconcrete may be used as vessel construction material. Any vessel used should not have anycorners and smooth surface is essential. The construction material must be non toxic andcorrosion proof.

Glass vessel (borosilicate glass)

Type I – glass vessel round or flat bottom with top plate. It can be sterilized by autoclaving and the largest diameter is 60cm.Type II – glass vessel flat bottom with top and bottom stainless steel plate. This type is used in insitu sterilization process and the largest diameter 30cm.


Stainless steelStainless steel is used as vessel construction material with the following modifications,

1. >4% chromium (atleast 10-13%) may be added

2. film of thin hydrous oxide - non-porous, continuous, self healing, corrosion resistance

3. inclusion of nickel - improves engineering

4. presence of molybdenum - resistance to halogen salts, brine, sea water

5. tungsten, silicone - improve resistance

Thickness of vessel should be increased with scale. Side plates have lower thickness than topand bottom plates. Top and bottom plate are hemispherical to withstand pressures.SEALING

Sealing between top plate and vessel is an important criteria to maintain airtightcondition, aseptic and containment. Sealing have to be done between three types of surfaces viz.between glass-glass, glass- metal and metal-metal. There are three types of sealing. They aregasket, lipseal and ‘O’ ring. This sealing ensures tight joint in spite of expansion of vesselmaterial during fermentation. The materials used for sealing may be fabric-nitryl or butylrubbers. The seals should be changed after finite time. There are two way of sealing in O ringtype simple sealing and double sealing with steam between two seals.BAFFLES:

Baffles are metal strips that prevent vortex formation around the walls of the vessel.

These metal strips attached radially to the wall for every 1/10th of vessel diameter. Usually 4

baffles are present but when the vessel diameter is over 3dm3 around 6-8 baffles are used. There

should be enough gap between wall and baffle so that scouring action around vessel is facilitated.This movement minimizes microbial growth on baffles and fermentation walls. If needed coolingcoils may be attached to baffles.

AERATION SYSTEM (SPARGER)

Sparger is a device for introducing air into fermenter. Aeration provides sufficientoxygen for organism in the fermenter. Fine bubble aerators must be used. Large bubbles willhave less surface area than smaller bubbles which will facilitate oxygen transfer to a greaterextent. Agitation is not required when aeration provides enough agitation which is the case Airlift fermenter. But this is possible with only for medium with low viscosity and low total solids.For aeration to provide agitation the vessel height/diameter ratio (aspect ration) should be 5:1.Air supply to sparger should be supplied through filter.

There are three types of sparger viz. porous sparger, orifice sparger and nozzle sparger.


1. Porous sparger: made of sintered glass, ceramics or metal. It is used only in lab scale-non

agitated vessel. The size of the bubble formed is 10-100 times larger than pore size.

There is a pressure drop across the sparger and the holes tend to be blocked by growth

which is the limitation of porous sparger.

2. Orifice sparger: used in small stirred fermenter. It is a perforated pipe kept below the

impeller in the form of crosses or rings. The size should be ~ ¾ of impeller diameter. Air

holes drilled on the under surfaces of the tubes and the holes should be atleast 6mm

diameter. This type of sparger is used mostly with agitation. It is also used with out

agitation in some cases like yeast manufacture, effluent treatment and production of SCP.

3. Nozzle sparger: Mostly used in large scale. It is single open/partially closed pipe

positioned centrally below the impeller. When air is passed through this pipe there is

lower pressure loss and does not get blocked.

4. Combined sparger agitator: This is air supply via hallow agitator shaft. The air is emitted

through holes in the disc or blades of agitator.

EXIT GAS COOLER

Similar to liebig condenser, condenses the moisture from the exhaust gas in the

fermenter. This removes as much moisture as possible from the gas leaving the fermenter and

prevent excess fluid loss.

AGITATION

Agitation provides uniform suspension of cells in homogenous nutrient medium. This

agitation provides bulk fluid and gas phase mixing, air dispersion, facilitates oxygen transfer and

heat transfer and uniform environment through out the vessel. There are four classes, namely

Disc turbine, Vaned disc, Open turbine of variable pitch and Marine impeller.

Disc turbine prevents flooding by air bubbles. Flooding occurs when the air bubble is not

properly dispersed the air pocket is formed one area. Flooded only at 120min/hour of air


discharge when disc turbine is used. When open turbine and propeller are used the medium is

flooded at 21min per hour of air discharge.

Difference between disc turbine and open turbine is as follows:

Disc turbine Open turbinePrevent flood by air bubbles till Prevent flooding only till120min/hour 21min/hourradial flow Axial flowDisc forces air to tip of agitator to disc is absentbe dispersed

TYPES OF FERMENTERS

The main function of a fermenter is to provide a controlled environment for the growthof microorganisms or animal cells, to obtain a desired product. Few of the bioreactor types arediscussed below:

STIRRED TANK FERMENTER

Microbial fermentations received prominence during 1940's namely for the production

of life saving antibiotics. Stirred tank reactor is the choice for many (more than 70%) though it is

not the best. Stirred tank reactor’s have the following functions: homogenization, suspension of

solids, dispersion of gas-liquid mixtures, aeration of liquid and heat exchange. The Stirred tank

reactor is provided with a baffle and a rotating stirrer is attached either at the top or at the

bottom of the bioreactor.

The typical decision variables are: type, size, location and the number of impellers;

sparger size and location. These determine the hydrodynamic pattern in the reactor, which in

turn influence mixing times, mass and heat transfer coefficients, shear rates etc. The

conventional fermentation is carried out in a batch mode. Since stirred tank reactors are

commonly used for batch processes with slight modifications, these reactors are simple in

design and easier to operate. Many of the industrial bioprocesses even today are being carried

out in batch reactors though significant developments have taken place in the recent years in

reactor design, the industry, still prefers stirred tanks because in case of contamination or any

other substandard product formation the loss is minimal

The batch stirred tanks generally suffer due to their low volumetric productivity. The

downtimes are quite large and unsteady state fermentation imposes stress to the microbial

cultures due to nutritional limitations. The fed batch mode adopted in the recent years


eliminates this limitation. The Stirred tank reactor’s offer excellent mixing and reasonably good

mass transfer rates. The cost of operation is lower and the reactors can be used with a variety

of microbial species. Since stirred tank reactor is commonly used in chemical industry the

mixing concepts are well developed. Stirred tank reactor with immobilized cells is not favored

generally due to attrition problems; however by separating the zone of mixing from the zone of

cell culturing one can successfully operate the system.

Fig.2 Stirred tank fermenter

AIR-LIFT FERMENTER

Airlift fermenter (ALF) is generally classified as pneumatic reactors without any

mechanical stirring arrangements for mixing. The turbulence caused by the fluid flow ensures

adequate mixing of the liquid. The draft tube is provided in the central section of the reactor.

The introduction of the fluid (air/liquid) causes upward motion and results in circulatory flow in

the entire reactor. The air/liquid velocities will be low and hence the energy consumption is

also low. ALFs can be used for both free and immobilized cells. There are very few reports on

ALFs for metabolite production. The advantages of Airlift reactors are the elimination of

attrition effects generally encountered in mechanical agitated reactors. It is ideally suited for

aerobic cultures since oxygen mass transfer coefficient are quite high in comparison to stirred

tank reactors. This is ideal for SCP production from methanol as carbon substrate. This is used

mainly to avoid excess heat produced during mechanical agitation.


Fig.3 Air-lift fermenter


FLUIDISED BED BIOREACTOR

Fluidized bed bioreactors (FBB) have received increased attention in the recent years

due to their advantages over other types of reactors. Most of the FBBs developed for biological

systems involving cells as biocatalysts are three phase systems (solid, liquid & gas). The

fundamentals of three phase fluidization phenomena have been comprehensively covered in

chemical engineering literature. The FBBs are generally operated in co-current upflow with

liquid as continuous phase and other more unusual configurations like the inverse three phase

fluidized bed or gas solid fluidized bed are not of much importance. Usually fluidization is

obtained either by external liquid re-circulation or by gas fed to the reactor. In the case of

immobilized enzymes the usual situation is of two-phase systems involving solid and liquid but

the use of aerobic biocatalyst necessitate introduction of gas (air) as the third phase. A

differentiation between the three phase fluidized bed and the airlift bioreactor would be made

on the basis that the latter have a physical internal arrangement (draft tube), which provides

aerating and non-aerating zones. The circulatory motion of the liquid is induced due to the draft

tube. Basically the particles used in FBBs can be of three different types: (i) inert core on which

the biomass is created by cell attachment. (ii) Porous particles in which the biocatalyst is

entrapped.(iii) Cell aggregates/ flocs (self-immobilization). In comparison to conventional

mechanically stirred reactors, FBBs provide a much lower attrition of solid particles. The

biocatalyst concentration can significantly be higher and washout limitations of free cell

systems can be overcome. In comparison to packed bed reactors FBBs can be operated with

smaller size particles without the drawbacks of clogging, high liquid pressure drop, channeling

and bed compaction. The smaller particle size facilitates higher mass transfer rates and better

mixing. The volumetric productivity attained in FBBs is usually higher than in stirred tank and

packed bed bioreactors. There are several successful examples of using FBBs in bioprocess

development.

Fig.5 Fluidised bed bioreactor


PACKED BED BIOREACTOR

Packed bed or fixed bed bioreactors are commonly used with attached biofilms especiallyin wastewater engineering. The use of packed bed reactors gained importance after the potentialof whole cell immobilization technique has been demonstrated. The immobilized biocatalyst ispacked in the column and fed with nutrients either from top or from bottom. One of thedisadvantages of packed beds is the changed flow characteristic due to alterations in the bedporosity during operation. While working with soft gels like alginates, carragenan etc the bedcompaction which generally occurs during fermentation results in high pressure drop across thebed. In many cases the bed compaction was so severe that the gel integrity was severelyhampered. In addition channeling may occur due to turbulence in the bed. Though packed bedsbelong to the class of plug flow reactors in which backmixing is absent in many of the packedbeds slight amount of backmixing occurs which changes the characteristics of fermentation.Packed beds arc generally used where substrate inhibition governs the rate of reaction. Thepacked bed reactors are widely used with immobilized cells. Several modifications such astapered beds to reduce the pressure drop across the length of the reactor, inclined bed, horizontalbed, rotary horizontal reactors have been tried with limited success.

Fig.6 Packed bed bioreactor

BUBBLE COLUMN FERMENTER

Bubble column fermenter is a simplest type of tower fermenter consisting of a tubewhich is air sparged at the base. It is an elongated non-mechanically stirred fermenter with anaspect ratio of 6:1. This type of fermenter was used for citric acid production.


14

Fig.7 Bubble column fermenter

CONTROL AND MONITORING FERMENTATION SYSTEM

The integral part of a high-quality bioreactor is a process controller. Such a controller iscommonly specially formed for a definite bioreactor brand. This is rather connected with thefact that microorganism cultivation processes have relatively high requirements in respect toprecision and sophistication. All this is despite the fact that almost all bioreactors monitor andregulate the same values actually invariably.

There are three types of sensors used in fermenter. They are,

In-line sensors form integral part of fermenter. The directly measured value controls the process.Eg. Antifoam probe

On-line sensors form integral part of fermenter. The measured value must be enteredinto control system to control process. Eg. Ion specific sensors, mass spectrophotometer.

Off-line sensors do not form integral part of fermenter. The measured value must be enteredinto control system for data collection.TEMPERATURE

Heat is generated from any fermentation process due to microbial activity and agitation.The heat control in small scale is carried out by thermostatically controlled bath, internalheating coils, heating jacket(water),silicon heating jacket ;large scale: inter coils and cold watercirculation. Cooling water is required less for bacteria but more for fungi (due to low optimumtemperature for growth).


