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Prescott’s Microbiology, 9th Edition 42 Biotechnology and Industrial Microbiology CHAPTER OVERVIEW Industrial microbiology exploits the range of genetic resources and natural products generated by microorganisms to produce compounds with industrial or medical application. Genetically engineered microorganisms can be used to increase the efficiency of the industrial processes and to produce new or modified products. Major products of industrial microbiology is discussed, including agricultural biotechnology and microbial energy conversion. LEARNING OUTCOMES After reading this chapter you should be able to: describe at least five compounds of industrial importance made by microbes define biocatalysis and explain its advantages over chemical synthesis compare the value of biofuel use with the challenges presented in its manufacture and transport discuss at least two types of biofuels explain the use of the term fermenter as it is used in an industrial setting describe two types of fermenters list three techniques used to optimize microbial output of industrial products compare and contrast three directed evolution technologies describe how metagenomics increases the pool of microbial products available for screening explain how the natural infection process used by Agrobacterium tumefaciens has been leveraged for the genetic modification of plants defend the use of Bt as a pesticide describe the use of microbes in nanotechnology explain the utility of biosensors CHAPTER OUTLINE I. Major Products of Industrial Microbiology A. Antibiotics 1. Predominantly produced by actinomycetes in the genus Streptomyces and by filamentous fungi 2. 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 B. Amino acids 1. Amino acids such as 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 C. Organic acids 1. These include citric, acetic, lactic, fumaric, and gluconic acids 1 © 2014 by McGraw-Hill Education. This is proprietary material solely for authorized instructor use. Not authorized for sale or distribution in any manner. This document may not be copied, scanned, duplicated, forwarded, distributed, or posted on a website, in whole or part. Prescott’s Microbiology, 9th Edition 2. Citric acid is used in large quantities by the food and beverage industry; produced largely by Aspergillus niger fermentation in which trace metals are limited to regulate glycolysis and the TCA cycle, thereby producing excess citric acid D. Biopolymers—microbially produced polymers 1. Used 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. Includes dextrans, polyesters, cellulose, and xantham gum E. Biosurfactants 1. Biosurfactants are biodegradable agents used for emulsification, increasing detergency, wetting and phase dispersion, as well as for solubilization 2. The most widely used biosurfactants are glycolipids, which are excellent dispersing agents; many have antimicrobial properties due to their amphipathic nature (disrupt membranes) F. Biocatalysts—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. Often used to carry out a minor modification of a biomolecule of commercial use G. Vaccines 1. Mining the genomic sequence of a pathogen allows reverse vaccinology where a possible antigen gene is identified in-silico and produced in a nonpathogenic organism 2. MenB (Neisseria meningitis serogroup B) vaccine was created this way and the technique is currently being applied to other pathogens such as Staphylococcus aureus and B. anthracis II. Biofuel Production A. Microbial energy conversion 1. Microbial transformation of organic materials into biofuels, such as ethanol and hydrogen that can be burned to fuel cars or other machines 2. Ethanol is currently used as a gasoline additive 3. Use of corn as a substrate for ethanol has escalated worldwide food prices 4. Biofuel has several disadvantages a. Absorbs water b. Cannot be shipped through existing pipelines c. Contains far less energy d. May consume more energy than is actually produced 5. Hydrogen as a biofuel compares favorably to ethanol and other fuels a. Has about three times more potential energy per unit weight than gas, it has the highest energy-content fuel available b. A diverse group of microbes produce hydrogen c. Can be produced through fermentation III. Growing Microbes in Industrial Settings A. Industrial microbiologists use the term “fermentation” primarily to refer to the mass culture of microorganisms; the term has many other meanings to other microbiologists B. Small-scale laboratory operations need to be scaled up to industrial-sized operations by maintaining culture conditions during the transition C. In stirred fermenters, all steps in growth and harvesting must be carried out aseptically and computers often are used to monitor microbial biomass, levels of critical metabolic products, pH, input and exhaust gas composition, and other parameters D. 