Industrial microbiology and biotechnology involve the use of microorganisms
to achieve specific goals
Biotechnology has developed rapidly due to the genetic modification
of microorganism, particularly by recombinant DNA technology
Choosing Microorganisms for Industrial Microbiology and Biotechnology
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; therefore, these traditional sources are still being searched
for new microorganisms
Genetic manipulation of microorganisms
Mutation-once a promising culture is found, it can be improved
by mutagenesis with chemical agents and UV light
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
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, but
these can result in unexpected changes in protein characteristics;
site-directed mutagenesis is important to field of protein engineering
Transfer of genetic information between different organisms
Combinatorial biology-transfer of genes (e.g., those for the
synthesis of a specific product) from one organism to another
Transfer of a gene into a different organism can improve production
efficiency and minimize purification of the product
Numerous vectors are available for transfer of genes
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
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 Envrironments
The term fermentation is primarily used by industrial microbiologists
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 these 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 media,
and surface-attached microorganisms (biofilms) in fixed and fluidized
bed reactors, where the media flows around the suspended particles
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 uses 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 and protect
spices from oxidation
Biosurfactants
Biosurfactants may replace chemically synthesized surfactants because
of increased biodegradability, which thereby creates better safety
for environmental applications
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
When freely suspended cells are used, the microbial biomass is
usually used once and then discarded; immobilized biocatalysts (cells
or enzymes) are attached to particulates so that they can be easily
recovered and used again; immobilized biocatalysts are used in the
bioconversion of steroids, degradation of phenol, and production of
antibiotics, organic acids, and metabolic intermediates; biocatalysts
are also used to recover precious metals from dilute-process streams
Microbial Growth in Complex Environments A. Industrial microbiology and
biotechnology can be carried out 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
B. 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 impacts rate of biodegradation (e.g.,
meta effect and preferential degradation on one isomer)
Microbial communities change in response to physical and chemical changes
in their environment; 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)
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)
C. 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
Marine environments-nutrients and substance that increase contact
between microorganisms and substrate are added
Subsurface environments-complicated by the limited permeability
of subsurface geological structures; frequently involves stimulation
of naturally occurring microbial communities by providing oxygen and
nutrients
Stimulating degradation with plants-phytoremediation is the use of
plants to stimulate the degradation, transformation or removal of compounds,
either directly or in conjunction with microorganisms; 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
D. Biodegradation and bioremediation can have negative effects that must be
controlled (e.g., unwanted degradation of paper, jet fuels, textiles and leather)
E. Addition of microorganisms to complex microbial communities
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"
Biotechnological Applications
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 developed to measure specific components in
beer, to monitor pollutants, to detect flavor compounds in foods,
and 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 microoarrays 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
