Industrial microbiology and biotechnology

Industrial
Microbiology and
Biotechnology

CO3:
Ability to define, describe and utilize
microbial growth in fermentation and
biological process

At the end of the chapter, the
student should be able to:
 discuss the sources of microorganisms for use in
industrial microbiology and biotechnology
 discuss the genetic manipulation of microorganism to
construct strains that better meet the needs of an
industrial or biotechnological process
 discuss the preservation of microorganisms
 describe the design or manipulation of environments in
which desired processes will be carried out

 discuss the management of growth
characteristics to produce the desired product
 list the major products or uses of industrial
microbiology and biotechnology
 discuss the use of microorganisms in
manufacturing biosensors, microarrays, and
biopesticides
 discuss the manipulation of microorganisms in
the environment to control biodegradation

Introduction
 Industrial microbiology and
biotechnology involve the use of
microorganisms to achieve specific
goals
 Biotechnology has developed rapidly
due to the genetic modification of
microorganisms, particularly by
recombinant DNA technology

Choosing
Microorganisms for
Industrial
Microbiology and
Biotechnology

 The characteristics of microbes that are
desirable to the industrial microbiologist
are:
 genetic stability,
 easy maintenance and growth, and
 amenability to procedures for extraction and
purification of desired product

 Finding microorganisms in nature
 major sources of microorganisms for use in
industrial processes are soil, water, and
spoiled bread and fruits;
 only a minor portion of microbial species in
most environments have been identified

Genetic manipulation of
microorganisms
1) Mutation—once a promising culture is
found, it can be improved by
mutagenesis with chemical agents and
UV light
2) Protoplast fusion
• Widely used with yeasts and molds,
especially if the microorganism is asexual or
of a single mating type; involves removal of
cell walls, mixing two different solutions of
protoplasts, and growth in selective media
• Can be done using species that are not
closely related

3) Insertion of short DNA sequences
• site-directed mutagenesis is used to insert
short lengths of DNA into specific sites in
genome of a microorganism;
• leads to small changes in amino acid
sequence that can result in unexpected
changes in protein characteristics;
• site-directed mutagenesis is important to the
field of protein engineering

4) Transfer of genetic information between
different organisms
• Combinatorial biology—transfer of genes
from one organism to another
• Can improve production efficiency and
minimize purification of the product
• Numerous vectors are available for transfer
of genes

5) Modification of gene expression
• Can involve modifying gene regulation to
overproduce a product
• Pathway architecture and metabolic
pathway engineering—intentional alteration
of pathways by inactivating or deregulating
specific genes
• Metabolic control engineering—intentional
alteration of controls for synthesis of a
product

6) Natural genetic engineering
• employs forced evolution and adaptive
mutations;
• specific environmental stresses are used to
force microorganism to mutate and adapt,
this creates microorganism with new
biological capabilities

Preservation of
microorganisms
 strain stability is of concern;
 methods that provide this stability are
lyophilization (freeze-drying) and
 storage in liquid nitrogen

Microorganism
Growth in
Controlled
Environments

 Industrial microbiologists use the term
fermentation primarily to refer to the
mass culture of microorganisms; the
term has many other meanings to other
microbiologists (table 42.7)

Medium development
 Low-cost crude materials are frequently used as
sources of carbon, nitrogen, and phosphorus;
these include crude plant hydrolysates, whey
from cheese processing, molasses, and by-
products of beer and whiskey processing
 The balance of minerals (especially iron) and
growth factors may be critical; it may be
desirable to supply some critical nutrient in
limiting amounts to cause a programmed shift
from growth to production of desired metabolites

Growth of microorganisms
in an industrial setting
 Physical environment must be defined (i.e.,
agitation, cooling, pH, oxygenation);
oxygenation can be a particular problem with
filamentous organisms as their growth
creates a non-Newtonian broth (viscous),
which is difficult to stir and aerate
 Attention must be focused on the above
physical factors to ensure that they are not
limiting when small-scale laboratory
operations are scaled up to industrial-sized
operations

