Industrial Production & Bioremediation

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 • 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 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