Industrial Production &

Bioremediation
Microbes for industrial production
Preservation of cultures
Methods of industrial production
Major products of industrial microbiology
Bioremediation
Biosensors & microarrays
Microbes for industrial production
Finding microorganisms in nature
Only a small percentage of microbial species
have been cultured
Bioprospecting: Hunting for new
microorganisms with potential for commercial
exploitation
Great deal of interest in microbes from extreme
environments
Challenge is to develop cost-effective
techniques for their culture
Microbes for industrial production
Genetic manipulation
Altering the characteristics of existing known
species to produce new and desirable
characteristics
Mutations can be induced with mutagenic
agents or UV irradiation
Example: Development of high-yield cultures of
Penicillium for penicillin production
Protoplast fusion can be used to fuse cells of
eukaryotic microbes and microbes that are not
phylogenetically related; used especially for
genetic manipulation in yeasts & molds
Microbes for industrial production
Genetic manipulation
Altering the characteristics of existing known
species to produce new and desirable
characteristics
Mutations can be induced with mutagenic
agents or UV irradiation
Example: Development of high-yield cultures of
Penicillium for penicillin production
Protoplast fusion can be used to fuse cells of
eukaryotic microbes and microbes that are not
phylogenetically related; used especially for
genetic manipulation in yeasts & molds
 Microbes for industrial production
Genetic manipulation
Altering the characteristics of existing known
species to produce new and desirable
characteristics
Mutations can be induced with mutagenic
agents or UV irradiation
Example: Development of high-yield cultures of
Penicillium for penicillin production
Protoplast fusion can be used to fuse cells of
eukaryotic microbes and microbes that are not
phylogenetically related; used especially for
genetic manipulation in yeasts & molds
Microbes for industrial production
Genetic manipulation
Altering the characteristics of existing known
species to produce new and desirable
characteristics
Mutations can be induced with mutagenic
agents or UV irradiation
Example: Development of high-yield cultures of
Penicillium for penicillin production
Protoplast fusion can be used to fuse cells of
eukaryotic microbes and microbes that are not
phylogenetically related; used especially for
genetic manipulation in yeasts & molds
Microbes for industrial production
Genetic manipulation
Altering the characteristics of existing known
species to produce new and desirable
characteristics
Mutations can be induced with mutagenic
agents or UV irradiation
Example: Development of high-yield cultures of
Penicillium for penicillin production
Protoplast fusion can be used to fuse cells of
eukaryotic microbes and microbes that are not
phylogenetically related; used especially for
genetic manipulation in yeasts & molds
 Microbes for industrial production
Genetic manipulation
Altering the characteristics of existing known
species to produce new and desirable
characteristics
Mutations can be induced with mutagenic
agents or UV irradiation
Example: Development of high-yield cultures of
Penicillium for penicillin production
Protoplast fusion can be used to fuse cells of
eukaryotic microbes and microbes that are not
phylogenetically related; used especially for
genetic manipulation in yeasts & molds
Microbes for industrial production
Genetic manipulation
Site-directed mutagenesis is the insertion of
short segments of DNA (using recombinant
DNA technology) into a gene to lead to desired
changes in its protein product
Recombinant DNA can be transferred between
different organisms, creating combinations of
genes with exhibit desired characteristics
Shuttle vectors: Vectors (such as bacterial plasmids)
that can replicate in more than one species
Expression vectors: Vectors that have transcriptional
promoters capable of mediating gene expression in
the target species.
Microbes for industrial production
Genetic manipulation
Gene expression can be modified by altering
transcriptional regulation, fusing proteins, and
removing feedback regulation controls
This is used for pathway architecture, or metabolic
pathway engineering, to increase or regulate
production.