AGITATION MEASURING AND CONTROLLING DEVICE

Agitation speed can be measured by power consumed by agitator shaft. Wattmeter isusually used in large scale process. It is a measure of power consumed for rotation of agitatorshaft. This measure is less accurate because power required to rotate against friction in thebearing is taken into consideration.

Torsion dynamometer is used in small scale. This has to be placed outside the vessel andless accurate due to friction. Strain gauges can be mounted on shaft within fermenter fromwhich electric signal is picked up through lead wires passing out of fermenter via an axial hole.

Tachometer can be used to control the agitation speed. The rate of rotation ismonitored either by electromagnetic induction or voltage generation or light sensing ormagnetic force. Final choice is made by the type of signal required to record or monitor thesignal. The agitator speed is also controlled by gear box usage, modifying the size of wheels anddrive belts and changing the drive motor.

FOAM SENSING

The appearance of foam is a very undesirable phenomenon, since, in the course of itsappearance, there is a risk to loose an essential part of the fermentation broth. During the foaming, it is not possible to perform high-quality analyses and measurements. For eliminationof foam, 2 methods or their combinations are commonly used:

1. Additional metering of antifoam, based on the information provided by the foamsensor. The given impulses are relatively low, with long pauses and a limited meteringtime. This additional control is necessary to avoid the possible overdose, since, in thiscase, the mass exchange parameters can decrease dramatically.

2. Mechanical metering of foam. For this purpose, an upper drive with a special disk-type or other type of the mechanical foam breaking mixer is installed in thebioreactor's upper cover. If an intensive foaming begins, then the mechanicalbreaking of foam will not help any more.

An optimal solution is the combination of both the parameters. The application ofVariant 1 is more widely used in laboratory bioreactors. Foam formation can be sensed by aprobe which is a stainless steel rod insulated except at tip and set at a defined level. Whenfoam touches the probe tip current passed through with foam as electrolyte and vessel asearth. This current actuates a vessel/pump to release antifoam into fermenter. Process timerare also included which ensures time gap for antifoam mixing in the medium and reducing foambefore next sensing occurs.


pH MONITORING DEVICES

A pH measurement is a determination of the activity of hydrogen ions in an aqueoussolution. Many important properties of a solution can be determined from an accuratemeasurement of pH, including the acidity of a solution and the extent of a reaction in thesolution. Many chemical processes and properties, such as the speed of a reaction and thesolubility of a compound, can also depend greatly on the pH of a solution. In applicationsranging from industrial operations to biological processes, it is important to have an accurateand precise measurement of pH

Most modern pH electrodes consist of a single combination reference and sensing

electrode instead of separate electrodes. This type of pH electrode is much easier to use andless expensive than the electrode pair. A combination electrode is functionally the same as an

electrode pair.


Batch culture is a closed culture system which contains initial, limited amount of

medium. As the growth of microorganism proceeds, the medium availability changes and hence

the organism goes through a number of phases as illustrated next section (2.7).

CONTINUOUS CULTURE

In continuous culture, the exponential growth phase of organism may be prolonged by

the addition of fresh medium to the vessel. The vessel should be designed in such a way that

the added volume displaced an equal volume of culture from the vessel. If medium is fed

continuously to such vessel at a suitable rate, a steady state is achieved eventually. Steady state

is formation of new biomass in the vessel is equivalent to the loss of cells from the vessel. The

medium flow into the vessel is related to the total volume of the medium in the vessel

expressed as dilution rate,


In other techniques, a fermenter variable, eg. turbidity or pH, will be monitored using anappropriate detector and the liquid flow rate will be automatically adjusted so as to maintainthe variable at a constant level. Examples of these types of continuous fermenters are the pH-stat, turbidostat and nutristat. Apart from the pH-stat, these reactors are however rarely usedas the necessary measurement-control systems are generally unreliable over long periods oftime.

A turbidostat is a continuous culturing method where the turbidity of the culture is heldconstant by manipulating the rate at which medium is fed. If the turbidity tends to increase, the

feed rate is increased to dilute the turbidity back to its setpoint. When the turbidity tends tofall, the feed rate is lowered so that growth can restore the turbidity to its set point.

The most widespread large scale application of continuous culture reactors is inwastewater treatment. Activated sludge plants, trickle bed filters, anaerobic digester and pondsall operate in an continuous manner. Cell immobilization is also often employed to improve theefficiency of the process.Continuous cultures are well established in the wastewater industryfor several reasons:

Unlike pure cultre microbial and animal cell systems, contamination is not a

consideration, as the wastewater feed will always contain microorganisms.

Continuous reactors have long been used in waste treatment and their use is not considered a risk.

Finally using batch cultures is simply not economically feasible. Wastewater flows areoften measured in mega litres per hour and batch reactors simply could not cope withthe load.

FED BATCH CULTURE

Two basic approaches to the fed-batch fermentation can be used: the constant volume

fed-batch culture or Fixed Volume Fed-Batch - and the Variable Volume Fed-Batch.

FIXED VOLUME FED-BATCH

In this type of fed-batch, the limiting substrate is fed without diluting the culture. Theculture volume can also be maintained practically constant by feeding the growth limitingsubstrate in undiluted form, for example, as a very concentrated liquid or gas (ex. oxygen).Alternatively, the substrate can be added by dialysis or, in a photosynthetic culture, radiationcan be the growth limiting factor without affecting the culture volume.


A certain type of extended fed-batch - the cyclic fed-batch culture for fixed volumesystems - refers to a periodic withdrawal of a portion of the culture and use of the residualculture as the starting point for a further fed-batch process. Basically, once the fermentationreaches a certain stage, (for example, when aerobic conditions cannot be maintained anymore)the culture is removed and the biomass is diluted to the original volume with sterile water ormedium containing the feed substrate. The dilution decreases the biomass concentration andresult in an increase in the specific growth rate. Subsequently, as feeding continues, the growthrate will decline gradually as biomass increases and approaches the maximum sustainable inthe vessel once more, at which point the culture may be diluted again.

ADVANTAGES AND DISADVANTAGES OF THE FED-BATCH REACTORS

Fed-batch fermentation is a production technique in between batch and continuous

fermentation. A proper feed rate, with the right component constitution is required during the

process.

Fed-batch offers many advantages over batch and continuous cultures. From theconcept of its implementation it can be easily concluded that under controllable conditions andwith the required knowledge of the microorganism involved in the fermentation, the feed ofthe required components for growth and/or other substrates required for the production of theproduct can never be depleted and the nutritional environment can be maintainedapproximately constant during the course of the batch. The production of by-products that aregenerally related to the presence of high concentrations of substrate can also be avoided bylimiting its quantity to the amounts that are required solely for the production of thebiochemical. When high concentrations of substrate are present, the cells get "overloaded",this is, the oxidative capacity of the cells is exceeded, and due to the Crabtree effect, productsother than the one of interest are produced, reducing the efficacy of the carbon flux. Moreover,these by-products prove to even "contaminate" the product of interest, such as ethanolproduction in baker's yeast production, and to impair the cell growth reducing the fermentationtime and its related productivity.

Sometimes, controlling the substrate is also important due to catabolic repression. Sincethis method usually permits the extension of the operating time, high cell concentrations canbe achieved and thereby, improved productivity [mass of product/(volume.time)]. This aspect isgreatly favored in the production of growth-associated products. Additionally, this methodallows the replacement of water loss by evaporation and decrease of the viscosity of the brothsuch as in the production of dextran and xanthan gum, by addition of a water-based feed. Aspreviously mentioned, fed-batch might be the only option for fermentations dealing with toxicor low solubility substrates.

When dealing with recombinant strains, fed-batch mode can guarantee the presence ofan antibiotic throughout the course of the fermentation, with the intent of keeping thepresence of an antibiotic-marked plasmid. Since the growth can be regulated by the feed, andknowing that in many cases a high growth rate can decrease the expression of encodedproducts in recombinant products, the possibility of having different feeds and feed modesmakes fed-batch an extremely flexible tool for control in these cases. Because the feed can alsobe multisubstrate, the fermentation environment can still be provided with required protease


inhibitors that might degrade the product of interest, metabolites and precursors that increasethe productivity of the fermentation.

Finally, in a fed-batch fermentation, no special piece of equipment is required inaddition to that one required by a batch fermentation, even considering the operatingprocedures for sterilization and the preventing of contamination. A cyclic fed-batch culture hasan additional advantage: the productive phase of a process may be extended under controlledconditions. The controlled periodic shifts in growth rate provide an opportunity to optimizeproduct synthesis, particularly if the product of interest is a secondary metabolite whosemaximum production takes place during the deceleration in growth.

MICROBIAL KINETICS

Growth is an increase in cellular constituents that may result in an increase in cell size,an increase in cell number, or both. When a microbiologist speaks of microbial growth it isusually increase in cell number that he/she is after. Consequently, there is a tendency formicrobiologists to follow microbial growth as populations rather than following the growth ofindividual cells. Microbiologists tend to be more interested in population sizes than the size(mass) of any individual cell. Typical measurement of microbial growth will be done over thespan of more than one microbial generation. An increase in cell number is an immediateconsequence of cell division. Increase in cell numbers occurs when microorganisms reproduceby a process like budding or binary fission. Budding is a form of reproduction in which a newcell is formed as an outgrowth from the parent cell, as in the case of yeast and some bacteria.The majority of bacteria reproduce by a mechanism termed binary fission.


Introduction To IBT B.Sc 6th SEM KLESNC

GROWTH CURVE

The population growth is studied by analyzing the growth curve of a microbial culture. Thestandard bacterial growth curve describes various stages of growth a pure culture of bacteria will gothrough, beginning with the addition of cells to sterile media and ending with the death of all of the cellspresent. The standard bacterial growth curve describes various stages of growth a pure culture ofbacteria will go through, beginning with the addition of cells to sterile media and ending with the deathof all of the cells present

Bacteria added to fresh media typically go through four more-or-less distinct phases of growth:

lag, exponential, stationary, and death.

Lag phase: The period of apparent inactivity in which the cells are adapting to a new environment andpreparing for reproductive growth. Cells are usually synthesizing new components. In practice, bacteriafrom one medium to another, where there are chemical differences between the two media, typicallyresults in a lag in cell division. This lag in division is associated with a physiological adaptation to thenew environment. Cells may increase in size during this time, but simply do not divide (by binaryfission). Lag phase varies considerably in length depending upon the condition of the microorganismsand the nature of the medium.

Log (exponential) phase: The period in which the organisms are growing at the maximal rate possiblegiven their genetic potential, the nature of the medium, and the conditions under which they aregrowing. Cells in optimum growth state, divide repeatedly by binary fission at maximal rate; thepopulation doubles in every generation time. Generation time, also called Doubling time, is the time ittakes a bacterium to do one binary fission starting from having just divided. Generation times varymarkedly with the species of microorganism and environmental conditions; they can range from 10minutes for a few bacteria to several days with some eukaryotic microorganisms. The population is mostuniform in terms of chemical and physical properties during this period.

Stationary phase: Eventually population growth ceases, and the growth curve becomes horizontal.

Increase in cell number due to cell divisions exactly balanced by a decrease in cell number due to

death. Cell death may result from Nutrient limitation & Toxic waste

HOD Dept of Biotechnology KLE SNC Page 40


DOWNSTREAM PROCESSING

INTRODUCTION

Industrial fermentations comprise both upstream (USP) and downstream processing (DSP) stages USPinvolves all factors and processes leading to and including the fermentation and consists of three mainareas: the producer organism, the medium and the fermentation process. DSP encompasses all processesfollowing the fermentation. In most cases this means recovery of a product from a dilute aqueoussolution.

