Industrial Industrial Microbiology and Microbiology and BiotechnologyBiotechnology

Khairul Farihan KasimKhairul Farihan Kasim

CO3: CO3:

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

At the end of the chapter, the At the end of the chapter, the student should be able to:student should be able to:

discuss the sources of microorganisms for use in discuss the sources of microorganisms for use in industrial microbiology and biotechnologyindustrial microbiology and biotechnology

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

discuss the preservation of microorganismsdiscuss the preservation of microorganisms

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

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

list the major products or uses of industrial list the major products or uses of industrial microbiology and biotechnologymicrobiology and biotechnology

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

discuss the manipulation of microorganisms in discuss the manipulation of microorganisms in the environment to control biodegradation the environment to control biodegradation

IntroductionIntroduction

Industrial microbiology and Industrial microbiology and biotechnology involve the use of biotechnology involve the use of microorganisms to achieve specific microorganisms to achieve specific goalsgoals

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

Choosing Choosing Microorganisms for Microorganisms for Industrial Industrial Microbiology and Microbiology and BiotechnologyBiotechnology

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

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

purification of desired productpurification of desired product

Finding microorganisms in natureFinding microorganisms in nature

major sources of microorganisms for use in major sources of microorganisms for use in industrial processes are soil, water, and industrial processes are soil, water, and spoiled bread and fruits; spoiled bread and fruits;

only a minor portion of microbial species in only a minor portion of microbial species in most environments have been identifiedmost environments have been identified

Genetic manipulation of Genetic manipulation of microorganismsmicroorganisms

1)1) Mutation—once a promising culture is Mutation—once a promising culture is found, it can be improved by found, it can be improved by mutagenesis with chemical agents and mutagenesis with chemical agents and UV lightUV light

2)2) Protoplast fusionProtoplast fusion• Widely used with yeasts and molds, Widely used with yeasts and molds,

especially if the microorganism is asexual or especially if the microorganism is asexual or of a single mating type; involves removal of of a single mating type; involves removal of cell walls, mixing two different solutions of cell walls, mixing two different solutions of protoplasts, and growth in selective mediaprotoplasts, and growth in selective media

• Can be done using species that are not Can be done using species that are not closely relatedclosely related

3)3) Insertion of short DNA sequencesInsertion of short DNA sequences• site-directed mutagenesis is used to insert site-directed mutagenesis is used to insert

short lengths of DNA into specific sites in short lengths of DNA into specific sites in genome of a microorganism; genome of a microorganism;

• leads to small changes in amino acid leads to small changes in amino acid sequence that can result in unexpected sequence that can result in unexpected changes in protein characteristics; changes in protein characteristics;

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

4)4) Transfer of genetic information between Transfer of genetic information between different organismsdifferent organisms

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

• Can improve production efficiency and Can improve production efficiency and minimize purification of the productminimize purification of the product

• Numerous vectors are available for transfer Numerous vectors are available for transfer of genesof genes

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

overproduce a productoverproduce a product• Pathway architecture and metabolic Pathway architecture and metabolic

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

• Metabolic control engineering—intentional Metabolic control engineering—intentional alteration of controls for synthesis of a alteration of controls for synthesis of a productproduct

6)6) Natural genetic engineeringNatural genetic engineering• employs forced evolution and adaptive employs forced evolution and adaptive

mutations; mutations;

• specific environmental stresses are used to specific environmental stresses are used to force microorganism to mutate and adapt, force microorganism to mutate and adapt, this creates microorganism with new this creates microorganism with new biological capabilitiesbiological capabilities

Preservation of Preservation of microorganismsmicroorganisms

strain stability is of concern; strain stability is of concern;

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

storage in liquid nitrogenstorage in liquid nitrogen

Microorganism Microorganism Growth in Growth in Controlled Controlled EnvironmentsEnvironments

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

Medium developmentMedium development Low-cost crude materials are frequently used as Low-cost crude materials are frequently used as

sources of carbon, nitrogen, and phosphorus; sources of carbon, nitrogen, and phosphorus; these include crude plant hydrolysates, whey these include crude plant hydrolysates, whey from cheese processing, molasses, and by-from cheese processing, molasses, and by-products of beer and whiskey processingproducts of beer and whiskey processing

