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  1. Introduction
    1. Industrial microbiology and biotechnology involve the use of microorganisms to achieve specific goals
    2. Biotechnology has developed rapidly due to the genetic modification of microorganism, particularly by recombinant DNA technology
  2. Choosing Microorganisms for Industrial Microbiology and Biotechnology
    1. Finding microorganisms in nature-major sources of microorganisms for use in industrial processes are soil, water, and spoiled bread and fruits; only a minor portion of microbial species in most environments have been identified; therefore, these traditional sources are still being searched for new microorganisms
    2. Genetic manipulation of microorganisms
      1. Mutation-once a promising culture is found, it can be improved by mutagenesis with chemical agents and UV light
      2. Protoplast fusion
        1. Widely used with yeasts and molds, especially if the microorganism is asexual or of a single mating type; involves removal of cell walls, mixing two different solutions of protoplasts, and growth in selective media
        2. Can be done using species that are not closely related
      3. Insertion of short DNA sequences-site-directed mutagenesis is used to insert short lengths of DNA into specific sites in genome of a microorganism; leads to small changes in amino acid sequence, but these can result in unexpected changes in protein characteristics; site-directed mutagenesis is important to field of protein engineering
      4. Transfer of genetic information between different organisms
        1. Combinatorial biology-transfer of genes (e.g., those for the synthesis of a specific product) from one organism to another
        2. Transfer of a gene into a different organism can improve production efficiency and minimize purification of the product
        3. Numerous vectors are available for transfer of genes
      5. Modification of gene expression
        1. Can involve modifying gene regulation to overproduce a product
        2. Pathway architecture and metabolic pathway engineering-intentional alteration of pathways by inactivating or deregulating specific genes
        3. Metabolic control engineering-intentional alteration of controls for synthesis of a product
      6. Natural genetic engineering-employs forced evolution and adaptive mutations; specific environmental stresses are used to force microorganism to mutate and adapt, this creates microorganism with new biological capabilities
    3. Preservation of microorganisms-strain stability is of concern; methods that provide this stability are lyophilization (freeze-drying) and storage in liquid nitrogen
  3. Microorganism Growth in Controlled Envrironments
    1. The term fermentation is primarily used by industrial microbiologists to refer to the mass culture of microorganisms; the term has many other meanings to other microbiologists (table 42.7)
    2. Medium development
      1. Low-cost crude materials are frequently used as sources of carbon, nitrogen, and phosphorus; these include crude plant hydrolysates, whey from cheese processing, molasses, and by-products of beer and whiskey processing
      2. The balance of minerals (especially iron) and growth factors may be critical; it may be desirable to supply some critical nutrient in limiting amounts to cause a programmed shift from growth to production of desired metabolites
    3. Growth of microorganisms in an industrial setting
      1. Physical environment must be defined (i.e., agitation, cooling, pH, oxygenation); oxygenation can be a particular problem with filamentous organisms as their growth creates a non-Newtonian broth (viscous), which is difficult to stir and aerate
      2. Attention must be focused on these physical factors to ensure that they are not limiting when small-scale laboratory operations are scaled up to industrial-sized operations
      3. Culture tubes, shake flasks, and stirred fermenters of various sizes are used to culture microorganisms
        1. In stirred fermenters, all steps in growth and harvesting must be carried out aseptically and computers are often used to monitor microbial biomass, levels of critical metabolic products, pH, input and exhaust gas composition, and other parameters
        2. Continuous feed of a critical nutrient may be necessary to prevent excess utilization, which could lead to production and accumulation of undesirable metabolic waste products
        3. Newer methods include air-lift fermenters, solid-state media, and surface-attached microorganisms (biofilms) in fixed and fluidized bed reactors, where the media flows around the suspended particles
        4. Dialysis culture systems allow toxic wastes to diffuse away from microorganisms and nutrients to diffuse toward microorganisms
      4. Microbial products are often classified as primary or secondary metabolites
        1. Primary metabolites are related to the synthesis of microbial cells in the growth phase; they include amino acids, nucleotides, fermentation end products, and exoenzymes
        2. Secondary metabolites usually accumulate in the period of nutrient limitation or waste product accumulation that follows active growth; they include antibiotics and mycotoxins
  4. Major Products of Industrial Microbiology
    1. Antibiotics
      1. Penicillin-careful adjustment of medium composition is used to slow growth and to stimulate penicillin production; side chain precursors can be added to stimulate production of particular penicillin derivatives; harvested product can then be modified chemically to produce a variety of semisynthetic penicillins
      2. Streptomycin is a secondary metabolite that is produced after microorganism growth has slowed due to nitrogen limitation
    2. Amino acids
      1. Amino acids such a lysine and glutamic acid are used as nutritional supplements and as flavor enhancers
      2. Amino acid production is usually increased through the use of regulatory mutants or through the use of mutants that alter pathway architecture
    3. Organic acids
      1. These include citric, acetic, lactic, fumaric, and gluconic acids
      2. Citric acid, which is used in large quantities by the food and beverage industry, is produced largely by Aspergillus niger fermentation in which trace metals are limited to regulate glycolysis and the TCA cycle, thereby producing excess citric acid
      3. Gluconic acid is also produced in large quantities by A. niger, but only under conditions of nitrogen limitation; gluconic acid is used in detergents
    4. Specialty compounds for use in medicine and health-include sex hormones, ionophores, and compounds that influence bacteria, fungi, amoebae, insects, and plants
    5. Biopolymers-microbially produced polymers
      1. Polysaccharides are uses as stabilizers, agents for dispersing particulates, and as film-forming agents; they also can be used to maintain texture in ice cream, as blood expanders and absorbents, to make plastics, and as food thickeners; also used to enhance oil recovery from drilling mud