The complexity of DSP is determined by the required purity of the product which is in turn determined byits application. The products of biotechnology include whole cells, organic acids, amino acids, solvents,antibiotics, industrial enzymes, therapeutic proteins, vaccines, gums etc.

The primary objective in industrial fermentation processes is to recover the product efficiently,reproducibly and safely to its required specification, while achieving maximum product yield at minimumrecovery costs. Fermentation factors affecting DSP include the properties of the microorganisms(particularly morphology, flocculation characteristics, size & cell wall rigidity).

These factors impact filterability, sedimentation and homogenization efficiency. The presence offermentation by-products, media impurities & additives like antifoam may interfere with DSP steps.Therefore, a holistic approach is required when developing a new industrial purification strategy.

The whole process, both upstream & downstream factors need to be considered, e.g., a cheap carbon andenergy source containing many impurities may provide initial cost savings but may necessitate increasedDSP costs. Hence overall cost savings may be achieved with a more expensive but purer substrate.

The five stages are: (1) Solid-Liquid Separation

(2) Release of Intracellular Products

(3) Concentration

(4) Purification by Chromatography and

(5) Formulation.






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Stage 1. Solid-Liquid Separation:

The first step in product recovery is the separation of whole cells (cell biomass) and other insolubleingredients from the culture broth (Note: If the desired product is an intracellular metabolite, it must bereleased from the cells before subjecting to solid-liquid separation). Some authors use the term harvestingof microbial cells for the separation of cells from the culture medium. Several methods are in use forsolid-liquid separation. These include flotation, flocculation, filtration and centrifugation.

Flotation:

When a gas is introduced into the liquid broth, it forms bubbles. The cells and other solid particles getadsorbed on gas bubbles. These bubbles rise to the foam layer which can be collected and removed. Thepresence of certain substances, referred to as collector substances, facilitates stable foam formation e.g.,long chain fatty acids, amines.

Flocculation:

In flocculation, the cells (or cell debris) form large aggregates to settle down for easy removal. Theprocess of flocculation depends on the nature of cells and the ionic constituents of the medium. Additionof flocculating agents (inorganic salt, organic polyelectrolyte, mineral hydrocolloid) is often necessary toachieve appropriate flocculation.

Filtration:

Filtration is the most commonly used technique for separating the biomass and culture filtrate. Theefficiency of filtration depends on many factors— the size of the organism, presence of other organisms,viscosity of the medium, and temperature. Several filters such as depth filters, absolute filters, rotarydrum vacuum filters and membrane filters are in use.

Depth Filters:

They are composed of a filamentous matrix such as glass wool, asbestos or filter paper. The particles aretrapped within the matrix and the fluid passes out. Filamentous fungi can be removed by using depthfilters.

Absolute Filters:

These filters are with specific pore sizes that are smaller than the particles to be removed. Bacteria from

culture medium can be removed by absolute filters.

Rotary Drum Vacuum Filters:

These filters are frequently used for separation of broth containing 10-40% solids (by volume) and

particles in the size of 0.5-10µm. Rotary drum vacuum filters have been successfully used for filtration of

yeast cells and filamentous fungi. The equipment is simple with low power consumption and is easy to

operate. The filtration unit consists of a rotating drum partially immersed in a tank of broth (Fig. 20.2). As

the drum rotates, it picks up the biomass which gets deposited as a cake on the drum surface. This filter

cake can be easily removed.



Membrane Filters:

In this type of filtration, membranes with specific pore sizes can be used. However, clogging of filters is a

major limitation. There are two types of membrane filtrations—static filtration and cross-flow filtration

(Fig. 20.3). In cross-flow filtration, the culture broth is pumped in a crosswise fashion across the

membrane. This reduces the clogging process and hence better than the static filtration.

Types of filtration processes:

There are 3 major types of filtrations based on the particle sizes and other characters (Table 20.1). These

are microfiltration, ultrafiltration and reverse osmosis.

Centrifugation:


http://cdn.biologydiscussion.com/wp-content/uploads/2015/09/clip_image00425.jpg




The technique of centrifugation is based on the principle of density differences between the particles to be

separated and the medium. Thus, centrifugation is mostly used for separating solid particles from liquid

phase (fluid/particle separation). Unlike the centrifugation that is conveniently carried out in the

laboratory scale, there are certain limitations for large scale industrial centrifugation.

However, in recent years, continuous flow industrial centrifuges have been developed. There is a

continuous feeding of the slurry and collection of clarified fluid, while the solids deposited can be

removed intermittently. The different types of centrifuges are depicted in Fig. 20.4, and briefly described

hereunder. T

ubular bowl centrifuge (Fig. 20.4A):

This is a simple and a small centrifuge, commonly used in pilot plants. Tubular bowl centrifuge can be

operated at a high centrifugal speed, and can be run in both batch or continuous mode. The solids are

removed manually.

Disc centrifuge (Fig. 20.4B):

It consists of several discs that separate the bowl into settling zones. The feed/slurry is fed through a

central tube. The clarified fluid moves upwards while the solids settle at the lower surface.

Multi-chamber centrifuge (Fig. 20.4C):

This is basically a modification of tubular bowl type of centrifuge. It consists of several chambers

connected in such a way that the feed flows in a zigzag fashion. There is a variation in the centrifugal

force in different chambers. The force is much higher in the periphery chambers, as a result smallest

particles settle down in the outermost chamber.

Scroll centrifuge or decanter (Fig. 20.4D):




It is composed of a rotating horizontal bowl tapered at one end. The decanter is generally used to

concentrate fluids with high solid concentration (biomass content 5-80%). The solids are deposited on the

wall of the bowl which can be scrapped and removed from the narrow end.

Stage 2. Release of Intracellular Products: As already stated, there are several biotechnological products (vitamins, enzymes) which are located

within the cells. Such compounds have to be first released (maximally and in an active form) for their

further processing and final isolation. The microorganisms or other cells can be disintegrated or disrupted

by physical, chemical or enzymatic methods. The outline of different techniques used for breakage of

cells is given in Fig. 20.5.

The selection of a particular method depends on the nature of the cells, since there is a wide variation in

the property of cell disruption or breakage. For instance, Gram-negative bacteria and filamentous fungi

can be more easily broken compared to Gram-positive bacteria arid yeasts.

Cell Disruption:

Physical methods of cell disruption:

The microorganisms or cells can be disrupted by certain physical methods to release the intracellular

products.

Ultra sonication:

Ultrasonic disintegration is widely employed in the laboratory. However, due to high cost, it is not

suitable for large-scale use in industries.

Osmotic shock:




This method involves the suspension of cells (free from growth medium) in 20% buffered sucrose. The

cells are then transferred to water at about 4°C. Osmotic shock is used for the release of hydrolytic

enzymes and binding proteins from Gram-negative bacteria.

Heat shock (thermolysis):

Breakage of cells by subjecting them to heat is relatively easy and cheap. But this technique can be used

only for a very few heat-stable intracellular products.

High pressure homogenization:

This technique involves forcing of cell suspension at high pressure through a very narrow orifice to come

out to atmospheric pressure. This sudden release of high pressure creates a liquid shear that can break the

cells.

Impingement:

In this procedure, a stream of suspended cells at high velocity and pressure are forced to hit either a

stationary surface or a second stream of suspended cells (impinge literally means to strike or hit). The

cells are disrupted by the forces created at the point of contact. Micro fluidizer is a device developed

based on the principle of impingement. It has been successfully used for breaking E. coli cells. The

advantage with impingement technique is that it can be effectively used for disrupting cells even at a low

concentration.

Grinding with glass beads:

The cells mixed with glass beads are subjected to a very high speed in a reaction vessel. The cells break

as they are forced against the wall of the vessel by the beads. Several factors influence the cell breakage-

size and quantity of the glass beads, concentration and age of cells, temperature and agitator speed. Under

optimal conditions, one can expect a maximal breakage of about 80% of the cells.

A diagrammatic representation of a cell disrupter employing glass beeds is shown in Fig. 20.6. It contains

a cylindrical body with an inlet, outlet and a central motor-driven shaft. To this shaft are fitted radial

agitators. The cylinder is fitted with glass beads. The cell suspension is added through the inlet and the

disrupted cells come out through the outlet. The body of the cell disrupter is kept cool while the operation

is on.




Mechanical and non-mechanical methods:

Among the physical methods of cell disruption described above, ultra sonication, high-pressure

homogenization, impingement and grinding with glass beads are mechanical while osmotic shock and

heat shock are non-mechanical. The chemical and enzymatic methods (described below) are non-

mechanical in nature.

Chemical methods of cell disruption:

Treatment with alkalies, organic solvents and detergents can lyse the cells to release the contents.

Alkalies:

Alkali treatment has been used for the extraction of some bacterial proteins. However, the alkali stability

of the desired product is very crucial for the success of this method e.g., recombinant growth hormone

can be efficiently released from E. coli by treatment with sodium hydroxide at pH 11.

Organic solvents:

Several water miscible organic solvents can be used to disrupt the cells e.g., methanol, ethanol,

isopropanol, butanol. These compounds are inflammable; hence require specialised equipment for fire

safety. The organic solvent toluene is frequently used. It is believed that toluene dissolves membrane

phospholipids and creates membrane pores for release of intracellular contents.

Detergents:

Detergents that are ionic in nature, cationic-cetyl trimethyl ammonium bromide or anionic-sodium lauryl

sulfate can denature membrane proteins and lyse the cells. Non-ionic detergents (although less reactive

than ionic ones) are also used to some extent e.g., Triton X-100 or Tween. The problem with the use of

detergents is that they affect purification steps, particularly the salt precipitation. This limitation can be

overcome by using ultrafiltration or ion-exchange chromatography for purification.

Enzymatic methods of cell disruption:

Cell disruption by enzymatic methods has certain advantages i.e., lysis of cells occurs under mild

conditions in a selective manner. This is quite advantageous for product recovery. Lysozyme is the most

frequently used enzyme and is commercially available (produced from hen egg white). It hydrolyses β-1,

4-glycosidic bonds of the mucopeptide in bacterial cell walls. The Gram- positive bacteria (with high

content of cell wall mucopeptides) are more susceptible for the action of lysozyme.

For Gram-negative bacteria, lysozyme in association with EDTA can break the cells. As the cell wall gets

digested by lysozyme, the osmotic effects break the periplasmic membrane to release the intracellular

contents. Certain other enzymes are also used, although less frequently, for cell disruption. For the lysis

of yeast cell walls, glucanase and mannanase in combination with proteases are used.

Combination of methods:



In order to increase the efficiency of cell disintegration in a cost-effective manner, a combination of

physical, chemical and enzymatic methods is employed.

Stage 3. Concentration: The filtrate that is free from suspended particles (cells, cell debris etc.) usually contains 80-98% of water.

The desired product is a very minor constituent. The water has to be removed to achieve the product

concentration. The commonly used techniques for concentrating biological products are evaporation,

liquid-liquid extraction, membrane filtration, precipitation and adsorption. The actual procedure adopted

depends on the nature of the desired product (quality and quantity to be retained as far as possible) and

the cost factor.

Evaporation:

Water in the broth filtrate can be removed by a simple evaporation process. The evaporators, in general,

have a heating device for supply of steam, and unit for the separation of concentrated product and vapour,

a condenser for condensing vapour, accessories and control equipment. The capacity of the equipment is

variable that may range from small laboratory scale to industrial scale. Some of the important types of

evaporators in common use are briefly described.

Plate evaporators:

The liquid to be concentrated flows over plates. As the steam is supplied, the liquid gets concentrated and

becomes viscous.

