Industrial Production & Bioremediation ?Microbes for industrial production ?Preservation of cultures...

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


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.


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 organism’s 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

Industrial Production & Bioremediation ?Microbes for industrial production ?Preservation of cultures...

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