Basic Knowledge about industrial microorganism. why industry choose microorganism rather than chemical. isolation technique of microorganism. source of microorganisms. Process of using microorganism. Disadvantages of using microorganisms in industry. Process of genetic modification of microorganisms. Storage process of microorganism. preservation methods of microorganism. Reculture methods of microorganism.

1. INDUSTRIAL
MICROORGANISMS

2. Presented by
Md. Rakibul Islam
M.Sc. Student
Dept. of Biotechnology and genetic Engineering
Islamic university, Kushtia.

3. What is Industrial Microorganism ?
 Industrial Microorganism is the branch of
biotechnology that applies in industry for creating
industrial products in mass quantities. Industrial
biotechnology or white biotechnology uses enzymes or
microorganisms to make biobased products such as
chemicals, food and feed, detergents, paper and pulp,
textile and bioenergy with less waste generation &
reduce energy consumption.
 E.g- Saccharomyces cerevisiae , Aspergillus Niger,
Acetobacter acete etc

4. Interaction between industrial
biotechnology & industrial
microorganism
 Low cost
 Ethical issue
 Genetically modifiable
Others
 Increase yield
 Make stable
 Purity
 Microorganisms purity
 Product purity
 More nutrient

5. Importance of Industrial
Microorganisms
 Safety
 Environment friendly
 Purity
 Cost effective
 Easily available

6. Feature of Industrial
Microorganism
Genetically stable
Efficiency
 Culture efficiency
 Product efficiency
Simple nutritional value
Cheap carbon and energy source
Genetically manupulatable
Addition
Deletion
Insertion
Safety
Easy product recovery
 Extracellular
 Intracellular
Profitable byproduct

7. Sources of Industrially important
Microorganisms
Way to collect of desired microorganisms
• The place where we can get M.Os readily &
• Get M.Os. from culturing sample
How to select the sources ?
 Study
 Select the area
 Cost
I. Culture cost
II. Maintenance cost
III. Production cost
 Environment
 Health

8. Sources of Industrially important
Microorganisms
Sources of industrially important microorganism:
a) Institution:
 Commercial source
e.g- IFO(Osaka, japan), ATCC(USA), CMI(England) etc.
 Educational source
I. Lab
II. University
 Personal source

9. Sources of Industrially important
Microorganisms
b)Natural sources:
 Soil
 Industrial area soil
 Municipal waste soil
 Farmland soil
 Water
 Lake & river water
 Industrial area sewage
 Municipal area sewage
 Food & vegetables
 Animal and plants

10. Screening of Microorganism
 The procedure of isolation, detection, and separation of
microorganism or metabolize of our interest from a mixed
population by using highly selective methods is called
screening. Which deals with low cost, rapid growth and
easy to handle.

11. Screening
1. Primary screening:
 Sample collection
 Culture
 Media formulation
 Inoculums preparation
 Incubation
 Activity test
 Characterization
 Identification

12. Screening
2. Secondary screening
 Commercial value
 Environment friendly
 New product
 Toxicity
 Pathogenicity
 Economical stability
 Scale up
 Genetic stability
 Culture condition and media composition
 Fermentation condition

13. Strain improvement
 The Science and technology of manipulating and
improving microbial strains, in order to enhance their
metabolic capacities for biotechnological applications, are
referred to as strain improvement.

14. Why strain improvement is
necessary ?
Strain improvement is necessary to
 Reduce production cost
 Media and product cost
 To ensure safety
 Toxicity
 Pathogenicity
 Increase efficiency
 Yield
 Purity
 Longevity
 Increase stability
 Genetic & continuous culture

15. Methods of strain improvement
 Recombinant DNA technology
 Mutation
 Recombination
 Hybridization

16. 1. Recombinant DNA technology
 Genetic engineering, also known as recombinant DNA
technology, molecular cloning or gene cloning.
Recombinant DNA Technology enables isolation of genes
from an organism, this gene can be amplified, studied,
altered & put into another organism.

17. Recombinant DNA procedure
 i. Cutting of donor DNA
 ii. Cloning of a gene
 iii. Transformation

19. 2. Mutation
 Mutation: A mutation is a sudden and heritable change in
the traits of an organisms. Application of mutagens to
induce mutation is called mutagenesis.
 Types of mutation:
1. Natural mutation
2. Induced mutation

20. 1. Natural mutation
 Natural mutation: Mutation occurring without any specific
treatment are called natural mutation. The causes of
spontaneous mutation which are thus for understood
include integration and excision of transposons along
with errors in the functioning of enzyme such as DNA
polymerase recombination enzymes and DNA repair
enzyme.

