Prescott's Microbiology,4/e

		Microbiology, 4/e Prescott, Harley, Klein
		Instructor Resources

44 Industrial Microbiology and Biotechnology

CHAPTER OVERVIEW

This chapter discusses the uses of microorganisms in controlled processes that are grouped under the heading of industrial microbiology and biotechnology. In these situations it is possible to choose the particular microorganism or microbial community used, and it is also possible to define and control the environment in which the activity will take place. The goals of the process will determine whether the environmental conditions will be held constant or changed. The use of genetically engineered microorganisms to increase the efficiency of the processes and to produce new or modified products is discussed, as is the integration of biological and chemical processes to achieve a desired objective.

CHAPTER OBJECTIVES

After reading this chapter you should be able to:

! discuss the use of microorganisms and the management of microbiological processes in industrial microbiology and biotechnology

! discuss the use of genetic engineering methodologies in constructing microorganisms with specific genetic characteristics to meet desired objectives

! describe the design of suitable environments in which to carry out the desired processes

! discuss the management of growth characteristics to produce the desired product

! discuss the major products of industrial microbiology and biotechnology

! discuss the use of recombinant DNA techniques in molecular biology

! discuss hypermutation and forced evolution as mechanisms of creating new microbes and microbial productsCevolutionary biotechnology

! discuss protein engineering

! discuss the use of DNA chips for rapid sequencing of DNA

! discuss natural attenuation (i.e., bioremediation without the addition of specific microorganisms)

! discuss the use of biopolymers and cyclodextrins in medical, industrial and consumer products

! discuss the concepts of bioenhancement and bioremediation, and their uses in industrial microbiology and biotechnology

! discuss the use of microbial immobilization and biocatalysts

! discuss the linkage of microorganisms with electronics to produce biosensors

! discuss reductive dehalogenation as a mechanism for managing the degradation of polychlorinated biphenyls (PCBs)

CHAPTER OUTLINE

I. Industrial Microbiology and the New Biotechnology

A. Development of microorganisms with usable metabolic activities

1. Mutation and screening from the available gene pool is the traditional approach; it is still important

2. Intentional transfer of genetic information by recombinant DNA technology is a relatively recent development that complements the traditional approach

3. Producing a desired product in usable amounts is the goal

B. Industrial fermentationCmass culture (aerobic or anaerobic) to produce a desired product in the desired quantities

II. Microbial Growth Processes

A. Microbial culture

1. Culture tubes, shake flasks, and stirred fermenters of various sizes are used

2. All steps in growth and harvesting must be carried out aseptically

3. Computers are often used to monitor microbial biomass, levels of critical metabolic products, pH, input and exhaust gas composition, and other parameters

4. 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

5. Dialysis culture systems allow toxic wastes to diffuse away from microorganisms and nutrients to diffuse toward microorganisms

6. Continuous culture techniques frequently improve output rates, but they are not always desirable for other reasons

B. Medium development and growth conditions

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. 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

4. Physical environment must be defined: agitation, cooling, pH, oxygenation changes; oxygenation can be a particular problem with filamentous organisms, which limit the ability to stir and aerate the medium, thereby creating what is called a non-Newtonian broth (viscous)

5. Attention must be focused on these physical factors to ensure that they are not limiting microbial growth; they can be very different from small-scale laboratory operations as scaleup procedures are employed

C. Strain selection, improvement, and preservation

1. Look for new strains with desired characteristics, which usually involve increased product formation

2. Traditional mutation and screening procedures can then be used to further increase efficiency

3. Protoplast fusion procedures can be used to introduce desirable genetic characteristics

4. Molecular techniques such as the use of the AGene Gun@ or artificial chromosomes are now being used to introduce desirable genetic characteristics

5. Preservation and strain stability are of concern; methods available include:

a. Lyophilization (freeze-drying) and storage in liquid nitrogen

b. Maintenance in water agar and common growth media

III. Major Products of Industrial Microbiology

A. Primary metabolites are related to the synthesis of microbial cells and are usually produced during trophophase (exponential growth); they include: amino acids, nucleotides, fermentation end products, and exoenzymes