Falling film evaporators:

In this case, the liquid flows down long tubes which gets distributed as a thin film over the heating

surface. Falling film evaporators are suitable for removing water from viscous products of fermentation.

Forced film evaporators:

The liquid films are mechanically driven and these devices are suitable for producing dry product

concentrates.

Centrifugal forced film evaporators:

These equipment evaporate the liquid very quickly (in seconds), hence suitable for concentrating even

heat-labile substances. In these evaporators, a centrifugal force is used to pass on the liquid over heated

plates or conical surfaces for instantaneous evaporation.

Liquid-Liquid Extraction:

The concentration of biological products can be achieved by transferring the desired product (solute) from

one liquid phase to another liquid phase, a phenomenon referred to as liquid-liquid extraction. Besides

concentration, this technique is also useful for partial purification of a product.



The efficiency of extraction is dependent on the partition coefficient i.e. the relative distribution of a

substance between the two liquid phases. The process of liquid-liquid extraction may be broadly

categorized as extraction of low molecular weight products and extraction of high molecular weight

products.

Extraction of low molecular weight products:

By using organic solvents, the lipophilic compounds can be conveniently extracted. However, it is quite

difficult to extract hydrophilic compounds. Extraction of lipophilic products can be done by the following

techniques.

Physical extraction:

The compound gets itself distributed between two liquid phases based on the physical properties. This

technique is used for extraction of non-ionising compounds.

Dissociation extraction:

This technique is suitable for the extraction of ionisable compounds. Certain antibiotics can be extracted

by this procedure.

Reactive extraction:

In this case, the desired product is made to react with a carrier molecule (e.g., phosphorus compound,

aliphatic amine) and extracted into organic solvent. Reactive extraction procedure is quite useful for the

extraction of certain compounds that are highly soluble in water (aqueous phase) e.g., organic acids.

Supercritical fluid (SCF) extraction:

This technique differs from the above procedures, since the materials used for extraction are supercritical

fluids (SCFs). SCFs are intermediates between gases and liquids and exist as fluids above their critical

temperature and pressure. Supercritical CO2, with a low critical temperature and pressure is commonly

used in the extraction. Supercritical fluid extraction is rather expensive, hence not widely used (SCF has

been used for the extraction of caffeine from coffee beans, and pigments and flavor ingredients from

biological materials).

Extraction of high molecular weight compounds:

Proteins are the most predominant high molecular weight products produced in fermentation industries.

Organic solvents cannot be used for protein extraction, as they lose their biological activities. They are

extracted by using an aqueous two-phase systems or reverse micelles formation.

Aqueous two-phase systems (ATPS):

They can be prepared by mixing a polymer (e.g., polyethylene glycol) and a salt solution (ammonium

sulfate) or two different polymers. Water is the main component in ATPS, but the two phases are not

miscible. Cells and other solids remain in one phase while the proteins are transferred to other phase. The



distribution of the desired product is based on its surface and ionic character and the nature of phases. The

separation takes much longer time by ATPS.

Reverse miceller systems:

Reverse micelles are stable aggregates of surfactant molecules and water in organic solvents. The proteins

can be extracted from the aqueous medium by forming reverse micelles. In fact, the enzymes can be

extracted by this procedure without loss of biological activity.

Membrane Filtration:

Membrane filtration has become a common separation technique in industrial biotechnology. It can be

conveniently used for the separation of biomolecules and particles, and for the concentration of fluids.

The membrane filtration technique basically involves the use of a semipermeable membrane that

selectively retains the particles/molecules that are bigger than the pore size while the smaller molecules

pass through the membrane pores.

Membranes used in filtration are made up of polymeric materials such as polyethersulfone and polyvinyl

di-fluoride. It is rather difficult to sterilize membrane filters. In recent years, micro-filters and ultrafiIters

composed of ceramics and steel are available. Cleaning and sterilization of such filters are easy. The other

types of membrane filtration techniques are described briefly.

Membrane adsorbers:

They are micro- or macro porous membranes with ion exchange groups and/or affinity ligands.

Membrane adsorbers can bind to proteins and retain them. Such proteins can be eluted by employing

solutions in chromatography.

Pervaporation:

This is a technique in which volatile products can be separated by a process of permeation through a

membrane coupled with evaporation. Pervaporation is quite useful for the extraction, recovery and

concentration of volatile products. However, this procedure has a limitation since it cannot be used for

large scale separation of volatile products due to cost factor.

Perstraction:

This is an advanced technique working on the principle of membrane filtration coupled with solvent

extraction. The hydrophobic compounds can be recovered/ concentrated by this method.

Precipitation:

Precipitation is the most commonly used technique in industry for the concentration of macromolecules

such as proteins and polysaccharides. Further, precipitation technique can also be employed for the

removal of certain unwanted byproducts e.g. nucleic acids, pigments.



Neutral salts, organic solvents, high molecular weight polymers (ionic or non-ionic), besides alteration in

temperature and pH are used in precipitation. In addition to these non-specific protein precipitation

reactions (i.e. the nature of the protein is unimportant), there are some protein specific precipitations e.g.,

affinity precipitation, ligand precipitation.

Neutral salts:

The most commonly used salt is ammonium sulfate, since it is highly soluble, nontoxic to proteins and

low-priced. Ammonium sulfate increases hydrophobic interactions between protein molecules that result

in their precipitation. The precipitation of proteins is dependent on several factors such as protein

concentration, pH and temperature.

Organic solvents:

Ethanol, acetone and propanol are the commonly used organic solvents for protein precipitation. They

reduce the dielectric constant of the medium and enhance electrostatic interaction between protein

molecules that lead to precipitation. Since proteins are denatured by organic solvents, the precipitation

process has to be carried out below 0°C.

Non-ionic polymers:

Polyethylene glycol (PEG) is a high molecular weight non-ionic polymer that can precipitate proteins. It

reduces the quantity of water available for protein solvation and precipitates protein. PEG does not

denature proteins, besides being non-toxic.

Ionic polymers:

The charged polymers such as polyacrylic acid and polyethyleneimine are used. They form complexes

with oppositely charged protein molecules that causes charge neutralisation and precepitation.

Increase in temperature:

The heat sensitive proteins can be precipitated by increasing the temperature.

Change in pH:

Alterations in pH can also lead to protein precipitation.

Affinity precipitation:

The affinity interaction (e.g., between antigen and antibody) is exploited for precipitation of proteins.

Precipitation by ligands:

Ligands with specific binding sites for proteins have been successfully used for selective precipitation.

Adsorption:



The biological products of fermentation can be concentrated by using solid adsorbent particles. In the

early days, activated charcoal was used as the adsorbent material. In recent years, cellulose-based

adsorbents are employed for protein concentration.

And for concentration of low molecular weight compounds (vitamins, antibiotics, peptides) polystyrene,

methacrylate and acrylate based matrices are used. The process of adsorption can be carried out by

making a bed of adsorbent column and passing the culture broth through it. The desired product, held by

the adsorbent, can be eluted.

Stage 4. Purification by Chromatography: The biological products of fermentation (proteins, pharmaceuticals, diagnostic compounds and research

materials) are very effectively purified by chromatography. It is basically an analytical technique dealing

with the separation of closely related compounds from a mixture. Chromatography usually consists of a

stationary phase and mobile phase.

The stationary phase is the porous solid matrix packed in a column (equilibrated with a suitable solvent)

on to which the mixture of compounds to be separated is loaded. The compounds are eluted by a mobile

phase.

A single mobile phase may be used continuously or it may be changed appropriately to facilitate the

release of desired compounds. The eluate from the column can be monitored continuously (e.g. protein

elution can be monitored by ultraviolet adsorption at 280 nm), and collected in fractions of definite

volumes.

The different types of chromatography techniques used for separation (mainly proteins) along with the

principles are given in Table 20.2. A large number of matrices are commercially available for purification

of proteins e.g., agarose, cellulose, polyacrylamide, porous silica, cross- linked dextran, polystyrene.

Some of the important features of selected chromatographic techniques are briefly described.



Gel-filtration chromatography:This is also referred to as size-exclusion chromatography. In this

technique, the separation of molecules is based on the size, shape and molecular weight. The sponge-like

gel beads with pores serve as molecular sieves for separation of smaller and bigger molecules. A solution

mixture containing molecules of different sizes (e.g. different proteins) is applied to the column and

eluted.

The smaller molecules enter the gel beads through their pores and get trapped. On the other hand, the

larger molecules cannot pass through the pores and therefore come out first with the mobile liquid (Fig.

20.7). At the industrial scale, gel-filtration is particularly useful to remove salts and low molecular weight

compounds from high molecular weight products.

Ion-exchange chromatography:

It involves the separation of molecules based on their surface charges. Ion-exchangers are of two types(cation- exchangers which have negatively charged groups like carboxymethyl and sulfonate, and anion-exchangers with positively charged groups like diethylaminoethyl (DEAE). The most commonly usedcation-exchangers are Dowex HCR and Amberlite IR, the anion-exchangers are Dowex SAR andAmberlite IRA.

In ion-exchange chromatography, the pH of the medium is very crucial, since the net charge varies withpH. In other words, the pH determines the effective charge on both the target molecule and the ion-exchanger. The ionic bound molecules can be eluted from the matrix by changing the pH of the eluant or





by increasing the concentration of salt solution. Ion-exchange chromatography is useful for thepurification of antibiotics, besides the purification of proteins.

Affinity chromatography:

This is an elegant method for the purification of proteins from a complex mixture. Affinitychromatography is based on an interaction of a protein with an immobilized ligand. The ligand can be aspecific antibody, substrate, substrate analogue or an inhibitor. The immobilized ligand on a solid matrixcan be effectively used to fish out complementary structures.

In Table 20.3, some examples of ligands used for the purification of proteins are given. The protein boundto the ligand can be eluted by reducing their interaction. This can be achieved by changing the pH of thebuffer, altering the ionic strength or by using another free ligand molecule. The fresh ligand used has tobe removed in the subsequent steps.

Hydrophobic interaction chromatography (HIC):

This is based on the principle of weak hydrophobic interactions between the hydrophobic ligands (alkyl,aryl side chains on matrix) and hydrophobic amino acids of proteins. The differences in the compositionof hydrophobic amino acids in proteins can be used for their separation. The elution of proteins can bedone by lowering the salt concentration, decreasing the polarity of the medium or reducing thetemperature.

Stage 5. Formulation:

Formulation broadly refers to the maintenance of activity and stability of a biotechnological productsduring storage and distribution. The formulation of low molecular weight products (solvents, organicacids) can be achieved by concentrating them with removal of most of the water. For certain smallmolecules, (antibiotics, citric acid), formulation can be done by crystallization by adding salts.

Proteins are highly susceptible for loss of biological activity; hence their formulation requires specialcare. Certain stabilizing additives are added to prolong the shelf life of protein. The stabilizers of proteinformulation include sugars (sucrose, lactose), salts (sodium chloride, ammonium sulfate), polymers(polyethylene glycol) and polyhydric alcohols (glycerol). Proteins may be formulated in the form ofsolutions, suspensions or dry powders.




Drying:

Drying is an essential component of product formulation. It basically involves the transfer of heat to a wetproduct for removal of moisture. Most of the biological products of fermentation are sensitive to heat, andtherefore require gentle drying methods. Based on the method of heat transfer, drying devices may becategorized as contact, convection, radiation dryers. These three types of dryers are commerciallyavailable.

Spray drying:

Spray drying is used for drying large volumes of liquids. In spray drying, small droplets of liquidcontaining the product are passed through a nozzle directing it over a stream of hot gas. The waterevaporates and the solid particles are left behind.