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 E. Microbial products often are 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 IV. Microorganisms Used in Industrial Microbiology A. Genetic manipulation of microorganisms 1. Mutagenesis—cultures can be improved by mutagenesis with chemical agents and UV light 2 © 2014 by McGraw-Hill Education. This is proprietary material solely for authorized instructor use. Not authorized for sale or distribution in any manner. This document may not be copied, scanned, duplicated, forwarded, distributed, or posted on a website, in whole or part. Prescott’s Microbiology, 9th Edition 2. Protoplast fusion—involves removal of cell walls (to create protoplasts), mixing two different solutions of protoplasts, and growth in selective media to prompt recombination to make useful industrial strains 3. Genetic transfer between different organisms—functional genes are cloned and inserted into new hosts where heterologous gene expression enables the production of specific proteins and products 4. Modification of gene expression—modification of regulatory molecules or endogenous promoters on DNA can increase gene expression 5. Directed evolution a. Construction of production strains by specifically targeting genes of interest for mutagenesis b. Site-directed mutagenesis changes nucleotide sequences using a PCR-based approach that uses methylation patterns to protect mutated gene copies; only a few nucleotides are changed at a time c. Combinatorial biosynthesis uses knowledge of pathways to create altered nonprotein products (e.g., polyketide antibiotics) d. Systematic evolution of ligands by exponential enrichment (SELEX) creates large populations of engineered RNA molecules (aptamers) for use in therapies e. Selecting the best variants is more rapid using high-throughput screening (HTS) methods often using robotic 96-well plate assays 6. Metagenomics—since most environmental microbes do not grow in the laboratory, bioprospecting is often done by examining metagenomic libraries to identify new versions of known genes and new genes through functional screening after expression in common laboratory microbes V. Agricultural Biotechnology A. The Ti plasmid from Agrobacterium tumefaciens is used to introduce genetic constructs into plant cells; T-DNA within the plasmid transfers genes to plant cells in a process similar to that of transposons B. Biopesticides and bioinsecticides—include uses bacteria, viruses, and fungi, and their genes 1. Bacillus thuringiensis—being used to control insects; accomplished by inserting toxinencoding gene into the plant or by production of a wettable powder that can be applied to agricultural crops; the toxin gene also has been introduced into crop plants VI. Microbes as Products A. Nanotechnology 1. Diatom shells have precise structures at the micrometer scale; these can be grown and the silicon oxides replaced by magnesium oxides 2. Magnetosomes formed by bacteria are minute, perfectly formed magnetic beads with a membrane envelope that can be used for drug delivery or diagnostic techniques B. 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 or are being developed to measure specific components in beer, to monitor pollutants, to detect flavor compounds in foods, and to study environmental processes such as changes in biofilm concentration gradients; they also are being used to detect glucose and other metabolites in medical situations and to combat bioterrorism 3. New immunochemical-based biosensors are being developed; these are used to detect pathogens, herbicides, toxins, proteins, and DNA CRITICAL THINKING 3 © 2014 by McGraw-Hill Education. This is proprietary material solely for authorized instructor use. Not authorized for sale or distribution in any manner. This document may not be copied, scanned, duplicated, forwarded, distributed, or posted on a website, in whole or part. Prescott’s Microbiology, 9th Edition 1. Discuss how regulatory mutants are used (and why they are necessary) in the production of amino acids. Cite a specific example and describe the nature of the regulatory changes. Why is it desirable to increase membrane permeability in these organisms as well? 2. Genetically engineered microorganisms have been developed for use as pesticides. What steps should be taken to test the safety of these organisms before releasing them for use in a natural environment? Justify your choices. If you do not think they should ever be used, state your reasons. 3. Search the cabinets and shelves in your home and identify products containing compounds made by microorganisms. List the product, the microbial contribution, and describe the function of the compound in the product. 4. Ethanol in the United States is prepared largely from corn sugar fermentation and subsequent distillation. It is added to many of the gasoline blends to boost octane. Using corn ethanol as a biofuel has received some criticism from economists who believe it drives the cost of food up and does not yield as much energy as corn requires external fertilization. There is much interest in utilizing non-foodstuff sources of cellulose such as switchgrass (Panicum virgatum) to yield sugar for fermentation, but the conversion of cellulose to sugar is currently a chemical process that does not scale up readily. Comment on how an industrial microbiological approach might be applied to this problem. CONCEPT MAPPING CHALLENGE Provide your own linking words to construct a concept map using the following words: Ethanol Methane Microbial fuel cell Directed evolution Aptamers Fermentation Antibiotics Bioprospecting Amino acids Hydrogen Primary metabolite Production strain Biofuels Anaerobic digester Secondary metabolite Organic acids Site directed mutagenesis Combinatorial biosynthesis Natural product 4 © 2014 by McGraw-Hill Education. This is proprietary material solely for authorized instructor use. Not authorized for sale or distribution in any manner. This document may not be copied, scanned, duplicated, forwarded, distributed, or posted on a website, in whole or part.