The balance of minerals (especially iron) and The balance of minerals (especially iron) and growth factors may be critical; it may be growth factors may be critical; it may be desirable to supply some critical nutrient in desirable to supply some critical nutrient in limiting amounts to cause a programmed shift limiting amounts to cause a programmed shift from growth to production of desired metabolitesfrom growth to production of desired metabolites

Growth of Growth of microorganisms in an microorganisms in an industrial settingindustrial setting

Physical environment must be defined (i.e., Physical environment must be defined (i.e., agitation, cooling, pH, oxygenation); oxygenation agitation, cooling, pH, oxygenation); oxygenation can be a particular problem with filamentous can be a particular problem with filamentous organisms as their growth creates a non-organisms as their growth creates a non-Newtonian broth (viscous), which is difficult to stir Newtonian broth (viscous), which is difficult to stir and aerateand aerate

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

Culture tubes, shake flasks, and Culture tubes, shake flasks, and stirred fermenters of various sizes stirred fermenters of various sizes are used to culture are used to culture microorganismsmicroorganisms In stirred fermenters, all steps in growth and harvesting must be carried In stirred fermenters, all steps in growth and harvesting must be carried

out aseptically and computers are often used to monitor microbial out aseptically and computers are often used to monitor microbial biomass, levels of critical metabolic products, pH, input and exhaust gas biomass, levels of critical metabolic products, pH, input and exhaust gas composition, and other parameterscomposition, and other parameters

Continuous feed of a critical nutrient may be necessary to prevent excess Continuous feed of a critical nutrient may be necessary to prevent excess utilization, which could lead to production and accumulation of undesirable utilization, which could lead to production and accumulation of undesirable metabolic waste productsmetabolic waste products

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

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

Microbial products are often Microbial products are often classified as primary or classified as primary or secondary metabolitessecondary metabolites

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

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

Major Products of Major Products of Industrial Industrial MicrobiologyMicrobiology

AntibioticsAntibiotics Penicillin—careful adjustment of medium Penicillin—careful adjustment of medium

composition is used to slow growth and to composition is used to slow growth and to stimulate penicillin production; side chain stimulate penicillin production; side chain precursors can be added to stimulate precursors can be added to stimulate production of particular penicillin derivatives; production of particular penicillin derivatives; harvested product can then be modified harvested product can then be modified chemically to produce a variety of chemically to produce a variety of semisynthetic penicillinssemisynthetic penicillins

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

Amino acidsAmino acids

Amino acids such a lysine and glutamic Amino acids such a lysine and glutamic acid are used as nutritional acid are used as nutritional supplements and as flavor enhancerssupplements and as flavor enhancers

Amino acid production is usually Amino acid production is usually increased through the use of regulatory increased through the use of regulatory mutants or through the use of mutants mutants or through the use of mutants

that alter pathway architecturethat alter pathway architecture

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

gluconic acidsgluconic acids

Citric acid, which is used in large quantities by the food Citric acid, which is used in large quantities by the food and beverage industry, is produced largely by and beverage industry, is produced largely by Aspergillus nigerAspergillus niger fermentation in which trace metals fermentation in which trace metals are limited to regulate glycolysis and the TCA cycle, are limited to regulate glycolysis and the TCA cycle, thereby producing excess citric acidthereby producing excess citric acid

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

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

Biopolymers—microbially Biopolymers—microbially produced polymers produced polymers

Polysaccharides are used as stabilizers, agents for Polysaccharides are used as stabilizers, agents for dispersing particulates, and as film-forming agents; dispersing particulates, and as film-forming agents; they also can be used to maintain texture in ice cream, they also can be used to maintain texture in ice cream, as blood expanders and absorbents, to make plastics, as blood expanders and absorbents, to make plastics, and as food thickeners; also used to enhance oil and as food thickeners; also used to enhance oil recovery from drilling mudrecovery from drilling mud

Cyclodextrins can modify the solubility of Cyclodextrins can modify the solubility of pharmaceuticals, reduce their bitterness, and mask pharmaceuticals, reduce their bitterness, and mask their chemical odors; can also be used to selectively their chemical odors; can also be used to selectively remove cholesterol from eggs and butter, to protect remove cholesterol from eggs and butter, to protect spices from oxidation, or as stationary phases in gas spices from oxidation, or as stationary phases in gas chromatographychromatography