Freeze-drying:

Freeze-drying or lyophilization is the most preferred method for drying and formulation of a wide-rangeof products—pharmaceuticals, foodstuffs, diagnostics, bacteria, viruses. This is mainly because freeze-drying usually does not cause loss of biological activity of the desired product.

Lyophilization is based on the principle of sublimation of a liquid from a frozen state. In the actualtechnique, the liquid containing the product is frozen and then dried in a freeze-dryer under vacuum. Thevacuum can now be released and the product containing vials can be sealed e.g., penicillin can be freezedried directly in ampules.

Integration of Different Processes:

It is ideal to integrate the fermentation and downstream processing to finally get the desired product.However, this has not been practicable for various reasons. Integration of certain stages in downstreamprocessing for purification of product has met with some success. For instance, protein concentration byextraction into two phase systems combined with clarification and purification can be done together.



FERMENTED FOODS

Introduction

Fermentation is an anaerobic oxidation of carbohydrates and carbohydrate like substances by the action of enzymes from microbes.

It is an energy yielding process The fermentation process is aided by the species of Lactobacillus,

Erwinia,Leuconostoc,Streptococcus,Penicillium. It is estimated that about 20-30% of the household budget is spent towards foods in the developed

countries. This may be a little less in the developing nations. Therefore, food and beverage biotechnology

occupies a prominent place world- over. There are records that man was making bread, wine, curd etc., as early as 4000 BC. These

processes, collectively referred to as traditional or old biotechnology With the advances made in microbiology and recently biotechnology, food and beverage

production is a major industry. Food biotechnology is also concerned with the improved quality, nutrition, consistency, colour,

safety and preservation of foods, besides making them available round the year In addition, modern biotechnological processes also take into account the health aspects of the

people. The production of fermented foods is variable. This depends on geographical region, availability

of raw materials, traditions and food habits of the people. A selected list of fermented foods along with raw materials and fermenting microorganisms is

given in Table



Advantages of Fermented Foods:

i. Enhanced nutritive value.

ii. Increased digestibility.

iii. Improved flavor and texture.

iv. Serve as supplements in preparing several dishes.

Fermented Dairy products: The fermented dairy products are produced by the action of Lactic acid bacteria that are naturally

present in milk . Normally the pasteurized milk is inoculated with starter culture during fermentation. The use of pasteurized milk reduces the growth of pathogen as well as suppresses the action of certain

enzymes in milk that hinder the fermentation process . The basic principle behind the production of various fermented dairy products is very similar.

Cheese:

Cheese production is the largest dairy industry in the world. There are around 1,000 types of different

cheeses.

They are broadly of two types — un-ripened cheeses (cottage cheese with low fat, cream cheese with

high fat) and ripened cheeses (hard cheese e.g. cheddar, blue cheese; soft cheese e.g. limburger,

camembert).



Irrespective of the type of the cheese, all of them are invariably made from the casein of milk that is

produced after separating the whey (liquid portion of milk). Milk from different animals can be used

e.g. sheep, cow, goat, buffalo.

Historical perspective:

The use of animal stomachs for carrying liquids is centuries old. When milk was transported in this

fashion, the formation of solids (that were tasty) was observed. The solids were concentrated after

draining liquids. These solids were salted and consumed later. A good example of food preservation, long

ago! We now know that this solid portion is the cheese. It is produced by the combined action of enzymes

(rennet) of the stomach living and the bacterial contamination.

Production process:

As already stated, cheese is produced from milk. This is carried out by a process of dehydration wherein

casein (milk protein) and fats are concentrated 5-15 fold. Cheese production is very complicated, and

broadly involves four stages- acidification of milk, coagulum formation, and separation of curd from

whey and ripening of cheese.

1. Acidification of milk:

By employing lactic acid bacteria (Streptococcus lactis, Lactobacillus lactis) the sugar of milk (lactose)

can be converted to lactic acid. This lowers the ph to around 4.6, and thus acidifies milk.

2. Coagulum formation:

When the acidified milk is treated with rennet (i.e. the enzyme chymosin of animal or fungal origin),

casein gets coagulated. Casein mainly consists of three components-insoluble α and β caseins and a k-

casein that keeps them in soluble state. By the action of chymosin, k-casein is degraded. Consequently, α

and β caseins and the degraded products of k casein combine to form a coagulum (curd) . This process of

coagulation is dependent on calcium ions.

3. Separation of curd from whey:

When the temperature of the coagulum is raised to around 40°C; the coagulum (curd) and whey (fluid

portion) get separated. The separated curd is cut into blocks, drained and pressed into different shapes.

4. Ripening of cheese:

The flavour of raw cheese (with rubber texture) such as cheddar is bland. Ripening imparts flavours,

besides making changes in its texture. The procedures adopted for ripening (or maturation) are highly

variable depending on the type of cheese to be prepared. The blocks of curd separated are subjected to the

action of proteases and/or lipases.



Alternatively, they may be inoculated with certain fungi (e.g. Penicillium roquefortii). The hydrolysis of

proteins and fats (either by enzymes or microorganisms) results in certain compounds which imparts

flavour to the cheese. Mild hydrolysis of fats (or cheese), usually carried out by lipases or Aspergillus

nigeror Mucor maihai results in butyric acid formation with characteristic flavour.

A diagrammatic representation of cheese production is depicted in

YOGURT:

Yoghurts are the traditional sour milk preparations that have become major dairy products.

Whole low fat and skim milks and even cream may be used to make yoghurt.

Yoghurt is produced by fermenting whole milk by employing a mixed culture of Lactobacillus

bulgaricus and Streptococcus thermophilus in 1:1 ratio

The milk is boile and cooled to prior inoculation .This is called as milk premix

This is then incubated with starter culture at 42C

The Lactobacillus bulgaricus produces mailnly acids ,where as the Streptococcus thermophiles

produces the flovoring agent like acetaldehyde.

Streptococcus thermophiles grows faster than Lactobacillus bulgaricus.The associate growth of two

organisms results in acid production at a rate greater than that produced them individually

The fermentation is allowed to continue for $ hours untll the desired flavor ,acidity and consistency

are attained.

A sharp tangy flavor is characteristic of Yoghurt is marked with sweetners like sucrose ,honey corn

sweetner ,lactose etc

Yoghurt is very delicious and in fact frozen yoghurt is becoming popular as an alternative to ice

cream.

BUTTER MILK: Buttermilk is the phospholipid rich liquid remaining after the cream is churned to produce butter. It is commonly produced by inoculating pasteurized skim milk with a selected starter culture. The liquid butter milk is a cultured milk. The fermentation is continued until the desired aroma ,acidity and flavor have been developed.



In the process of manufacturing, starter culture of Streptococcus lactis or Streptococcus cremoris isadded to the milk to produce acids(Lactic acid).

This is followed by addition of cultures of Leuconostoc citrovorum and L. dextranicum thet impartscharacteristics aroma and flavor.

The product is incubated at 20C untill the pH drops to 4.6 Further the flavor is enhanced by the addition of citric acid ,salt or butter granules. The actual process of manufacture of culture butter milk involves basically three

steps:Pasteurization,Homogenization,CulturingThe cultured butter milk can be stored for 20-28 daysunder refrigeration .

IDLI-

Idli is a fermented food of India which is prepared by steaming a fermented blackgram (Phaseolus mungo

L.) and rice (Oryza sativa L.) batter. It makes an important contribution to the diet as a source of protein,

calories and vitamins, especially B-complex vitamins, compared to the raw unfermented ingredients. It

can be produced locally and used as a dietary supplement in developing countries to treat people suffering

from protein calorie malnutrition and kwashiorkor. Other legumes such as soybeans and Great Northern

beans could be substituted for black gram in preparation of a idli. Further research is needed regarding the

increase of methionine content during idli fermentation, by which pathway methionine is synthesized, and

identification and isolation of microorganisms responsible for methionine production or synthesis.

TEMPEH- Tem

peh or tempe isa

traditional Indonesian soy product, that is made from fermented soybeans.


https://en.wikipedia.org/wiki/Indonesian_cuisine

https://en.wikipedia.org/wiki/Fermentation_(food)


It is made by a natural culturing and controlled fermentation process that binds soybeans into acake form.

A special fungus is used, which has the Latin name Rhizopus oligosporus, usually marketed underthe name tempeh starter.

Tempeh begins with whole soybeans, which are softened by soaking, and dehulled, then partlycooked. Specialty tempeh may be made from other types of beans, wheat, or may include amixture of beans and whole grains.

The principal step in making tempeh is the fermentation of soybeans which undergo inoculationwith Rhizopus spp. molds, a type of filamentous fungus most widely used for the production oftempeh A fermentation starter containing the spores of fungus Rhizopus oligosporus or Rhizopusoryzae is mixed in The beans are spread into a thin layer and are allowed to ferment for 24 to 36hours at a temperature around 30°C (86°F). The soybeans have to cool down to allow sporegermination and abundant growth of mycelium. Later, the temperature of the beans will naturallyrise and rapid mold growth happens for around 4 hours. As mold growth declines, the soybeansshould be bound into a solid mass by the mycelium. In good tempeh, the beans are knittedtogether by a mat of white mycelium. Typically, tempeh is harvested after 48 hours offermentation with its distinguishable whitish color, firm texture, and nutty flavor. Extendedfermentation time results in an increase in pH and undesirable color darkening in the tempeh.

During the fermentation process, optimal time of fermentation, temperature, oxygen, humidity,and pH levels are required to encourage the growth of the Rhizopus mold, while discouraging thegrowth of undesired microorganisms. The pH level should be kept around 3-5 by adding amild acidulant such as vinegar, lactic acid, or acetic acid, thereby favoring mold growth andrestricting the growth of spoilage microorganisms. Oxygen is required for Rhizopus spp. growth,but should be maintained at low levels to prevent the production of undesired microorganisms.Under conditions of lower temperature, or higher ventilation, gray or black patches of spores mayform on the surface—this is not harmful, and should not affect the flavor or quality of the tempeh.This sporulation is normal on fully mature tempeh. A mild ammonia smell may accompany goodtempeh as it ferments, but it should not be overpowering.

Traditional tempeh is often produced in Indonesia using Hibiscus tiliaceus leaves. The undersidesof the leaves are covered in downy hairs (known technically as trichomes) to which themold Rhizopus oligosporus can be found adhering in the wild. Soybeans are pressed into the leaf,and stored. Fermentation occurs resulting in tempeh. In particular, the tempeh undergoes salt-freeaerobic fermentation.


https://en.wikipedia.org/wiki/Bean

https://en.wikipedia.org/wiki/Soybean

https://en.wikipedia.org/wiki/Rhizopus_oligosporus

https://en.wikipedia.org/wiki/Hibiscus_tiliaceus

https://en.wikipedia.org/wiki/Indonesia

https://en.wikipedia.org/wiki/Ammonia

https://en.wikipedia.org/wiki/Spore

https://en.wikipedia.org/wiki/Acetic_acid

https://en.wikipedia.org/wiki/Lactic_acid

https://en.wikipedia.org/wiki/Vinegar

https://en.wikipedia.org/wiki/Acidulant

https://en.wikipedia.org/wiki/Mycelium

https://en.wikipedia.org/wiki/Mycelium

https://en.wikipedia.org/wiki/Fahrenheit

https://en.wikipedia.org/wiki/Celsius

https://en.wikipedia.org/wiki/Rhizopus_oryzae

https://en.wikipedia.org/wiki/Rhizopus_oryzae


https://en.wikipedia.org/wiki/Fungus

https://en.wikipedia.org/wiki/Fermentation_starter

https://en.wikipedia.org/wiki/Rhizopus

https://en.wikipedia.org/wiki/Fermentation

https://en.wikipedia.org/wiki/Whole_grain

https://en.wikipedia.org/wiki/Wheat


https://en.wikipedia.org/wiki/Soybean

https://en.wikipedia.org/wiki/Fermentation_(food)


ENZYME BIOTECHNOLOGY

Introduction: All the biochemical reactions in living organisms at body temperatureproceed at a rapid rate. Such reactions are would have been slow if they had not beencatalysed by the ‘biocatalysts’ known as enzymes. Thus enzymes can be defined as ‘themolecules that catalyse various biochemical reactions without themselves undergoing anychemical change.