      2. Cyclodextrins can modify the solubility of pharmaceuticals, reduce their bitterness, and mask their chemical odors; can also be used to selectively remove cholesterol from eggs and butter and protect spices from oxidation
    6. Biosurfactants
      1. Biosurfactants may replace chemically synthesized surfactants because of increased biodegradability, which thereby creates better safety for environmental applications
      2. The most widely used biosurfactants are glycolipids, which are excellent dispersing agents
    7. Bioconversion processes-microbial transformations or biotransformations
      1. Microorganisms are used as biocatalysts; bioconversions are frequently used to produce the appropriate stereoisomer, are very specific, and can be carried out under mild conditions
      2. When bioconversion reactions require ATP or reductants, an energy source must be supplied
      3. When freely suspended cells are used, the microbial biomass is usually used once and then discarded; immobilized biocatalysts (cells or enzymes) are attached to particulates so that they can be easily recovered and used again; immobilized biocatalysts are used in the bioconversion of steroids, degradation of phenol, and production of antibiotics, organic acids, and metabolic intermediates; biocatalysts are also used to recover precious metals from dilute-process streams
  5. Microbial Growth in Complex Environments A. Industrial microbiology and biotechnology can be carried out in natural environments; in these environments, complete control of the process is not possible; processes carried out in natural environments include:
    1. Biodegradation, bioremediation and environmental maintenance processes
    2. Addition of microorganisms to soils or plants for improvement of crop production
    B. Biodegradation using natural microbial communities
    1. Biodegradation has at least three definitions
      1. A minor change in an organic molecule, leaving the main structure still intact
      2. Fragmentation of a complex organic molecule in such a way that the fragments could be reassembled
      3. Complete mineralization
    2. Some organic molecules exhibit recalcitrance; they are not immediately biodegradable
    3. Degradation of a complex compound such as a halogenated compound occurs in stages
      1. Dehalogenation often occurs faster under anaerobic conditions; humic substances may facilitate this stage
      2. Subsequent steps usually proceed more rapidly in the presence of oxygen
    4. Structure and stereochemistry impacts rate of biodegradation (e.g., meta effect and preferential degradation on one isomer)
    5. Microbial communities change in response to physical and chemical changes in their environment; these can impact rate and extent of biodegradation (e.g., repeated contact with a herbicide leads to the adaptation of the microbial community and a faster rate of degradation)
    6. Land farming-waste material is degraded after incorporation into soil or as it flows across soil surface
    7. Biodegradation does not always reduce environmental problems (e.g., partial degradation can produce equally hazardous or more hazardous substances)
    8. Biodegradation can cause damage and financial losses (e.g., corrosion of metal pipes in oil fields)
    C. Changing environmental conditions to stimulate biodegradation
    1. Engineered bioremediation-addition of oxygen or nutrients to stimulate degradation activities of microorganisms
    2. Stimulating hydrocarbon degradation in waters and soils
      1. Marine environments-nutrients and substance that increase contact between microorganisms and substrate are added
      2. Subsurface environments-complicated by the limited permeability of subsurface geological structures; frequently involves stimulation of naturally occurring microbial communities by providing oxygen and nutrients
    3. Stimulating degradation with plants-phytoremediation is the use of plants to stimulate the degradation, transformation or removal of compounds, either directly or in conjunction with microorganisms; transgenic plants can be used
    4. Stimulation of metal bioleaching from minerals-involves the use of acid-producing bacteria to solubilize metals in ores; may require addition of nitrogen and phosphorous if they are limiting
    D. Biodegradation and bioremediation can have negative effects that must be controlled (e.g., unwanted degradation of paper, jet fuels, textiles and leather) E. Addition of microorganisms to complex microbial communities
    1. Addition of microorganism without considering protective microhabitats
      1. Often fails to produce long-lasting increases in rates of biodegradation; this may be due to three factors:
        1. Attractiveness of laboratory grown microbes as a food source for predators
        2. Inability of microorganisms to contact the compounds to be degraded
        3. Failure of the microorganisms to survive
      2. "Toughening" microorganisms by starvation before they are added has increased microbial survival somewhat, but has not solved the problem
    2. Addition of microorganisms considering protective microhabitats-adding microorganisms with materials that provide protection and/or supply nutrients
      1. Living microhabitats-include surfaces of a seed, a root, or a leaf
      2. Inert microhabitats-include microporous glass or "clay hutches"
  6. Biotechnological Applications
    1. Biosensors
      1. Biosensors make use of microorganisms or microbial enzymes that are linked to electrodes in order to detect specific substances by converting biological reactions to electric currents
      2. Biosensors have been developed to measure specific components in beer, to monitor pollutants, to detect flavor compounds in foods, and to detect glucose and other metabolites in medical situations
      3. New immunochemical-based biosensors are being developed; these are used to detect pathogens, herbicides, toxins, proteins, and DNA
    2. Microarrays
      1. Arrays of genes that can be used to monitor gene expression in complex biological systems
      2. Commercial microoarrays are now available for Saccharomyces cerevisiae and Escherichia coli
    3. Biopesticides
      1. Bacteria-(e.g., Bacillus thuringiensis) are being used to control insects; accomplished by inserting toxin-encoding gene into plant or by production of a wettable powder that can be applied to agricultural crops
      2. Viruses-nuclear polyhedrosis viruses (NPV), granulosis viruses (GV), and cytoplasmic polyhedrosis viruses (CPV) have potential as bioinsecticides
      3. Fungi-fungal biopesticides are increasingly being used in agriculture
  7. Impacts of Microbial Biotechnology
    1. Ethical and ecological considerations are important in the use of biotechnology
    2. Industrial ecology-discipline concerned with tracking the flow of elements and compounds through biosphere and anthrosphere

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