Culture tubes, shake flasks, and
stirred fermenters of various sizes
are used to culture microorganisms
 In stirred fermenters, all steps in growth and harvesting must be carried out
aseptically and computers are often used to monitor microbial biomass,
levels of critical metabolic products, pH, input and exhaust gas
composition, and other parameters
 Continuous feed of a critical nutrient may be necessary to prevent excess
utilization, which could lead to production and accumulation of undesirable
metabolic waste products
 Newer methods include air-lift fermenters, solid-state fermentation, and
fixed and fluidized bed reactors, where the media flows around the
attached or suspended microorganisms, respectively
 Dialysis culture systems allow toxic wastes to diffuse away from
microorganisms and nutrients to diffuse toward microorganisms

Microbial products are often
classified as primary or
secondary metabolites
 Primary metabolites are related to the
synthesis of microbial cells in the growth
phase; they include amino acids, nucleotides,
fermentation end products, and exoenzymes
 Secondary metabolites usually accumulate in
the period of nutrient limitation or waste
product accumulation that follows active
growth; they include antibiotics and mycotoxins

Major Products of
Industrial
Microbiology

Antibiotics
 Penicillin—careful adjustment of medium
composition is used to slow growth and to
stimulate penicillin production; side chain
precursors can be added to stimulate
production of particular penicillin derivatives;
harvested product can then be modified
chemically to produce a variety of
semisynthetic penicillins
 Streptomycin is a secondary metabolite that is
produced after microorganism growth has
slowed due to nitrogen limitation

Amino acids
 Amino acids such a lysine and glutamic
acid are used as nutritional
supplements and as flavor enhancers
 Amino acid production is usually
increased through the use of regulatory
mutants or through the use of mutants
that alter pathway architecture

Organic acids
 These include citric, acetic, lactic, fumaric, and
gluconic acids
 Citric acid, which is used in large quantities by the food
and beverage industry, is produced largely by
Aspergillus niger fermentation in which trace metals
are limited to regulate glycolysis and the TCA cycle,
thereby producing excess citric acid
 Gluconic acid is also produced in large quantities by A.
niger, but only under conditions of nitrogen limitation;
gluconic acid is used in detergents

 Specialty compounds for use in
medicine and health—include sex
hormones, ionophores, and compounds
that influence bacteria, fungi, amoebae,
insects, and plants

Biopolymers—microbially
produced polymers
 Polysaccharides are used as stabilizers, agents for
dispersing particulates, and as film-forming agents;
they also can be used to maintain texture in ice
cream, as blood expanders and absorbents, to make
plastics, and as food thickeners; also used to
enhance oil recovery from drilling mud
 Cyclodextrins can modify the solubility of
pharmaceuticals, reduce their bitterness, and mask
their chemical odors; can also be used to selectively
remove cholesterol from eggs and butter, to protect
spices from oxidation, or as stationary phases in gas
chromatography

Biosurfactants
 Biosurfactants are biodegradable
agents used for emulsification,
increasing detergency, wetting and
phase dispersion, as well as for
solubilization
 The most widely used biosurfactants
are glycolipids, which are excellent
dispersing agents

Bioconversion processes—
microbial transformations or
biotransformations
 Microorganisms are used as biocatalysts;
bioconversions are frequently used to
produce the appropriate stereoisomer; are
very specific, and can be carried out under
mild conditions
 When bioconversion reactions require ATP
or reductants, an energy source must be
supplied

Microbial Growth
in Complex
Natural
Environments

 Microorganisms can be used to carry out
desirable processes in natural
environments; in these environments,
complete control of the process is not
possible; processes carried out in natural
environments include:
 Biodegradation, bioremediation, and
environmental maintenance processes
 Addition of microorganisms to soils or plants for
improvement of crop production

Biodegradation using natural
microbial communities
 Biodegradation has at least three
definitions
 A minor change in an organic molecule,
leaving the main structure still intact
 Fragmentation of a complex organic
molecule in such a way that the fragments
could be reassembled
 Complete mineralization