BiosurfactantsBiosurfactants

Biosurfactants are biodegradable agents Biosurfactants are biodegradable agents used for emulsification, increasing used for emulsification, increasing detergency, wetting and phase detergency, wetting and phase dispersion, as well as for solubilizationdispersion, as well as for solubilization

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

Bioconversion processes—Bioconversion processes—microbial transformations or microbial transformations or biotransformationsbiotransformations

Microorganisms are used as biocatalysts; Microorganisms are used as biocatalysts; bioconversions are frequently used to bioconversions are frequently used to produce the appropriate stereoisomer; are produce the appropriate stereoisomer; are very specific, and can be carried out under very specific, and can be carried out under mild conditionsmild conditions

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

Microbial Microbial Growth in Growth in Complex Natural Complex Natural EnvironmentsEnvironments

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

Biodegradation, bioremediation, and Biodegradation, bioremediation, and environmental maintenance processesenvironmental maintenance processes

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

Biodegradation using Biodegradation using natural microbial natural microbial communitiescommunities

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

leaving the main structure still intactleaving the main structure still intact Fragmentation of a complex organic Fragmentation of a complex organic

molecule in such a way that the fragments molecule in such a way that the fragments could be reassembledcould be reassembled

Complete mineralizationComplete mineralization

Some organic molecules exhibit Some organic molecules exhibit recalcitrance; they are not immediately recalcitrance; they are not immediately biodegradablebiodegradable

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

anaerobic conditions; humic substances anaerobic conditions; humic substances may facilitate this stagemay facilitate this stage

Subsequent steps usually proceed more Subsequent steps usually proceed more

rapidly in the presence of oxygenrapidly in the presence of oxygen

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

Microbial communities change in Microbial communities change in response to addition of inorganic and response to addition of inorganic and organic substrates; these can impact rate organic substrates; these can impact rate and extent of biodegradation (e.g., and extent of biodegradation (e.g., repeated contact with a herbicide leads repeated contact with a herbicide leads to the adaptation of the microbial to the adaptation of the microbial community and a faster rate of community and a faster rate of degradation—acclimation)degradation—acclimation)

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

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

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

Changing environmental Changing environmental conditions to stimulate conditions to stimulate biodegradationbiodegradation

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

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

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

Stimulation of metal bioleaching from Stimulation of metal bioleaching from minerals—involves the use of acid-minerals—involves the use of acid-producing bacteria to solubilize metals in producing bacteria to solubilize metals in ores; may require addition of nitrogen ores; may require addition of nitrogen and phosphorous if they are limitingand phosphorous if they are limiting

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

Addition of microorganisms to Addition of microorganisms to complex microbial communities—complex microbial communities—bioaugmentationbioaugmentation

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

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

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

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

Biotechnological Biotechnological ApplicationsApplications

BiosensorsBiosensors Biosensors make use of microorganisms or microbial Biosensors make use of microorganisms or microbial

enzymes that are linked to electrodes in order to detect enzymes that are linked to electrodes in order to detect specific substances by converting biological reactions specific substances by converting biological reactions to electric currents to electric currents

Biosensors have been or are being developed to Biosensors have been or are being developed to measure specific components in beer, to monitor measure specific components in beer, to monitor pollutants, to detect flavor compounds in foods, and to pollutants, to detect flavor compounds in foods, and to study environmental processes such as changes in study environmental processes such as changes in biofilm concentration gradients; they are also being biofilm concentration gradients; they are also being used to detect glucose and other metabolites in used to detect glucose and other metabolites in medical situationsmedical situations

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

MicroarraysMicroarrays

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

Commercial microarrays are now Commercial microarrays are now available for available for Saccharomyces cerevisiae Saccharomyces cerevisiae andand Escherichia coli Escherichia coli

Biopesticides Biopesticides Bacteria—(e.g., Bacteria—(e.g., Bacillus thuringiensisBacillus thuringiensis) are being ) are being

used to control insects; accomplished by used to control insects; accomplished by inserting toxin-encoding gene into plant or by inserting toxin-encoding gene into plant or by production of a wettable powder that can be production of a wettable powder that can be applied to agricultural crops applied to agricultural crops

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

Fungi—fungal biopesticides are increasingly Fungi—fungal biopesticides are increasingly being used in agriculturebeing used in agriculture

Impacts of Microbial Impacts of Microbial BiotechnologyBiotechnology

Ethical and ecological considerations are Ethical and ecological considerations are important in the use of biotechnologyimportant in the use of biotechnology

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