B. Secondary metabolites have no direct relationship to synthesis of cell materials and natural growth and are usually produced in idiophase (a period after active growth); they include: antibiotics and mycotoxins

C. Microorganisms in balanced growth generally do not accumulate cellular products in amounts beyond those needed for growth; therefore, in industrial fermentations the cell is often tricked into producing large excesses of the desired compounds

D. Antibiotics

1. Penicillin

a. Produced best when growth is not too rapid

b. Sidechain precursors can be added to stimulate production of a particular penicillin derivative

c. 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 if the nitrogen concentration is limited

E. Amino acidsClysine and glutamic acid are needed in large amounts; they are primary metabolites whose accumulation can be increased through the use of regulatory mutants that cannot limit their production and that have increased membrane permeability, which facilitates release of the accumulated products

F. 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

G. Bioconversion processes (microbial transformations or biotransformations) are frequently used instead of chemical transformation because of the high specificity of enzyme-mediated (biocatalyst) conversions in producing the appropriate stereoisomer and the mild conditions in which they can be carried out

1. Freely suspended cells are usually used only once and then discarded

2. Immobilized biocatalysts (cells or enzymes) are attached to particulates so that they can be easily recovered and used again

3. Immobilized biocatalysts are used in the bioconversion of steroids, degradation of phenol, and production of antibiotics, organic acids, and metabolic intermediates

4. Biocatalysts are also used to recover precious metals from dilute-process streams

IV. Molecular Biology and Biotechnology

A. Biochemical markers such as nutrient requirements or antibiotic resistance are often used to identify mutant or recombinant organisms

B. Modification of gene expression involves making specific changes in control regions so that desired enzymes are expressed constitutively, which leads to increases in product formation

C. Design of proteins and peptides

1. Site-directed mutagenesis and chemical synthesis of DNA can be used to tailor specific proteins (protein engineering); yeasts are becoming of increasing importance in the production of medically important proteins

2. It is possible to create enzymes that modify Aunnatural substrates@ (e.g., improve transformation of previously recalcitrant molecules)

3. Metabolic engineering involves the restructuring of pathways with engineered proteins to control production of end products and other small metabolites

4. Synthetic medical peptides that promote healing and blood coagulation, assist in treating cancer and AIDS, influence sexual dysfunction, and influence other processes have been developed

D. Hypermutation and Evolutionary Biotechnology

1. Forced mutation (evolution) is used to produce microbes with new degradative capabilities or which can produce compounds with new and unique properties

2. Optimizes production of new macromolecules at faster rates than normally occur in nature

E. DNA ChipsCcomputer chips designed with a layer of probes which allow detection of specific sequences and their statistical processing; used for sequencing and the detection of mutations; may lead to the development of chip-based technologies

F. Vectors for product expression

1. Vectors include artificial chromosomes

2. The gene for foot-and-mouth disease virus antigen has been incorporated into E. coli; the gene product can then be used for vaccine production

3. Other substances can be produced in other organisms, either in larger quantities than those produced by the original organism, or in some specifically modified form

4. Transgenic plants have been used for large-scale production of a variety of desired metabolic products and have been produced by shooting the DNA into the plant using the AGene Gun@

V. Other Microbial Products

A. BioinsecticidesCbacteria (e.g., Bacillus thuringiensis), viruses, and fungi are processed to form a wettable powder that can be applied to agricultural crops, where they are ingested by the insects, releasing toxins into the gut or otherwise killing the insect

1. B. thuringiensis releases a protoxin that is activated by a protease enzyme

2. Six of the active toxin units integrate into the membrane of a midgut cell forming a hexagonal-shaped pore leading to loss of osmotic balance and to cell lysis

3. This will only happen if a specific plasmid is present in the bacterium

4. The gene for the toxin has been successfully introduced into tomato plants that have subsequently become pest-resistant

5. Efforts are underway to make such transgenic plant modifications more cost-effective for routine production of commercial crops

6. Viruses such as nuclear polyhedrosis viruses (NPV), granulosis viruses (GV), and cytoplasmic polyhedrosis viruses (CPV) have potential as bioinsecticides