History: The term ‘enzyme’ was introduced by Kuhne in 1878, although the firstobservation of enzyme activity in a tube was reported by Payen and Persoz in 1833.During 1890s Fisher suggested the ‘lock and key’ model of enzyme action, while amathematical model of enzyme action was proposed by Michaelis and Menten in 1913. In1926, James Sumner crystallized for the first time an enzyme (urease). The transition statetheory of enzyme action was put forth by Pauling in 1948, and in 1951 Paulin and Coreydiscovered the α-helix and β-sheet structure. Sanger, in 1953, determined the amino acidsequence of a protein (insulin). In 1986, Cech discovered the catalytic RNA, while Lernerand Schutlz developed catalytic antibodies.

Chemical Nature of Enzymes

All enzymes are proteins except a small group of catalytic RNA. Such RNA are calledRibozymes. The catalytic activity of an enzyme depends on its conformation. Thus if anenzyme is denatured it loses its biological activity.

Some enzymes do not require any chemical group for their activity except amino acidresidues, such enzymes are referred to as simple protein enzymes. Example: Urease,Amylase.

Whereas some enzymes require an additional non protein component known as prostheticgroup. The protein part of such enzymes is known as Apoenzyme or Apoprotein. The twoparts together constitute the Holoenzyme.

Apoenzyme + Prosthetic Group = Holoenzyme

When the prosthetic group is inorganic ion, it is known as co factor. When the prostheticgroup is an organic molecule it is called co enzyme.

General Characteristics of Enzyms

1. Proteinaceous Nature: most enzymes are proteins.2. Catalytic Power: Enzymes display enormous catalytic power. Catalytic power isdefined as the number of substrate molecules converted to products per unit time when the



enzyme is fully saturated with the substrate. Experiments havebeen shown that theefficiency of the enzyme catalysed reactions is greater by as much as 108 times than theuncatalysed reaction. The catalytic power is measured by Turn Over Number.3. Specificity of enzyme action: Enzymes are highly specific which catalyse only onetype of chemical reaction of a single substrate or a group of related substrate.4. Sensitivity of enzymes: Enzymes are highly sensitive to changes in pH andtemperature. Enzymes are denatured at higher temperature of 60-70o C.5. Reversibility of Enzyme action: Most of the reactions catalysed by enzymes arereversible and the enzymes can catalyse the reactions in both directions. Ex: Lipase cancatalyse not only the hydrolysis of fats into fatty acids and glycerol but also can synthesizefats from fatty acids and glycerol.6. Regulation of enzyme activity: Enzymes are inhibited by specific molecules. Thisphenomenon is important in regulation of their catalytic activities.7. Active Site: The enzyme catalysed reactions occur at an asymmetric cleft of theenzyme called the active site at which the substrate will bind and get converted intoproducts.

Specificity of Enzyme Activity:

The enzymes are highly specific in their actions which act upon only one substrate or agroup of structurally related substrates. The different types of enzyme specificities are asfollows:

1. Absolute Specificity: Some enzymes will act only on particular substrate. Suchenzymes are said to exhibit absolute specificity. Ex: Carbonic anhydrase that catalysessynthesis of carbonic acid.2. Group Specificity: Few enzymes act on structurally related group of compounds.Ex: Hexokinase enzyme assists the transfer of phosphate group from ATP to many hexosesugars such as galactose, glucose, fructose and mannose.3. Stereochemical Specificity: Many enzymes exhibit stereochemical specificity. Ifthe substrate can exist in two stereochemical forms, only one isomer can be acted upon bythe enzyme. Ex: Fumarase which catalyses the reaction involving interconversion offumarate and malate. The enzyme Fumarase can act on the trans isomer Fumarase but noton the cis isomer Malate.

Nomenclature of Enzymes

Generally enzymes are named by adding a suffix ‘ase’to the name of their substrate or aword describing the nature of reaction it catalyses. For example: Urease catalyses thehydrolysis of urea.



However names of some enzymes neither indicate the substrate nor indicate the nature ofthe reaction. For example: Pepsin, Trypsin.

With the ever increasing new discoveries on enzymes, a systematic naming andclassification is necessary. The International Union of Biochemists (IUB) has set u anEnzyme Commission (EC) in 1961 suggesting guidelines for enzyme nomenclature andclassification. According to the EC each enzyme is assigned with a Common name(Trivial Name), a Systematic name (Indicating the nature of reaction catalysed, reactant,product), and EC number consisting of four digits. For example: 1.1.1.3 - the first numberrefers to the enzyme class, the second digit to the subclass,the third sub-sub class and thefourth to the serial number of the enzyme within the sub-subclass.

According to Enzyme Commission, enzymes are classified into six major classes whichare as follows:

1. Oxidoreductases: They take part in oxidation and reduction reactions or transfer ofelectrons.Oxidoreductases are of three types— oxidases, dehydrogenases and reductases, e.g.,cytochrome oxidase (oxidises cytochrome), succinate dehydrogenase, nitrate reductase. 2. Transferases:They transfer a group from one molecule to another e.g., glutamate- pyruvate transaminase(transfers amino group from glutamate to pyruvate during synthesis of alanine). Thechemical group transfer does not occur in the Free State.

3. Hydrolases:They catalyse hydrolysis of bonds like ester, ether, peptide, glycosidic, С-С, С halide, P—N, etc. which are formed by dehydration condensation. Hydrolases break up largemolecules into smaller ones with the help of hydrogen and hydroxyl groups of watermolecules. The phenomenon is called hydrolysis. Digestive enzymes belong to this group,e.g., amylase (hydrolysis of starch), sucrase, lactase.

4. Lyases:The enzymes cause cleavage, removal of groups without hydrolysis, addition of groups todouble bonds or removal of a group producing double bond, e.g., histidine decarboxylase


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(breaks histidine to histamine and CO2), aldolase (fructose-1, 6-diphosphate to dihydroxyacetone phosphate and glyceraldehyde phosphate).

Fructose 1, 6-diphosphate – aldolase → Dihydroxy acetone phosphate + Glyceraldehydephosphate.

5. Isomerases:The enzymes cause rearrangement of molecular structure to effect isomeric changes. Theyare of three types, isomerases (aldose to ketose group or vice-versa like glucose 6-phosphate to fructose 6-phosphate), epimerases (change in position of one constituent orcarbon group like xylulose phosphate to ribulose phosphate) and mutases (shifting theposition of side group like glucose-6-phosphate to glucose-1- phosphate).

6. Ligases (Synthetases):The enzymes catalyse bonding of two chemicals with the help of energy obtained fromATP resulting in formation of such bonds as С-О, С-S, С-N and P-O, e.g., Pyruvatecarboxylase. It combines pyruvic acid with CO2 to produce oxaloacetic acid.

Active Site or Active Spot:The whole of enzyme molecule is not active in catalysing a chemical reaction. Only asmall portion of it is active. It is called active site or active spot. An enzyme may have oneto several active sites. An active site or spot is an area of the enzyme which is capable ofattracting and holding particular substrate molecules by its specific charge, size and shapeso as to allow the chemical change.

It fails to recognise other molecules. Active site consists of a few amino acids and theirside groups which are brought together in a particular fashion due to secondary andtertiary folding of a protein molecule.

The active site is a 3 D cleft containing amino acid residues from different parts of theenzyme. Active site possesses two groups: Binding group and catalytic group

Binding group: it helps in the tight binding of the substrate onto the enzyme resulting inthe formation of Enzyme – Substrate Complex.



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Catalyic Group: it promotes the conversion of Enzyme – Substrate Complex to products.

Due to the specific 3D conformation of the enzyme side chains, distantly located aminoacids are clustered together to form an active site.

Common Features of an active site

The important common features of an active site are as follows:

The active site takes up a relatively small part of the total volume of the enzyme. Active sites are clefts or crevices. Active sites bond with substrates with weak forces. Active site is a 3D entity. The specificity of binding depends on the defined arrangement of atoms in an activesite of an enzyme.

Theories of interaction between Active sites and Substrates

Two important models have been postulated in order to explain the interactions betweenthe enzyme and substrate in transition state. They are as follows:

LOCK AND KEY MODEL

This theory was proposed by Emil Fischer in the year 1894.

According to this model, the substrate and the active site of the enzyme arecomplementary to each other so that they fit together just like a lock and key. He proposedthat the active site of the enzyme is rigid. This type of interaction was proposed in case ofenzymes known to exhibit absolute specificity.



However a mojor demerit of tis model is the rigid active site.Because in later work, the Xray crystallographic studies revealed that the conformation of free enzyme is differentfrom that of the enzyme bound to a substrate. Hence another model was put forth in theyear 1963.

INDUCED FIT THEORY

This theory was proposed by D E Koshland in 1963. According to this model, the enzymedoesnot retain its original shape and structure. That is, the substrate binds at the active siteeffectively by inducing conformational change in the active site of the enzyme. Thismechasim explains whhy enzymes become inactive upon denturation. Denaturationdestroys the preffered 3-D structure of the enzyme so that substrate cannot induceconfomational change in the enzyme to fit itself at the active site.

INDUSTRIAL PRODUCTION OF ENZYMES

Enzymes are obtained form animal tissues,plants, bacteria and fungi including yeast. Thebulk enzymes both in terms of quality and variety are derived from microorganisms,higher plants being the distant second and animals being the last important.

Most of the enzymes are used by food industry. Therefore, initially pant and animalenzymes were preffered over microbial enzyms mainly for considerations of safety and thefear of contamination by microorganisms, toxins etc.

But increased demands, shortagesin supplies of enzymes from plant and animal sourcesand difficulties in maintainng a continued supply of these enzymes prompted a muchcloser and much progmatic evaluation of the microbialenzymes.These enzymes havefound increasing applications even in such areas where enzymes of animal origin wereonce exclusively used. For example Cheese Production: the enzyme Rennet orChymotrypsin(an asrartic proteinase) produced by Mucor meihei is now used widely forcheese productionin place of rennet from calf stomach which was onnce extensively used.



Advantages of Microbial Enzymes

Microbes as a source of enzymes have the advantage of large scale production byfermetaton, ease in isolation of especially those enzymes that are excreated into themedium (ex- hydrolases), the variety of enzymes produced and the ease in the geneticmanipulation to enhance the enzyme yeilds and even to modify the enzyme using therDNA technology if requird.

AMYLASES

Amylase is an ezyme that catalyses the hydrolysis of starch into sugars. Amylase ispresent in the saliva of humans and some other mammals where it begins the process ofdigestion. Foods that contain large amounts of starch but little sugar such as rice andpotato may acquire a sweet taste as they re chewed because amylase degrades some oftheir strch to sugar.

Plants and some bacteria also produce amylase. All amylases are glycoside hydrolases.

The α amylases are abundantly found in case of plants, animals and bacteria, generally theα amylases hydrolyse the α,1-4 glycosidic linkage. Generally when strch undergoeshydrolysis in the presence of α amylase, it forms oligosaccharide and maltose. The fungalamylase is inactivated at about 50-60oC while the bacterial α amylase can be used at higtemperature ie.,˃ 80oC.

β Amylase is usually produced by most of higher plants. This enzyme hydrolyses starchinto dextrin and maltose.

