Industrial Production Bioremediation Microbes for industrial production Preservation

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 G Only a small percentage of microbial species have been cultured G Bioprospecting: Hunting for new microorganisms with potential for commercial exploitation G Great deal of interest in microbes from extreme environments G Challenge is to develop cost-effective techniques for their culture

Microbes for industrial production ? Genetic manipulation G Altering the characteristics of existing known species to produce new and desirable characteristics G Mutations can be induced with mutagenic agents or UV irradiation 6 Example: Development of high-yield cultures of Penicillium for penicillin production G 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 G 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 G Recombinant DNA can be transferred between different organisms, creating combinations of genes with exhibit desired characteristics 6 Shuttle vectors: Vectors (such as bacterial plasmids) that can replicate in more than one species 6 Expression vectors: Vectors that have transcriptional promoters capable of mediating gene expression in the target species.

Microbes for industrial production ? Genetic manipulation G Gene expression can be modified by altering transcriptional regulation, fusing proteins, and removing feedback regulation controls 6 This is used for pathway architecture, or metabolic pathway engineering, to increase or regulate production. G Natural genetic engineering 6 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 with 50% glycerol ? Drying ? Lyophilization (freeze drying) ? Ultracold freezing

Methods of industrial production ? Medium development G Lower-cost ingredients, such as crude plant or animal by-products, are used for costeffectiveness G 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 G 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) G Mixing, aeration, p. H control, foaming, & formation of filamentous growth or biofilms are significant issues in scaleup

Methods of industrial production ? Methods for mass culture G Batch fermentation G Continuous culture (chemostat) G Lift-tube fermentation G Solid-state fermentation G Fixed-bed reactors G Fluidized-bed reactor G Dialysis culture unit

Methods of industrial production ? Primary & secondary metabolites G Primary metabolites are produced during the growth phase of the microbe. Examples: amino acids, nucleotides, fermentation end products, and many types of enzymes G Secondary metabolites accumulate during periods of nutrient limitation and waste buildup. Examples: many antibiotics and mycotoxins

Major products ? Antibiotics G Examples: penicillin & streptomycin G The yield of both of these antibiotics are optimized by nutrient limitation (carbon & nitrogen) ? Recombinant DNA products G Proteins produced from genes introduced into microbes via recombinant DNA techniques, such as enzymes, peptide hormones, recombinant vaccines

Major products ? Amino acids G 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 G Lysine is produced by a Corynebacterium glutamicum strain in which homoserine lactone synthesis is blocked

Major products ? Other organic acids G Acetic acid, citric acid, fumaric acid, gluconic acid, itaconic acid, kojic acid, lactic acid ? “Speciality” compounds G A variety of drugs (cholesterol drugs, immunosuppressants, antitumor drugs), ionophores, enzyme inhibitors, pesticides ? Biopolymers G Microbial-produced polymers, mostly polysaccharides, useful as thickening or gelling agents in foods, pharmaceuticals, paints, etc.

Major products ? Biosurfactants G Microbial-produced detergents, such as glycolipids; used in bioremediation applications such as oil spill cleanups ? Bioconversions G 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 G Includes: 6 minor changes in organic molecules, leaving the main structure still intact 6 fragmentation of an organic molecule into smaller organic molecules, still resembling the original structure 6 complete mineralization of an organic molecule to CO 2 G Recalcitrant compounds are organic compounds that are resistant to biodegradation

Bioremediation ? Biodegradion in natural communities G Halogenated compounds, especially halogenated aromatic compounds (such as polychlorinated biphenyls) are often recalcitrant G The presence of halogens in a meta position makes the compound more recalcitrant G Often one stereoisomer of an organic compound will be biodegradable, while another isomer will be recalcitrant G Specific organisms in an environment may be able to degrade recalcitrant compounds, at varying rates depending on the conditions

Bioremediation ? Biodegradation in natural communities G Sometimes partial degradation of a compound may yield compounds that are worse; for example, trichloroethylene can be degraded to form highly carcinogenic vinyl chloride G Another example of detrimental biodegradation is microbial corrosion of metal pipes

Bioremediation ? Stimulating biodegradation G Biodegradation by naturally-occurring organisms may be stimulated by 6 Adding essential nutrients to the contaminated area 6 Providing aeration or limiting aeration, depending on whether the contamination is better degraded under aerobic or anaerobic conditions 6 Using plants and the microbial communities of their rhizospheres (phytoremediation) 6 Using microbes for metal bioleaching from minerals

Bioremediation ? Bioaugmentation G Adding microbes not normally found in an environment to try to alter or accelerate the biodegradation process G When the microbes are added without consideration of their “normal” habitat (e. g. , just adding a pure culture), there may be shortterm improvement but the added microbe usually fails to establish a stable population G 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 G 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) G 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 G 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