7. An important commercial viral pesticide (Elcar) is being used to control the cotton bollworm, Heliothis zea

8. Fungal biopesticides are increasingly being used in agriculture

9. The modification of baculoviruses to produce a potent scorpion toxin that is active against insect larvae is one of the most exciting advances in this field

B. Biopolymers include microorganism-produced polysaccharides that can be used to modify the flow characteristics of liquids and to serve as gelling agents, particulate dispersing agents, and film-forming agents; they also can be used to maintain texture in ice cream and to enhance oil recovery from drilling muds by increasing water contact and displacement of oil; cyclodextrins can modify the solubility of pharmaceuticals, reduce bitterness, mask odors, selectively remove cholesterol and protect spices from oxidation

C. 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

3. Biosurfactants have been used with the Exxon-Valdez oil spill

4. In bioremediation programs, the biosurfactant-producing microorganism can be added directly to an oil-containing geological structure; the biosurfactant will then be produced in situ; this may improve oil recovery, particularly from older fields

VI. Biodegradation and its managementCmicrobial-mediated destruction of paper, paint, metals, textiles, concrete, and other materials

A. Biodegradation stimulationCapplication of biodegradation to bioremediation efforts (removal of environmental pollutants using microorganisms); early work involved modification of the environment

1. Stimulation of an oil spill degradationCgenetically engineered Pseudomonas species have augmented hydrocarbon degradation plasmids; clean up is enhanced by the addition of certain nutrients to stimulate a hydrocarbon metabolism; use of this technology was part of a multiple approach strategy in the clean-up of the Exxon-Valdez spill in 1989

2. GEMs, although potentially beneficial, have generally not proven to be effective

a. They are used as a food source by protozoan predators

b. They frequently do not come in contact with the compounds to be degraded

c. They are unable to survive and compete with indigenous microbes

3. Biodegradation enhancement has recently taken advantage of Natural Attenuation; the environment is modified to promote biodegradation by indigenous microbes

4. Prolonged contact with Anon degradable@ materials such as PCBs may lead to the development of mutated organisms that can now degrade that material through a process called reductive dehalogenation

5. Subsurface biodegradation enhancementCinvolves similar principles as the oil-spill clean-up, but is complicated by the limited permeability of subsurface geological structures; frequently involves stimulation of naturally occurring microbial communities

6. Bioleaching of metalsCinvolves the use of Thiobacillus ferroxidans to solubilize copper in low-grade ores; soluble copper sulfate is formed and is then precipitated by reaction with elemental iron; the same process can be used to solubilize uranium

B. Contol of BiodeteriorationClimits undesired degradation

1. Jet fuelsCfungi will grow at the water-hydrocarbon interface; this can cause pump clogging; this is alleviated by growth inhibitors, filtering, and frequent cleaning of components

2. PaperCmicrobial growth causes loss of paper strength, discoloration, and increased deterioration; it is controlled by use of biocides and by washing equipment with hot alkali

3. Computer chipsCmicrobial contamination can decrease service life; the use of ultrapure water during the manufacturing of chips helps to alleviate the problem

4. PaintsCcontrolled by metal biocides as well as other biocides; common in limiting microbial growth in paints

5. Textiles and leatherCcontrolled by the use of fungicides

6. MetalsCmicrobial-mediated corrosion is particularly problematic in iron pipes; control is under investigation but not readily available yet

7. ConcreteCcan be dissolved by Thiobacillus spp. that produces sulfuric acid; this is controlled by organic inhibitors and the use of cements that do not contain appreciable levels of oxidizable sulfur compounds

VII. Biosensors

A. Biosensors involve the use of microorganisms or microbial enzymes linked to electrodes in order to detect specific substances by converting biological reactions to electric currents which can then be measured

B. 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

C. New biosensors using monoclonal and polyclonal antibodies with ssDNA-binding proteins are used to detect pathogens, herbicides, toxins, proteins and DNA

VIII. Impacts of Microbial BiotechnologyCethical and ecological considerations are important in the use of biotechnology

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