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 Recombinant DNA products 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 “Speciality” compounds Biopolymers 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 Bioconversions 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 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 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