Glucoamylase also reffered to as amyloglycosidase hydrolyses starch into glucose.

MICROBIAL PRODUCTION OF AMYLASE

Microbial production of amylase generally involves the following steps:

Microbial Source Selection of Microorganisms Preparation of inoculum Media preparation Process of fermentation Enzyme recovery from submerged culture Recovery of enzyme from the surface culture method



1. Microbial Source: Basically enzymes are produced from plants or animals ormicroorganisms. Bacteria used for amylase production include Bacillus coagulans,Bacillus licheniformis. Fungal amylases are produced by Aspergillus niger, Aspergillusoryzae.2. Selection of Microorganisms: This is the most important step in the production ofamylases. To slect he microorganisms the following points must be kept in mind.

The enzyme produced by the enzyme should be extracellular. This makes it easy oextract the enzyme. The microbe should be capable of growing on cheaper raw mwterials Only the desired enzyme has to be produced Microbe should be ffree from activity of toxins.

3. Preparation of Inoculum: a selected strain of Aspergillus oryzae is revived on wortagar slant and maintained till spores (conidia) are formed.4. Media preparation: the production of the enzyme amylase requires two types ofmedia- Surface Culture Media and Submerged Culture Media. For Surface Culture Mediawheat bran is used as raw mwterial. For Submerged Culture Medim corn strch is used asraw material. The prepared medium is sterilized at 121oC and 15lbs for 20 minutes.5. Process of ferentation: Most of the commercially important enzymes are usuallyproduced by two kinds of fermentation processes: a) Surface Culture Fermentation b)Submerged Culture Fermentation. However submerged culture fermentation for aerobicmicroorganisms is now widely used. In case of submerged culture method, the enzymefermentation is usually carried out in the closed tank fermenters which vary between 1000-30,000 gallons in capacity.6. A) Enzyme Recovery from Submerged Culture: For enzyme recovery the fermentedliquor is subjected to rapid cooling at 5oC. To remove insoluble products like microbialcells centrifugation is done. Since amylase is secreted into the medium it remains in thefermented broth after the biomass has been removed. The broth is then terated withcalcium phosphate to remove any residues. In order to obtain high purity of the enzyme, itis usually precipitated with ethyl alcohol or propane.

B) Enzyme recovery from Surface Culture: The microbial biomass is removed bycentrifugation at high speeds (40,000-50,000g) under refrigerated conditions. In order tocomplement centrifugation, microfiltration and ultra filtration have emerged. Thesupernatant is decanted and equal volume of alcohol is mixed. The enzyme obtained isrecovered by filtration. Then the filtrate is dried at about 4oC in a drier.

USES OF AMYLASE



It is used extensively in baking

It is used as meat tenderiser

It is useful in beer production during mashing

Amylase is used in the manufacture of maltose which is the main component ofmaltosugar syrup. Maltosugar syrup is used as a sweetener in food industry.

Amylases are used in the manufacture of dextrins. Dextrins are used as glossing agents.

APPLICATIONS OF ENZYMES

In recent times, interest has been sustained in using enzymes to speed up several industrialprocesses. The understanding of the structure and functions of enzymes combined withimproved methods of their handling have given a boost to enzyme applications in diversefields like food, detergent, leather, pharmaceutical and paper industry.

APPLICATIONS OF ENZYMES IN DETERGENT INDUSTRY

Enzymes derived from animal pancreas were used more than 70 years ago to removeblood stains from butcher’s apron, without weakening the cloth. These enzymes were triedas laundry aids, but it was found that they were inactivated by soap.

In the 1970s proteolytic enzymes (proteases) from bacteria which could retain theiractivity in the presence of detergents and hot water were found and were added todetergents. When this was first tried, workers in the detergent factories developedrespiratory problems and skin irritations. The illness was found to be due to the enzymemolecules that were in the aerosol and were breathed by the factory workers.

Today, enzymes are placed inside some coated granules and mixed with detergents so thatthe enzyme molecules are released along with the detergents during washing process.

Modern high value detergents contain proteases and lipases. These enzymes are used forfood and blood stain remval from clothing which mainly contain proteins and lipids.

Conventional detergents are just surfactants producing foam which physically remove dirtand other deposited materials from clothes. But enzyme action gives the detergents thatextra power to remove food stains and spots due to milk, egg, blood or sweat.



Proteases used in the detergent industry have to withstand and be active under alkalineconditions of pH 8-10. Many bacteria produce alkaline proteases which are active at highpH. Bacillus subtilis, Bacillus licheniformis and Rhizopus species are used for theproduction of alkaline proteases and lipases respectively.

Recent examples of second generation detergent enzymes include amylases that haveenhanced activity at lower temperatures and alkaline pH, while maintaining the necessarystability under detergent conditions. Amylase is used as a starch stain remover inlaundry pre soaks.


StockCulture

Shake flaskCulture

Seed fermenter

Medium sterilization

Medium Formulation

Medium raw materials

Production fermenter

Product purification

Product packaging

Product extraction

Effluent tracement

Cell free supernatant

Biomass

Cell separation

Inoculum Development

Culture Fluid


Upstream Processing (USP)

Upstream Processing (USP): Types of Fermenters – Typical, Airlift, Tower and Bubble-up Fermenter. Solid

Substrate, Submerged, continuous fermentation & shake flask fermentation. Fermentation Media-Natural

and Synthetic Media, Sterilization techniques-Heat, Radiation and Filtration methods. Process of Aeration,

Agitation, Temperature regulation and Foam control. Immobilized enzyme and cell bioreactors.

Upstream Processing:

Terms and Definitions:

Industrial Biotechnology deals with the study, utilization and manipulations of those microorganisms

capable of economically producing desirable substances or changes in substances and the control of

undesirable microorganisms.

Fermentation is a process mediated by microorganisms in which a product of economic value is

obtained.

In Metabolism Fermentation refers to energy generating process where organic compound acts as both

electron donor and acceptor.

In context to industrial biotechnology, fermentation is defined as the process by which large quantities of

the cells are grown under aerobic or anaerobic conditions.

A. Upstream processing involves all factors and processes leading to the product and including the

fermentation. It consists of three main areas.

i. Obtaining a suitable microorganism for the particular productii. Strain improvement to increase the productivity and yieldiii.Preparation of suitable inoculums and this is followed by fermentation media and fermentation

process.

Figure. Stages of Fermentation



B. Major Characters of suitable industrial microorganisms:

The most suitable important factor for the success of any fermentation industry is the microbial strain. It

should have the following characteristics.

High yielding strain. Stable biochemical characters. Should not produce undesirable substances. Easily cultivable on a large scale. Should be a stress tolerant strain. Utilization of a wide range of low cost and readily available carbon sources. Amenability to genetic manipulation. Safety, non-pathogenicity and should not produce toxic agents, unless there is the target product. Ready harvesting from the fermentation.

C. Screening of industrially important microorganisms:

Suitable high yielding strain should be selected from the natural sources like soil. The process of

microbial strain from the nature are called screening. There are two types of screening. They are primary

screening and secondary screening.

Primary Screening

This consists of simple tests to isolate microbes from the nature with desirable property. It removes

worthless microbes using simple fundamental criteria. It is performed by crowded plate technique,

auxanography, enrichment enrichment culture techniques and use of an indicator dye. Primary screening



of an antibiotic producer is done by crowed plate technique. It is the simplest screening technique

employed in detecting and isolating antibiotic producers.

It is done by diluting sample of the source material for the antibiotic producing microorganisms, followed

by spreading the dilution on the agar plates, incubating at specified environment. Colonies showing

activity is indicated by the presence of a zone of inhibition surrounding the colony. Such a colony is sub-

cultured to a similar medium and purified.

Secondary Screening

Secondary screening is strictly essential in any systematic screening programme intended to isolate

industrially useful microorganisms, since primary screening merely allows the detection and isolation

microbes that possess potentially interesting industrial applications. Secondary screening helps in

detecting in really useful microorganisms in fermentation processes. This can be realised by a careful

understanding of the following points associated with secondary screening:

1. It is very useful in sorting out microorganisms that have real commercial value from many isolates

obtained during primary screening.

2. It provides information whether the product produced by microorganisms is a new one or not.

3. It gives an idea about economic position of fermentation process involving the use of newly

discovered culture.

4. It helps in providing information regarding the product yield potentials of different isolates.

5. It determines the optimum conditions for growth or accumulation of a product associated with a

particular culture.

6. It provides information pertaining to the effect of different components of a medium.

7. It detects gross genetic instability in microbial cultures.

8. It gives information about the number of products produced in single fermentation.



D. Fermentation media:

Those components that support the growth of microbe and induce product formation are called

fermentation media. Submerged fermentation requires liquid media.Solid Substrate fermentation

requires solid or semisolid medium. Fermentation media must satisfy all the nutritional requirements of

the microorganism and fulfil the technical objectives of the process.

The nutrients should be formulated to promote the synthesis of the target product, either cell biomass or

specific metabolite. Media used in cultivation of microorganisms must contain all elements in a form

suitable for the synthesis of cell substance and for the production of metabolic products.

Media requirements and media formulations:

Most fermentations, except those involving solid substrates, require large quantities of water in which the

medium is formulated. General media requirements include a carbon source, sources of nitrogen,

phosphorus and sulphur. Minor and trace elements must also need and some microorganisms require

vitamins, such as biotin and riboflavin.Usually, media incorporate buffers or the pH is controlled by acid

and alkali additions. Antifoam agents also required.

The main factors that affect the final choice of the individual raw materials are as follows.

1. Cost and availability: ideally, materials should be inexpensive and of consistent quality and year

round availability.

2. Ease of handling in solid or liquid forms, along with associated transport and storage costs, e.g.

requirements for temperature control.

3. Sterilization requirements and any potential denaturation problems.

4. Formulation, mixing, complexing and viscosity characteristics that may influence agitation, aeration

and foaming during fermentation and downstream processing stages.

5. The concentration of target product attained, its rate of formation and yield per gram of substrate

utilized.

6. The levels and range of impurities and the potential for generating further undesired products during

the process.

7. Overall health and safety implications.



Some of the frequently used substrates in industrial fermentation

In general, production media is used in the liquid state to facilitate the fermentation process. Each

compound production needs individual medium. The production medium must have a suitable chemical

composition. It should contain a source a carbon, nitrogen, growth factors and mineral salts. Medium

should supply for precursor compound for the production of compound. Medium should have buffering

activity to maintain pH. Antifoaming agents are needed in the medium to avoid the problems of foaming.

The raw materials required for designing of the production medium should be freely available in large

quantities at reasonable price.

Mostly agricultural products are utilized as a source of raw materials. There are five major categories of

raw materials.

Those are

(i) Saccharine materials (Pure glucose or sucrose, cane and beeet molasses, sugarcane juice, cheese)

(ii) Starchy materials (cereals and root tubers

(iii) Cellulosic materials(sulphite waste liquor, wood molasses, rice straw)

(iv) Hydrocarbon and vegetable oils (oleic acid, gas oil)

(v) Nitrogenous materials (corn steep liquor, soya bean meal, pharma media, distilled soluble,

ammonium salts, urea or gaseous ammonia, peptone, yeast extract).

Malt extract, an aqueous extract of malted barley, is an excellent substrate for many fungi, yeast and

actinomycetes. Dry malt extract consists of about 90-92% carbohydrates and is composed of

hexoses (glucose and fructose), disaccharides (maltose and sucrose), trisaccharides (maltotriose) and

dextrins.nitrogenous substances present in malt extract include proteins, peptides, amino acids,

purines, pyrimidines and vitamins. The aminoacids composition of different malt extracts varies

according to the grain used, but proline always makes up about 50% of the total amino acids present.