21. 2. Induced mutation
 Induced mutation: Mutation are resulting due to a
certain treatment are known as induced mutation.
 Induced mutation also classified into two type:
I. Physical mutation
II. Chemical mutation

22. Induced mutation
 Physical mutation caused by physical agents include X ray, gamma
ray and ultraviolet radiations. X rays and gamma ray break the covalent
bonds in DNA molecules thereby producing fragment. Double stand
break result in major structural changes such as translocation, inversion
or similar chromosomal mutation.
 Chemical mutation is the interaction of certain chemical compounds
and cell metabolism may result in genetic changes in DNA structure,
affecting one or more genes. Some chemical mutagenic agents are
HNO2(nitrous acids), Alkylating agents, formaldehyde, benzene, DDT.

23. Results of mutation
a)Genome mutation: : Mutation occur in chromosome level is
called genome mutation.
b) Chromosome mutation: when mutation occur in gene level it
is called chromosome mutation. ( eg: deletion ,inversion,
duplication or translation)
c)Gene or point mutation: May result from changes in the base
sequence in a gene.
-Transition: purine-purine (A-G) and Pyrimidine-Pyrimidine (C-T)
-Transversion: Pyrimidine–purine or purine-pyrimidine

24. Results of mutation
 Frameshift: When one nucleotide or more is inserted ,deleted
thus altering the reading frame in the following transcription
and translation processes and lead to a changed amino acid
sequence in the resulting protein.

25. 3.Recombination
 Recombination: when sequence change with any process
that is called recombination. Recombination is used for both
genetic analysis as well as strain improvement.
 Recombination is two types
I. Sexual
II. Parasexual

26. Recombination
 Sexual: Some fungi used industrially have a complete sexual
cycle. In these organisms, nuclear fusion results after the fusion
of hypae has led to a mix together of nuclei in the heterokaryotic
mycelium.
 Parasexual: Some of the most economically useful fungi such
as penicillium chrysogenum and cephalosporium acremonium
do not have sexual recombination . In parasexuality , the fusion
of two hypae of equal or different polarity results in a mycelium
with nuclei of both parent strain.

27. Others types
 Homologous: recombination occur in same locus is called
homologous recombination.
 Heterologous: recombination occur in foreign locus is
called heterologous recombination.

28. 4. Hybridization
 Hybridization: the process of an animal or plant breeding
where protoplast fused with an individual of another species or
variety is called hybridization.
 Types of hybridization-
a) Interspecies
b) Intraspecies

29. Hybridization
 Interspecies: This includes crosses between different species of the
same genus or of different genera. When two species of the same genus
are crossed, it is known as inter-species hybridization.
E.g- E.coli with bacillus.
 Intraspecies: The parents involved in hybridization belong to the same
species; they may be two strains, varieties or races of the same species.
It is also known as intraspecies hybridization.
e.g- hybridization between two E.coli

30. Preservation
 Preservation usually involves preventing the growth of bacteria,
fungi (such as yeasts), and other microorganisms, as well as
retarding the oxidation of fats which cause rancidity.
 Methods of preservation
i. Sub-Culturing
ii. cryopreservation
iii. With Mineral Oil
iv. Storage under Liquid Nitrogen
v. Freeze drying

31. 1. Repeated Sub-Culturing
 This is the most common, simplest and routine method of
preservation of microorganisms.
 Selected microorganisms are initially grown on agar
slants. After sufficient growth has taken place, they are
transferred to fresh medium before they lose their
viability.
 The appropriate time period for such transfer ranges from
a week to few months (generally four to eight months).

32. Repeated Sub-Culturing
 Advantages:
 1. This method is cheap,
 2. Needs no special equipment,
 3. Recommended for small collection centers and
 4. Retrieval easy
 Disadvantages:
 1. Change in physiological and genetical characters and
 2. Time consuming.

33. Preservation with Mineral Oil
 In this method tubes with sterile agar slants are inoculated with a
given culture.
 The tubes are incubated till sufficient growth of the given microbe
takes place.
 The grown up culture is covered with a suitable mineral oil to a
depth of about 1 cm above the top of the slanted surface using sterile
technique.
 Thus, over laid cultures can be stored at room temperature or
preferably at low temperature by about 15°C.

34. With Mineral Oil
 Advantages:
 This method of maintenance has the unique advantage that you can
remove some of the growth under the oil with a transfer needle,
inoculate a fresh medium and still original culture can be preserved.
It is easy to control mites problem.
 Disadvantages:
 1. Chances of air-borne contamination during sub-culturing are
more,
 2. Chances of mutations are more and
 3. Retarded growth or inability to sporulate on retrieval.

35. Storage under Liquid Nitrogen
 This method is also called as cryogenic storage method, because a
cryoprotective agent in the form of 10% glycerol is used. Industrially useful
microorganisms are stored under very low temperature ranging from -150°C
– 196°C.
 In this method ranging, low temperatures are created by employing liquid
nitrogen. Metabolic activities of microorganisms are reduced considerably
at this low temperature.
 This method is generally employed for the preservation of fungi,
bacteriophages, viruses, algae, yeasts, animal and plant cells, and tissue
cultures.