Natural genetic engineering
Growing cultures under marginal (stressful)
growth conditions and selecting for new strains
(spontaneous mutations) that have increased growth
in those conditions
 Preservation of cultures
Periodic transfer + refrigeration
Mineral oil slant + refrigeration
Washed culture + refrigeration
Freezing
Freezing with 50% glycerol
Drying
Lyophilization (freeze drying)
Ultracold freezing
Methods of industrial production
Medium development
Lower-cost ingredients, such as crude plant or
animal by-products, are used for cost-
effectiveness
Manipulating the levels of a limiting nutrient
may be critical to trigger or optimize the
production of a desired product
Methods of industrial production
Scaleup
Successive optimization of growth & product
yield from a small scale (such as a shaking
flask or small fermenter) to a large scale (such
as industrial scale fermenters)
Mixing, aeration, pH control, foaming, &
formation of filamentous growth or biofilms are
significant issues in scaleup
Methods of industrial production
Methods for mass culture
Batch fermentation
Continuous culture (chemostat)
Lift-tube fermentation
Solid-state fermentation
Fixed-bed reactors
Fluidized-bed reactor
Dialysis culture unit
Methods of industrial production
Primary & secondary metabolites
Primary metabolites are produced during the
growth phase of the microbe. Examples: amino
acids, nucleotides, fermentation end products,
and many types of enzymes
Secondary metabolites accumulate during
periods of nutrient limitation and waste
buildup. Examples: many antibiotics and
mycotoxins
 Major products
Antibiotics
Examples: penicillin & streptomycin
The yield of both of these antibiotics are
optimized by nutrient limitation (carbon &
nitrogen)
Recombinant DNA products
Proteins produced from genes introduced into
microbes via recombinant DNA techniques,
such as enzymes, peptide hormones,
recombinant vaccines
 Major products
Amino acids
Glutamic acid (monosodium glutamate) is
produced by regulatory mutants of
Corynebacterium glutamicum that have a
modified Krebs cycle that can be manipulated
to shift -ketoglutarate to glutamate
production
Lysine is produced by a Corynebacterium
glutamicum strain in which homoserine lactone
synthesis is blocked
 Major products
Other organic acids
Acetic acid, citric acid, fumaric acid, gluconic
acid, itaconic acid, kojic acid, lactic acid
Speciality compounds
A variety of drugs (cholesterol drugs,
immunosuppressants, antitumor drugs),
ionophores, enzyme inhibitors, pesticides
Biopolymers
Microbial-produced polymers, mostly
polysaccharides, useful as thickening or gelling
agents in foods, pharmaceuticals, paints, etc.
 Major products
Biosurfactants
Microbial-produced detergents, such as
glycolipids; used in bioremediation applications
such as oil spill cleanups
Bioconversions
Using a microbe as a biocatalyst to convert a
substrate into a desired product; for example, in
the modification of steroid hormones
 Bioremediation
Biodegradion in natural communities
Includes:
minor changes in organic molecules, leaving the
main structure still intact
fragmentation of an organic molecule into smaller
organic molecules, still resembling the original
structure
complete mineralization of an organic molecule to
CO2
Recalcitrant compounds are organic compounds
that are resistant to biodegradation
 Bioremediation
Biodegradion in natural communities
Halogenated compounds, especially
halogenated aromatic compounds (such as
polychlorinated biphenyls) are often recalcitrant
The presence of halogens in a meta position
makes the compound more recalcitrant
Often one stereoisomer of an organic
compound will be biodegradable, while another
isomer will be recalcitrant
Specific organisms in an environment may be
able to degrade recalcitrant compounds, at
varying rates depending on the conditions
 Bioremediation
Biodegradation in natural communities
Sometimes partial degradation of a compound
may yield compounds that are worse; for
example, trichloroethylene can be degraded to
form highly carcinogenic vinyl chloride
Another example of detrimental biodegradation
is microbial corrosion of metal pipes
 Bioremediation
Stimulating biodegradation
Biodegradation by naturally-occurring
organisms may be stimulated by
Adding essential nutrients to the contaminated area
Providing aeration or limiting aeration, depending
on whether the contamination is better degraded
under aerobic or anaerobic conditions
Using plants and the microbial communities of their
rhizospheres (phytoremediation)
Using microbes for metal bioleaching from
minerals
 Bioremediation
Bioaugmentation
Adding microbes not normally found in an
environment to try to alter or accelerate the
biodegradation process
When the microbes are added without
consideration of their normal habitat (e.g.,
just adding a pure culture), there may be short-
term improvement but the added microbe
usually fails to establish a stable population
Better results are may be seen when the added
organisms microenvironment (nutrients,
oxygen, aeration, etc.) are included in the
bioaugmentation strategy
 Biosensors & microarrays
Biosensors
Devices in which a biospecific molecule (e.g., a
monoclonal antibody or a hormone receptor
protein) is attached to a transducer (often a
piezoelectrically-active quartz chip)
When the biosensor binds to its target, it slighty
twists the transducer, creating a small
electrical current that can be amplified,
detected, and measured
 Biosensors & microarrays
Microarrays
A series of microscopic DNA spots on a glass,
plastic, or silicon backing; used to monitor
levels of gene expression for thousands of
genes simultaneously, or to determine
differences in genotype