Sterilization of Media.

Successful fermentation usually needs the sterilization of the equipment, medium, air, and subsequent

maintenance of sterility of the whole system. UV, chemical agents are employed in the sterilization

process. Fermenter is sterilized by steam. Boiling and steam under pressure are used to sterilize

fermentation medium. Air can sterilized by filtration, UV, heat and chemical agents.

E. FERMENTOR - Design and Role Of Different Parts of Fermentor

In fermentation industries, microbes are to be grown in specially designed vessels loaded with

particulars type of nutritive media. These vessels are referred to as Fermentor or Bioreactors.

Fermentors are complicated in design, because the must provide for the control and observation of many



facts of microbial growth and biosynthesis. The design of the fermentor depends upon the purpose for

which it is to be utilised. Industrial fermentors are designed to provide best possible growth and

biosynthesis conditions for industrially important microorganisms and allows ease of manipulation for all

operations associated with the use of the fermentors. The fermentor used for a particular process and

should posses following characters.

A simple fermenter with temperature, pH, and oxygen control.

Characteristics of an indeal fermenter of an ideal fermentor or bioreactor.

There cannot be a fermentor ideal for almost all the fermentation processes. It should have the following

characteristics.

1. Material used in the fabrication of fermentor should be strong enough to withstand the interior

pressure due to the fermentations media.

2. It should be resistant to corrosion and free from any toxic effect for the microbial culture.

3. A fermentor should permit easy control of contaminating microbes.

4. It should be provided with the inoculation point for aseptic transfer of inoculum.

5. Should be equipped with the aerating device (spargers).

6. Should be equipped with a stirring deice for uniform distribution of air, nutrients and

microbes(Impellers).



7. There should be provision of baffles to avoid vortex formation.

8. Fermentor should be provided with a sampling valve for aseptic withdrawing of sample for

different laboratory tests.

9. Fermentor should possess adevice for controlling temperature.

10. Fermentor should be provided with with pH controlling device for monitoring and maintaining

pH.

11. Should be provided with a facility for intermittent addition of antifoam agents for controlling

foam formation.

12. There should be provision for feeding certain media components during the progress of

fermentation (precursors).

13. A drain at the bottom is essential for the removal of the completed fermentation broth for further

processing.

14. A main hole should be provide at the top of fermentor for access inside the fermentor for different

purposes like repairing and thorough cleaning of fermentors between runs.

15. A exit valve should be provided at the top for the exit of metabolic gases produced during

fermentation processes.

F. TYPES OF FERMENTOR

Batch Fermentor

Batch reactors are simplest type of reactor. Bacth fermentors are used to carry out microbiological

processes on batch basis. They are available with varying capacities. The capacity of the fermentor amy

range from a few hundred to several thousand gallons. They are four types batch fementor. They are as

follows.

i. Small laboratory fermentors.

ii. Pilot plant fermentors.

iii.Large industrial fermentors.

iv.Horton spheres.

The small laboratory fermentor ranges from 1-2 liters with a maximum up to 12-15liters. Pilot plant

fermentors have a total volume of 25-100 gallons upto 2000 gallons total volume. Larger fermentors

range from 5000 or 10000 gallons of total volume to approximately 1,00,0000 gallons. Horton spheres are

rarely employed with a size range of 2,50,000 to 5,00,000 gallons total capacity. Actually the working

volume in a fermentor is always less than that of the total volume. In other words, head space is left at the

top of the fermentor above the level of fermentation media. The reason for keeping a head space is to



allow aeration, splashing and foaming of the aqueous medium. This head space usually occupies a fifth to

a quarter or more of the volume of the fermentor. Batch fermentation is carried out by the reactor is filled

with medium and fermentation is allowed to proceed. When the fermentation has finished the contents are

emptied for downstream processing. The reactor is then cleaned, refilled re-inoculated and the

fermentation process starts again.

Components of the batch fermentor

pH control: pH control is achieved by acid or alkali addition, which is controlled by an auto-titrator . the

autotitrator in turn is connected to a pH probe.

Temperature Control: It is achieved by a water jacket around the vessel. This is often supplemented by

the use of internal coils, in order to provide sufficient heat transfer surface.

Agitation: The agitating device consists of a strong and straight shaft to which impellors are fitted. An

impellor, in turn consists of a circular disc to which blades are fitted with bolts. Different types of blades

are available and are used according to the requirements.

Aeration: usually the aerating device consists of a pipe with minute holes, through which pressurized air

escapes into the aqueous medium in the medium in the form of tiny air bubbles. This aeration device is

called a ‘SPARGER’. The size of the holes in a sparger ranges from 1/64 to 1/32 of an inch or larger.

Foam control: Aeration and agitation of liquid medium can cause the production of foam. This is

particularly true for the media containing high levels of proteins or peptides. If the foam is not controlled,

it will rise in the head space of the tank and be forced from the tank along with the exit valve. This

condition often causes contamination of the fermentation from organisms picked up by breaking of some

of the foam which then drains back into the tank. Excessive foaming also causes other problems for

fermentation.

Types of fermentation process

Varieties of the processes are followed in fermentation industry depends on the media used and type of

operations. Based on the nature of media used the process of fermentation are classified as surface

fermentation, submerged fermentation and solid state fermentation.

Surface fermentations

It is as simplest process by which a culture is allowed to grow on the surface of the medium and allowed

the fermentation process. It is vat process. Eg. Vinegar Production.

Submerged Fermentation

In the normal fermentation process the fermentation medium is in liquid condition and also known as

submerged fermentation. Eg. Enzyme Production.

Solid-Substrate Fermentation



Solid substrate fermentation involves the growth of microorganisms on solid, normally organic materials

in the absence of free water. The substrates used are cereal grains, brans, legumes and lignocellulosic

materials, such as straw, wood chippings etc. Solid-substrate fermentation lack the sophisticated control

mechanisms. Solid substrate fermentations are the most suitable methods for the production of fungal

products. Fungi do not form spores in submerged fermentations. Solid-substrate fermentations are

normally involves multistep processing.

1. Pre-treatment of substrate that often requires mechanical, chemical or biological processing.

2. Hydrolysis of polymeric substrates, e.g. polysaccharides and proteins.

3. Utilization of hydrolysis products.

4. Separation and purification of end products.

Based on the type of operation in liquid media, the process of fermentation is of three types. They are

Batch Fermentation, Continuous Fermentation and Fed-Batch Fermentation.

Batch fermentation is dynamic process that is never in a steady state.

In batch processes the critical parameter is gas exchange or balance

between respiration rate and oxygen transfer. Ni batch operation the

sterilized media components are supplied at the beginning of the

fermentation with no additional feed after inoculation. When cells

are grown in a batch reactor, they go through a series of stages known

as lag phase, exponential phase, stationary phase and death phase.

Continuous Fermentation: in this method fresh media is continuously

added and bioreactor fluid is continuously removed. As a result, cells

continuously receive fresh medium and products and waste products and cells are continuously removed

for processing. The reactor can thus be operated for long periods of the time without having to be shut

down.

Fed batch fermentationis the most common type of process used in industry. In this process, fresh

media is added continuously or periodically to the bioreactor. There is continuous removal of product or

medium/ cells like continuous process. The fermenter is emptied or partially emptied when reactor is full

or fermentation is finished.

G. THEMAJOR TYPES OF BIOREACTORS USED IN

SUBMERGED PROCESS.


Stirred Tank Reactors (STR) Airlift Reactors (ALR) and An airlift fermenter with draft tube.

b) fixed bed bioreactors

a) Fluidized bed bioreactors

A continuous flow stirred tank reactor.Parameters such as pH and concentrationsof specific metabolites are closelymonitored to ensure the maintenance ofoptimum conditions. Outlets allow for thecollection of samples during fermentationas well as the collection of cells andmedium at the conclusion of the reaction.Addition and collections are carried outunder aseptic conditions.




a) Stirred Tank Reactors (STR)

In These reactors, mechanical stirrers (using impellers) are used to mix the reactor to distributed heat and

materials (such as oxygen and substrates) Bubble column reactors. These are tall reactors which use air

alone to mix the contents.

b) Airlift Reactors (ALR)

These reactors are similar to bubble column reactors, but differ by the fact that they contain a draft tube.

The draft tube is typically an inner tube which improves circulation and oxygen transfer and equalizes

shear forces in the reactor.

c) Fluidised Bed Reactors (FBR)

In fluidised bed reactors, cells are “immobilized” small particles which move with the fluid. The small

particles create a surface area for cells to stick to and enable a high rate of transfer of oxygen and

nutrients to the cells.

d) Packed Bed reactors (PBR)

In packed bed reactors, cells are immobilized on large particles. These particles do not move with liquid.

Packed bed reactors are simple to construct and operate but can suffer from blockages and from poor

oxygen transfer.

e) Flocculated Bed Reactors (FBR)

Flocculated cell reactors retain cells by allow them to flocculate. These reactors are used mainly in

wastewater treatment.

f) Stirred Tank Bioreactors (STB)

A stirred tank reactor is the simplest type of reactor. It is composed of a reactor and mixer such as a

stirrer, a turbine wing or a propeller. This reactor is useful for substrate solution of high viscosity and for

immobilized enzymes with relatively low activity.

H.BIOREACTORS USED FOR SOLID-SUBSTRATE FERMENTATIONS.

Most solid-substrate fermentations are batch processes.

1. Rotating drum fermenters, comprising a cylindrical vessel of

around 100L capacities mounted on its side onto rollers that both

support and rotate the vessel. These fermenters are used in enzyme and

microbial biomass production. There main disadvantage is that the drum

is filled to only 30% capacity, otherwise mixing is inefficient.



2. Tray fermenters,which are used extensively for the

production of fermented oriental foods and enzymes.

Their substrates are spread onto each tray to a depth of

only a few centimetres and stacked in chamber

through which humidified air is circulated. These

systems require numerous trays and large volume

incubation chambers of up to 150m3Capacities.

3. Bed systems consisting of a bed of substrate up to 1M deep,

through which humidified air is continuously forced fro below.

4. Column bioreactors, consisting of a glass or plastic column, into

which the solid substrate is loosely packed, surrounded by a jacket

that provides a means of temperature control. These vessels are used

to produce organic acids, ethanol and biomass.

5. Fluidized bed reactors, which provides continuous agitation with

forced air to prevent adhesion and aggregation of substrate particles. These

systems have been particularly useful for biomass production for

animal feed.Column bioreactors

Anaerobic Reactors:

Anaerobic reactors are generally used for the production of methane rich biogas from manure (Human

and animal) and crop residues. They utilise mixed methanogenic bacterial cultures which are

characterised by defined optimal temperature ranges for growth. These mixed cultures allow digesters to

be operated over a wide temperature range i.e. above 0°C up to 60°C.

References for IBT

Textbook of Biotechnology ,Dubey R. C.S. Chand and Company, New Delhi5th Revised EditionBiotechnologyJohn E. SmithCambridge University Press5thEdition

FermentationMicrobiologyAndBiotechnologyE.M.T. El-Mansi, C.F.A. Bryce, B. DahhouS. Sanchez, A.L. Demain, A.R. AllmanCRC Press, Taylor and Francis Group3rd Edition

Industrial microbiology, Casida, Jr, L.ENew Age international Pvt. Ltd New Delhi.1st Edition Textbook of Microbiology,Dubey R. C.S. Chand and Company, New Delhi5th Revised Edition Microbiology,Pelczar, M.J., Chan E.C.S. and Krieg, N.R.Tata McGraw Hill Book Co. New York.

5th Edition Essentials of Microbiology ,Dr. S. Rajan and Mrs. R. Selvi Christy,Anajana Book House, Chennai ,1st Edition




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