36. Storage under Liquid Nitrogen
Advantages:
 1. Viable cultures may be preserved for many years by this method,
 2. Though the equipment is costly, the process is economical.
 3. The cultures remain viable under these conditions for 10-30 years
without undergoing any change in their characteristics.
Disadvantages:
 1. Evaporation of liquid nitrogen and replacement of lost liquid
nitrogen regularly and periodically.
 2. The method is relatively expensive.

37. Freeze drying
 Freeze drying involves freezing of a culture followed by its drying under
vacuum which results in the temporary inhibition of metabolic activities of
microorganisms.
 1. The organism is allowed to grow to the maximum stationary phase on a
suitable sterilized medium.
 2. The cells are suspended in a protective medium like milk, serum or
sodium glutamate.
 3. A few drops of suspension are transferred to a glass ampoule.
 4. The ampoules are then frozen by immersing into a freezing mixture of
dry ice and alcohol at -78°C and are subjected to high vacuum until
evaporation takes place completely.
 5. The ampoules are then sealed and stored in a refrigerator

38. Freeze drying
Advantages:
 1. Culture once dried needs no further attention
 2. It needs very cheap storage equipment like refrigerator and
 3. It is easy to transport freeze-dried ampoules to far off places
in large numbers in relatively small boxes.
Disadvantages:
 1. This is expensive
 Need expertise

39. Isolation of industrially important
microorganism
 The term isolation refers to the separation of a strain from a natural,
mixed population of living microbes, as present in the environment,
for example in water or soil flora, or from living beings with skin
flora, oral flora or gut flora, in order to identify the microbe(s) of
interest.
Microorganism isolation has 4 different techniques
i. Crowded plate technique
ii. Auxanography
iii. Enrichment culture
iv. Indicator system

40. Crowded plate technique
This technique is primarily employed for detecting those
microorganisms, which are capable of producing antibiotics.
The steps of crowed plate technique are given bellow:
 This technique starts with the selection of a natural substrate like soil
or other source consisting of microorganisms.
 Then serial dilution of the soil or other source material for antibiotic
producing microorganism is made.
 Then pouring and spreading of dilution soil samples 0.1ml on nutrient
agar plate for 24-48 hours that gives 300 to 400 or more colonies per
plate.

41. Crowded plate technique
 Colonies showing antibiotic activity are indicated by zone of
inhibition around the colony.
 Such colonies are sub culture and purified by streak before
making stock culture.
 The purified cultures are than tested to find the microbial
inhibition spectrum.

42. Auxanography technique
 Auxanography technique: This technique is employed for the
detection and isolation of microorganisms capable of producing
certain extracellular substances such as growth stimulating factors
like amino acids, vitamins etc.
This technique has two major steps are:-
A) Preparation of first plate:
 A filter paper is put across the bottom of petri dish.
 The nutrient agar is prepared and poured on the paper disc.
 Allowed to solidify
 Soil sample is diluted and proper dilutions are inoculated

43. Auxanography technique
B)Preparation of second plate:
 A minimal media lacking the growth factors is prepared and seeded
with the test organism.
 The seeded medium is poured onto fresh petri plate and the plate
allowed to set.
 The agar in first plate is then lifted and placed on the second plate
without inverting.
 The growth factor produced on agar can diffuse into the lower layer
containing test organism
 The zones of stimulated growth of test organism around colonies is an
indication that organism produce growth factor extracellularly.

44. Enrichment culture
 Enrichment culture is the use of certain growth media to
favor the growth of a particular microorganism over others by
enriching a component for the microorganism of interest. This
is generally done by introducing nutrients or environmental
conditions that only allow the growth of an organism of
interest.
 Such as, Skim milk agar is used to selectively isolate protease
producing species. Alkaline Peptone Water is used for the
cultivation of vibrio.

45. Enrichment culture
It consists of following steps:
a.) Nutrient broth is inoculated with microbial source material
and incubated.
b.) A small portion of all inoculums is plated onto the solid
medium and well isolated colonies are obtained.
c.) Suspected colonies from the plate are sub cultured on fresh
media and subjected for further testing.

47. Indicator system
 Indicator system: Microorganisms capable of producing acids or
amines from natural sources can be detected using this method by
incorporating certain pH indicator dyes such as neutral red or
bromothymol blue into nutrient agar medium. The change in the
color of a particular dye in the vicinity of a colony will indicate the
ability of that colony to produce an organic acid or base.
The steps of indicator system are given bellow:
 The pH indicating dye may be used for detecting microorganisms
that are capable of producing organic acid

48. Indicator system
 These dyes undergo color changes according to its pH
 Dye such as neutral red , bromothymol blue are added to the poorly
buffered nutrient agar media.
 Colonies are sub cultured to make stock culture.
 Further testing is needed since inorganic acids, base are also
metabolic products of microbial growth.
 Incorporation of CaCO3 in medium is also used to screen organic
acid producing microbes on basis of formation of clear zone of
dissolved CaCO3 around the colony.