 Some organic molecules exhibit
recalcitrance; they are not immediately
biodegradable

 Degradation of a complex compound
such as a halogenated compound occurs
in stages
 Dehalogenation often occurs faster under
anaerobic conditions; humic substances
may facilitate this stage
 Subsequent steps usually proceed more
rapidly in the presence of oxygen

 Structure and stereochemistry impact
rate of biodegradation (e.g., meta effect
and preferential degradation of one
isomer)
 Microbial communities change in
response to addition of inorganic and
organic substrates; these can impact rate
and extent of biodegradation (e.g.,
repeated contact with a herbicide leads
to the adaptation of the microbial
community and a faster rate of
degradation—acclimation)

 Land farming—waste material is
degraded after incorporation into soil or
as it flows across soil surface
 Biodegradation does not always reduce
environmental problems (e.g., partial
degradation can produce equally
hazardous or more hazardous
substances)
 Biodegradation can cause damage and
financial losses (e.g., corrosion of metal
pipes in oil fields)

Changing environmental
conditions to stimulate
biodegradation
 Engineered bioremediation—addition of
oxygen or nutrients to stimulate degradation
activities of microorganisms
 Stimulating hydrocarbon degradation in waters
and soils—usually involves addition of
nutrients and substances that increase contact
between microorganisms and substrate to be
degraded; can also involve aeration or creating
anoxic conditions

 Stimulating degradation with plants—
phytoremediation is the use of plants to
stimulate the extraction, degradation,
adsorption, stabilization or volatilization
of contaminants; transgenic plants can
be used
 Stimulation of metal bioleaching from
minerals—involves the use of acid-
producing bacteria to solubilize metals in
ores; may require addition of nitrogen
and phosphorous if they are limiting

 Biodegradation and bioremediation can
have negative effects that must be
controlled (e.g., unwanted degradation
of paper, jet fuels, textiles and leather)

Addition of microorganisms to complex
microbial communities—
bioaugmentation
 Addition of microorganism without considering protective
microhabitats
 Often fails to produce long-lasting increases in rates of biodegradation;
this may be due to three factors:
 Attractiveness of laboratory grown microbes as a food source for predators
 Inability of microorganisms to contact the compounds to be degraded
 Failure of the microorganisms to survive
 “Toughening” microorganisms by starvation before they are added has
increased microbial survival somewhat, but has not solved the problem
 Addition of microorganisms considering protective microhabitats—
adding microorganisms with materials that provide protection and/or
supply nutrients
 Living microhabitats—include surfaces of a seed, a root, or a leaf
 Inert microhabitats—include microporous glass or “clay hutches”

Biosensors
 Biosensors make use of microorganisms or microbial
enzymes that are linked to electrodes in order to detect
specific substances by converting biological reactions
to electric currents
 Biosensors have been or are being developed to
measure specific components in beer, to monitor
pollutants, to detect flavor compounds in foods, and to
study environmental processes such as changes in
biofilm concentration gradients; they are also being
used to detect glucose and other metabolites in
medical situations
 New immunochemical-based biosensors are being
developed; these are used to detect pathogens,
herbicides, toxins, proteins, and DNA

Microarrays
 Arrays of genes that can be used to
monitor gene expression in complex
biological systems
 Commercial microarrays are now
available for Saccharomyces cerevisiae
and Escherichia coli

Biopesticides
 Bacteria—(e.g., Bacillus thuringiensis) are being
used to control insects; accomplished by
inserting toxin-encoding gene into plant or by
production of a wettable powder that can be
applied to agricultural crops
 Viruses—nuclear polyhedrosis viruses (NPV),
granulosis viruses (GV), and cytoplasmic
polyhedrosis viruses (CPV) have potential as
bioinsecticides
 Fungi—fungal biopesticides are increasingly
being used in agriculture

Impacts of Microbial
Biotechnology
 Ethical and ecological considerations are
important in the use of biotechnology
 Industrial ecology—discipline concerned
with tracking the flow of elements and
compounds through biosphere and
anthrosphere

Industrial microbiology and biotechnology

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