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Industrial Microbiology of Molds

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INDUSTRIAL MICROBIOLOGY
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Page 1: Industrial Microbiology of Molds

INDUSTRIAL MICROBIOLOGY

Page 2: Industrial Microbiology of Molds

• Industrial microbiology uses microorganisms, typically

grown on a large scale, to produce valuable commercial

products or to carry out important chemical

transformations.

• The actual reactions carried out by microorganisms in

industrial microbiology are called biocatalysis.

• Originated with alcoholic fermentation processes.

– Later on, processes such as production of

pharmaceuticals, food additives, enzymes, and

chemicals were developed

Page 3: Industrial Microbiology of Molds
Page 4: Industrial Microbiology of Molds

• An industrial microorganism must:– produce the product of interest in high yield;

– grow rapidly on inexpensive culture media available in bulk

quantities;

– be amenable to genetic manipulation; and,

– if possible, be nonpathogenic. • Microbial products of industrial interest include

– Enzymes– Antibiotics, steroids, alkaloids – Food additives– Commodity chemicals

• Inexpensive chemicals produced in bulk• Include ethanol, citric acid, and many others

Page 5: Industrial Microbiology of Molds

• A primary metabolite is a kind of metabolite that is

directly involved in normal growth, development, and

reproduction. Eg: alcohol, lactic acid etc

• Secondary metabolites are organic compounds that

are not directly involved in the

normal growth, development, or reproduction of an

organism. Unlike primary metabolites, absence of

secondary metabolites does not result in immediate

death.

• Primary metabolites are produced during active cell

growth, and secondary metabolites are produced near

the onset of stationary phase

Page 6: Industrial Microbiology of Molds

• Secondary metabolites– Not essential for growth– Formation depends on growth conditions– Produced as a group of related compounds– Often produced by spore-forming microbes during

sporulationPrimarymetabolite Secondary

metabolite

Alcohol

Penicillin

Cells

Sugar

Cells

Sugar

Time Time

Alc

ohol

, sug

ar, o

r cel

l num

ber

Peni

cilli

n, s

ugar

, or c

ell n

umbe

r

Page 7: Industrial Microbiology of Molds

• Many economically valuable microbial products are

secondary metabolites.

• Humans use secondary metabolites as food flavours,

medicines etc.

Antibiotic Producing microorganism Cephalosporin Cephalosporium acrimonium Chloramphenicol Streptomyces venezuelae Erythromycin Streptomyces erythreus Griseofulvin Penicillium griseofulvin Penicillin Penicillium chrysogenum Streptomycin Streptomyces griseus Tetracycline Streptomyces aureofaciens Gentamicin Micromonospora purpurea

Page 8: Industrial Microbiology of Molds

INDUSTRIAL USES OF MOLDS

Page 9: Industrial Microbiology of Molds

PENICILLIN• A class of antibiotics that comes from mold.

• All penicillin like antibiotics inhibit synthesis of peptidoglycan,

an essential part of the cell wall and lyses it.

• They do not interfere with the synthesis of other intracellular

components.

• These antibiotics do not affect human cells because human

cells do not have cell walls.

• Penicillin include ampicillin, phenoxymethylpenicillin,

amoxicillin etc.

• Penicillin is active against Gram positive bacteria

Page 10: Industrial Microbiology of Molds

• Some members (e.g. amoxicillin) are also effective

against Gram negative bacteria

• Penicillin was the first important commercial product

produced by an aerobic, submerged fermentation

• Used as input material for some semi synthetic

antibiotics.

• Aerobic processes require mechanisms for stirring and

aeration.

• Industrial fermentors can be divided into two major

classes, those for anaerobic processes and those for

aerobic processes

Page 11: Industrial Microbiology of Molds
Page 12: Industrial Microbiology of Molds

• Table 30.1 shows fermentor sizes for various industrial processes.

Page 13: Industrial Microbiology of Molds

• The major steps in the commercial production if pencillin are:– Preparation of inoculum– Preparation of sterilization of medium– Inoculation of medium of the fermenter– Forced aeration with sterile air during

incubation– Removal of the mold mycelium after

fermentation– Extraction and purification of pencillin

PRODUCTION OF PENICILLIN

Page 14: Industrial Microbiology of Molds

• Raw Materials– Carbon Sources: lactose acts as a very satisfactory compound.

6% is used. Glucose and sucrose can be used.

– Nitrogen Sources: Corn steep liquor can be used. Ammonium

acetate and ammonium sulphate can be used.

