Industrial Microbiology and Biotechnology

JAMMALA VAMSIKRISHNA

CO3:

Ability to define, describe and utilize microbial growth in fermentation and biological process

At the end of the chapter, the student should be able to: discuss the sources of microorganisms for use in

industrial microbiology and biotechnology

discuss the genetic manipulation of microorganism to construct strains that better meet the needs of an industrial or biotechnological process

discuss the preservation of microorganisms

describe the design or manipulation of environments in which desired processes will be carried out

discuss the management of growth characteristics to produce the desired product

list the major products or uses of industrial microbiology and biotechnology

discuss the use of microorganisms in manufacturing biosensors, microarrays, and biopesticides

discuss the manipulation of microorganisms in the environment to control biodegradation

Introduction Industrial microbiology and

biotechnology involve the use of microorganisms to achieve specific goals

Biotechnology has developed rapidly due to the genetic modification of microorganisms, particularly by recombinant DNA technology

Choosing Microorganisms for Industrial Microbiology and Biotechnology

The characteristics of microbes that are desirable to the industrial microbiologist are:

genetic stability, easy maintenance and growth, and amenability to procedures for extraction and

purification of desired product

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

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• 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

• 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 that can result in unexpected changes in protein characteristics;

• site-directed mutagenesis is important to the field of protein engineering

4) Transfer of genetic information between different organisms

• Combinatorial biology—transfer of genes from one organism to another

• Can improve production efficiency and minimize purification of the product

• Numerous vectors are available for transfer of genes

5) Modification of gene expression• Can involve modifying gene regulation to

overproduce a product• Pathway architecture and metabolic

pathway engineering—intentional alteration of pathways by inactivating or deregulating specific genes

• 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

Preservation of microorganisms

strain stability is of concern;

methods that provide this stability are lyophilization (freeze-drying) and

storage in liquid nitrogen

Microorganism Growth in Controlled Environments

Industrial microbiologists use the term fermentation primarily to refer to the mass culture of microorganisms; the term has many other meanings to other microbiologists (table 42.7)

Medium development 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

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

Growth of microorganisms in an industrial setting

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

Attention must be focused on the above physical factors to ensure that they are not limiting when small-scale laboratory operations are scaled up to industrial-sized operations

Culture tubes, shake flasks, and stirred fermenters of various sizes are used to culture microorganisms 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

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

Newer methods include air-lift fermenters, solid-state fermentation, and fixed and fluidized bed reactors, where the media flows around the attached or suspended microorganisms, respectively

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

Microbial products are often classified as primary or secondary metabolites

Primary metabolites are related to the synthesis of microbial cells in the growth phase; they include amino acids, nucleotides, fermentation end products, and exoenzymes

Secondary metabolites usually accumulate in the period of nutrient limitation or waste product accumulation that follows active growth; they include antibiotics and mycotoxins

Major Products of Industrial Microbiology

Antibiotics 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

Streptomycin is a secondary metabolite that is produced after microorganism growth has slowed due to nitrogen limitation

Amino acids Amino acids such a lysine and glutamic

acid are used as nutritional supplements and as flavor enhancers

Amino acid production is usually increased through the use of regulatory mutants or through the use of mutants that alter pathway architecture

Organic acids These include citric, acetic, lactic, fumaric, and

gluconic acids

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

Gluconic acid is also produced in large quantities by A. niger, but only under conditions of nitrogen limitation; gluconic acid is used in detergents

Specialty compounds for use in medicine and health—include sex hormones, ionophores, and compounds that influence bacteria, fungi, amoebae, insects, and plants

Biopolymers—microbially produced polymers

Polysaccharides are 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

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, to protect spices from oxidation, or as stationary phases in gas chromatography

Biosurfactants Biosurfactants are biodegradable

agents used for emulsification, increasing detergency, wetting and phase dispersion, as well as for solubilization

The most widely used biosurfactants are glycolipids, which are excellent dispersing agents

Bioconversion processes—microbial transformations or biotransformations

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

When bioconversion reactions require ATP or reductants, an energy source must be supplied

Microbial Growth in Complex Natural Environments

Microorganisms can be used to carry out desirable processes in natural environments; in these environments, complete control of the process is not possible; processes carried out in natural environments include:

Biodegradation, bioremediation, and environmental maintenance processes

Addition of microorganisms to soils or plants for improvement of crop production

Biodegradation using natural microbial communities

Biodegradation has at least three definitions A minor change in an organic molecule,

leaving the main structure still intact Fragmentation of a complex organic

molecule in such a way that the fragments could be reassembled

Complete mineralization

Some organic molecules exhibit recalcitrance; they are not immediately biodegradable

Degradation of a complex compound such as a halogenated compound occurs in stages Dehalogenation often occurs faster under

anaerobic conditions; humic substances may facilitate this stage

Subsequent steps usually proceed more

rapidly in the presence of oxygen

Structure and stereochemistry impact rate of biodegradation (e.g., meta effect and preferential degradation of one isomer)

Microbial communities change in response to addition of inorganic and organic substrates; 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—acclimation)

Land farming—waste material is degraded after incorporation into soil or as it flows across soil surface

Biodegradation does not always reduce environmental problems (e.g., partial degradation can produce equally hazardous or more hazardous substances)

Biodegradation can cause damage and financial losses (e.g., corrosion of metal pipes in oil fields)

Changing environmental conditions to stimulate biodegradation

Engineered bioremediation—addition of oxygen or nutrients to stimulate degradation activities of microorganisms

Stimulating hydrocarbon degradation in waters and soils—usually involves addition of nutrients and substances that increase contact between microorganisms and substrate to be degraded; can also involve aeration or creating anoxic conditions

Stimulating degradation with plants—phytoremediation is the use of plants to stimulate the extraction, degradation, adsorption, stabilization or volatilization of contaminants; transgenic plants can be used

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

Biodegradation and bioremediation can have negative effects that must be controlled (e.g., unwanted degradation of paper, jet fuels, textiles and leather)

Addition of microorganisms to complex microbial communities—bioaugmentation

Addition of microorganism without considering protective microhabitats Often fails to produce long-lasting increases in rates of biodegradation;

this may be due to three factors: Attractiveness of laboratory grown microbes as a food source for predators Inability of microorganisms to contact the compounds to be degraded Failure of the microorganisms to survive

“Toughening” microorganisms by starvation before they are added has increased microbial survival somewhat, but has not solved the problem

Addition of microorganisms considering protective microhabitats—adding microorganisms with materials that provide protection and/or supply nutrients Living microhabitats—include surfaces of a seed, a root, or a leaf Inert microhabitats—include microporous glass or “clay hutches”

Biotechnological Applications

Biosensors 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

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 are also being used to detect glucose and other metabolites in medical situations

New immunochemical-based biosensors are being developed; these are used to detect pathogens, herbicides, toxins, proteins, and DNA

Microarrays

Arrays of genes that can be used to monitor gene expression in complex biological systems

Commercial microarrays are now available for Saccharomyces cerevisiae and Escherichia coli

Biopesticides 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

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

Fungi—fungal biopesticides are increasingly being used in agriculture

Impacts of Microbial Biotechnology

Ethical and ecological considerations are important in the use of biotechnology

Industrial ecology—discipline concerned with tracking the flow of elements and compounds through biosphere and anthrosphere