– Mineral Sources: minerals such as magnesium, phosphorous,

sulphur, pottasium, zinc and copper are essential for penicillin

production. Some of these are supplied by corn steep liquid.

Page 15: Industrial Microbiology of Molds

• Fermentation Process– The medium is inoculated with a suspension of

penicillium chrysogenum spores.

– The medium is constantly agitated, aerated and the

molds grows throughout.

– After above 8 days, pH rises above 8.0 and the

growth is complete and the penicillin production

ceases.

Page 16: Industrial Microbiology of Molds
Page 17: Industrial Microbiology of Molds

• Downstreaming process– The product should be so pure so that it is dissolved

and separated as potassium salts to separate it from

other substances in the medium.

– The first step in product recovery is the separation of

whole cells and other insoluble ingredients from the

culture broth by centrifugation and filtration.

– pH is adjusted to 2-2.5 with the help of phosphoric

acids or sulphuric acids.

– This pH will help certain organic solvents to separate

from aqueous solutions.

Page 18: Industrial Microbiology of Molds

• This step has to be carried out quickly as penicillin is

very unstable at low pH

• Antibiotic is then extracted back to a buffer solution with

pH 7.5. These shifts between the water and the solvent

purifies penicillin.

• The crude penicillin from the solvent is then treated with

sodium hydroxide and charcoal and then further

sterilized.

• Pure metal salts of penicillin can be sterilized by dry heat

method.

Page 19: Industrial Microbiology of Molds

• Further Processing– For use the antibiotic is packed in sterile vials as

powder or suspension.

– For oral use it is now tabletted now with a film coating

Page 20: Industrial Microbiology of Molds

Main Stages of Penicillin Production

• A medium of corn steep liquor, lactose, salts and other

ingredients is mixed, sterilised, cooled and pumped into

the fermenter

• After 40 hrs, penicillin starts to secreted by the fungus.

• The mould mycelium is filtered from the harvested

product.

• Penicillin is extracted in the organic solvent, butyl

acetate, in which it is dissolved.

Page 21: Industrial Microbiology of Molds

• Potassium

salts are

added and a

penicillin

precipitate is

formed, this is

washed and

dried.

Page 22: Industrial Microbiology of Molds

PRODUCTS

• The resulting penicillin can be chemically and

enzymatically modified to make a variety of penicillins

with slightly different property.

• These semi synthetic penicillins include penicillin V,

penicillin O, ampicillin and amoxycillin.

Page 23: Industrial Microbiology of Molds
Page 24: Industrial Microbiology of Molds

CITRIC ACID

• Weak organic acid found in fruits.

• Produced by fermentation and suitable pH is around 3-6.

• Application In Industry– Beverages

– Food

– Pharmaceutical

– Agriculture

– Metal Industry Structural Formula of Citric Acid

Page 25: Industrial Microbiology of Molds

SOURCE OF RAW MATERIALS

Beet Molasses• the source of sugar for

microbial production of citric acid

• low cost and high sugar content

• low content of trace metals• acts as carbon source of the

fermentation

Microorganism• mycomycetes of A.niger

species can produce high yield• consequence of incomplete

respiration

Aspergillus Niger• filamentous ascomycete fungus• maintained at pH 4.5 and

temperature at 5 °C• The best strain for citric acid

production

Page 26: Industrial Microbiology of Molds

INDUSTRIAL PRODUCTION OF CITRIC ACID

• Citric acid production is mixed growth associated mainly

takes place under nitrogen and phosphate limitations

after growth has ceased.

• Medium requirements for high production:– Carbon Source: molasses or sugar solution

– Na-ferrocyanide is added to reduce iron and manganese

– High DO concentration

– High sugar concentration

– pH < 2

– Temperature : 30 degree celsius

Page 27: Industrial Microbiology of Molds

• 5.25X106 A. niger spores/L may be introduced to the

fermentor.

• Aeration is provided to the fermentator. Temperature is

controlled by cooilng coils.

• Agitated at a speed of 50-100 rpm to avoid damage to

the spores.

Page 28: Industrial Microbiology of Molds

• Downstreaming Process– Two major purification processes involved:– Filtration and precipitation

React citric acid with calcium carbonate

Filter precipitate calcium citrate

React precipitate with sulphuric acid

Filter precipitate calcium sulphate

Purified citric acid

Page 29: Industrial Microbiology of Molds

• Citric acid broth from the fermentator is highly

contaminated with left over biomass, sugars, salts and

water.

• First the citric acid must be reacted with calcium

carbonate to neutralize the broth and form the insoluble

precipitate calcium citrate.

• Calcium citrate contains 74% of citric acid.

• It is then washed, heated and filtered to remove any no:

of contaminats.

• To crack calcium citrate, sulphuric acid is used.

Page 30: Industrial Microbiology of Molds

• The reaction will produce free citric acid and a new

product, calcium sulphate, which will be removed later.

• In the filter calcium sulfate is washed away and left over

biomass is removed.

• Again the contamination that were present in the

fermentation broth can be removed by ultrafiltration or

nanofiltration.

• Citric acid can be produced in two forms: anhydrous and

monohydrate.

• These forms may require additional purification steps to

reach the desired purity.

Page 31: Industrial Microbiology of Molds

• Monohydrate:

– Contains one water molecule for every citric acid

molecule

– Requires repeated crystallisation until the water

content reaches 7.5-8.8%.

• Anhydrous:

– Processed to remove all water from final product.

– Prepared by dehydrating monohydrate citric acid at a

temperature above 36.6 degree celsius

Page 32: Industrial Microbiology of Molds

• Once the product has achieved desired impurity, it can

be sent for packaginf and distribution• APPLICATIONS

– Flavour and preservative in food items– Used in cleaning product and sodas– Used to remove scale from boilers– Used to soften water– Emulsifying agent

Page 33: Industrial Microbiology of Molds

Main stages in the process

1. Inoculation of Aspergillus Niger2. Fermentation of Citric Acid3. Biomass Removal 4. Liquid-liquid extraction5. Crystallization 6. Drying

Page 34: Industrial Microbiology of Molds

ENZYMES

• It is industrially feasible to concentrate and purify

enzymes from cultures of molds of Aspergillus,

Pencillium and Rhizopus.

• Mold enzymes; amylases, invertase, proteases and

pectinases are useful in processing wide variety of

materials

Page 35: Industrial Microbiology of Molds

PRODUCTION OF ENZYMES

• The large scale production of enzymes involves culturing

micro-organisms in chambers called FERMENTERS or

BIOREACTORS

• COMMERCIAL ENZYME PRODUCTION - AN EXAMPLE

• Pectin is an insoluble substance found in the cell walls of

plants

• Pectinase is obtained from the fungus Aspergillus niger

PRODUCTION OF PECTINASE

Page 36: Industrial Microbiology of Molds

PRODUCTION OF PECTINASE

Aspergillus niger is grown ina fermenter with a source of

nitrogen, with sucrose as the carbon source and the substrate

pectin to stimulate pectinaseproduction by the fungus

Filtration or centrifugation to obtaina cell-free system containing

pectinase in solution

Evaporate to concentrate the enzyme

Precipitate the pectinaseout of the solution and

filter the solid

Dry and purify the crudepectinase

Pure, powdered pectinase

Page 37: Industrial Microbiology of Molds

IMMOBILIZATION OF ENZYMES• Immobilisation of enzymes is an important technique

used in industry as it enables economical operation of a

process and protection of enzymes during their use

• The costs associated with the use of enzymes for

industrial purposes can also be reduced by immobilising

the enzymes

• Enzymes are immobilised by binding them to, or trapping

them in a solid support

• Various methods for immobilising enzymes are available

Page 38: Industrial Microbiology of Molds

Enzymes are held on to a solidsupport (matrix) by weak forcessuch as hydrogen bonding

Enzymes are trapped withinthe structure of a solid polymer(usually in the form of beads)– the enzyme is trapped ratherthan bound

Methods for Immobilising Enzymes

Page 39: Industrial Microbiology of Molds

Enzymes are covalently bondedto a matrix such as celluloseor collagen

Another more expensive method involvesenzymes which are both covalently bondedto, and cross-linked within, a matrix

Cross-linking and covalent bonding maycause some enzymes to lose their catalyticactivity especially if the active site is involvedin forming the linkages

Page 40: Industrial Microbiology of Molds

ADVANTAGES OF IMMOBILIZED ENZYMES

• Compared with free enzymes in solution, immobilised

enzymes have a number of advantages for use in industrial

processes

• The stability of many enzymes is increased when they are in

an immobilised state; they are less susceptible to changes in

environmental conditions such as temperature and pH

fluctuations

• Immobilised enzymes can be recovered and re-used,

reducing overall costs

Page 41: Industrial Microbiology of Molds

• The products of the reaction are not contaminated with

enzyme eliminating the need to undertake costly

separation of the enzyme from the product

• Immobilising enzymes allows for continuous production

of a substance

• Biosensors are electronic monitoring devices that make

use of an enzyme’s specificity and the technique of

enzyme immobilisation


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