BBS3010 Molecular and applied microbiology

Module: Industrial microbiology

Topics Introduction Microbial contamination in industry Microbial biotechnology Industrial processes

Reading: Madigan, et al. (2000) Brock Biology of Microorganisms, 9th edition, Chapters 11, 16

Introduction

Microorganisms: importance in industry

1. Negative or detrimental effects: contamination, infection and spoilage of processes and products

2. Positive effects: produce valuable products or useful services

Effects of microorganisms in industry

Detrimental Causative agents of diseases Causing food poisoning and spoilage Affecting water quality Deterioration of materials and products Biological warfare

Beneficial (Microbial Biotechnology) Producing therapeutic compounds, foods and beverages, additives Pollution control Alternative fuels Agriculture Uses in genetic engineering and modern biotechnology

Topic 1. Microbial contamination in industryMicrobiology impacts on many industries, particularly manufacturing industries, and these include: Food: raw, cooked, processed, meats, vegetables, dairy, etc. Medical: devices, equipment Pharmaceutical: medicines, injectable, topical, oral, ophthalmic, ear, etc Cosmetic: topical (lotions, creams, shampoos, make up, etc) Biotechnology: cell cultures, media Chemical: fouling, biodegradable compounds

A. Problems caused by microorganisms in industry

The presence of large numbers of microorganisms can have a major effect on the quality of preparation and result in contaminated products. The major effects on the products are:

1. Spoilage: changes in appearance 2. Infection: growth of large numbers of pathogenic microorganisms3. Intoxication: growth of pathogenic microorganisms and formation of a toxin4. Ineffective product: microorganisms grow and breakdown ingredients5. Contaminated raw materials: potential for contaminating the final product6. Process contamination: contamination of the production process and equipment

B. Regulatory and advisory bodies

Regulatory and advisory bodies ensure that the safety and quality of products is maintained. They put in place the following: GMP: good manufacturing practice GLP: good laboratory practice Set limits and guidelines on products

Some of the more important regulatory and advisory bodies include:

Therapeutic goods administration (TGA)

The TGA is a federal government body that controls the quality, safety, efficacy and distribution of medicines and medical devices. Its regulatory framework is designed to ensure public health and safety.

The TGA Laboratories (TGAL) carry out independent chemical, microbiological, molecular biology, immunobiology, biomaterials testing. In particular, microbiological testing includes testing of non-sterile pharmaceuticals microbial loading and pathogens, and sterile pharmaceuticals and devices for any contamination.

National registration authority (NRA)

The NRA is the equivalent of the TGA for animals and the veterinary industry.

Australia New Zealand Food Authority (ANZFA)

ANZFA have the responsibility of developing, varying and reviewing standards for food available in Australia and New Zealand; coordinating national food surveillance and recall systems; and conducting research, assessing policies about imported food and developing codes of practice with industry.

National association of testing authorities (NATA)

NATA is a nationwide provider of laboratory accreditation. It aims at ensuring that a high standard of laboratory testing is maintained. In doing so, it conducts audits to ensure laboratories and staff are maintaining a high standard of testing and analysis.

Others include:

Environment protection agency (EPA): Controls microbial levels in waters, wastewaters, recreational waters and other pollution and contamination issues

Food and drug administration (FDA): body controlling food and therapeutic goods in the USA

US, British and European Pharmacoepia: associations which publish standards and procedures for testing and analysing 1000s of pharmaceutical products

C. Microbiology testing programs

Most manufacturing industries have testing programs to comply with regulatory bodies, to ensure products are safe and of acceptable quality, to ensure their quality control procedures are effective.

SamplingSampling is a critical step in any study within microbiology: medical, environmental, food, etc. If you don’t sample correctly then you won’t get a correct diagnosis of the microbial ecology of the particular environment, that is types and numbers present. It needs to give effective representation of the product or batch of product. Important factors to consider in sample collection are that the numbers and activity of the microorganisms are not altered, there is no contamination, and that samples are correct stored.

Sample processingLaboratory procedures used to determine the presence or numbers of microorganisms in samples. All sampling is aimed at optimum recovery of microorganisms. Microorganisms in samples are

usually present both in inappropriate numbers (too low or high) and with many other microorganisms, and may require concentration, dilution, selective and/or differential media or resuscitation of injured or stressed cells

1. General microbiological screening Measuring total numbers of microorganisms in samples of products (batches) Include: total (standard) plate count, yeast and mould, coliforms Limits usually allow some presence depending on the product (e.g. non sterile products

<100/g)

2. Pathogens Analysis of specific disease-causing microorganisms and the basis of product safety Generally limits are set at 0 presence Examples: Salmonella, Staphylococcus aureus, Clostridium perfringens, E.coli,

Enterococcus, Pseudomonas aeruginosa, Bacillus cereus, Legionella

3. Specialised tests

Sterility test (TGO 11) Sterile pharmaceuticals for humans and animals usually injectable substances, eye

preparations Large number of samples (2-20% of batches) added to aerobic and anaerobic liquid media at

different temperatures

Preservative efficacy test This test determines the antimicrobial effectiveness of a preservative in a pharmaceutical or

cosmetic product Along with chemical stability tests on the active ingredient, determines the shelf life of a

product Limits depend on the type of product

TGA disinfectant test (TGO 54) Testing the effectiveness and grade of a disinfectant, sterilants and antibiotics Bacteriocidal (antibacterial), sporicidal, fungicidal, virucidal, tuberculocidal Disinfectants can be hospital grade, household grade or antiseptic

Endotoxin testing The lipopolysaccharide of gram negative bacteria is often highly toxic to animals and

humans (endotoxin) Especially important for pharmaceuticals like antibiotics, intravenous solutions Test is by chromogenic LAL (Limulus amebocyte lysate) assay:

Ames test A microbial assay for detecting hazardous chemicals: mutagenicity and

carcinogenicity It uses a reverse or back mutation assay in histidine requiring Salmonella typhimurium

mutants, where the chemical induces his- to his+ reversion caused

by base changes or frameshift mutations within the bacterial genome Genetic toxicity is evidenced by an increase in the number of bacterial revertants

D. Quality control

Quality control covers all methods and operations and the documentation of these. It is aimed at consistent and reproducible production or testing every time, and to protect the manufacturer or testing lab legally. Regulatory authorities require that stringent controls are in place for quality.

The areas covered in a microbiology laboratory include:

Environmental monitoringAir and surfaces can contain many microorganisms that can contaminate industrial processes. Factory hygiene tests are done to assess the levels on floors, walls, equipment, drains, foods, etc.

Culture collectionEnsures that cultures should are readily available, genetically stable and stored. Pure cultures of microorganisms are maintained and preserved as reference, controls and comparisons with other organisms or reference, teaching or research purposes.

MediaEliminate the generation of erroneous test method data due to failure in the performance of media. Ensures that media is made up properly, components are not too old, they are properly sterilised, etc.

MethodsAll methods must be validated and documented in full.

EquipmentShould be tested and calibrated.

StaffProficient in the testing carried out.

Validation

MethodsEnsures that a method is giving the correct answer with respect to sensitivity, reproducibility and repeatability

ProductsEnsures that compounds are not interfering with the testing procedure and the ability to detect microorganisms in test methods.

Microbial control in industry

SterilisationImpossible to sterilise all products due to the heat lability of ingredients, it is time consuming, and can be expensive.

DisinfectionRestricted to production facility and equipment because disinfectants are often toxic chemicals that cannot be added to products.

Monitoring programsPreventative measures, that show if quality control process are working correctly and used in the prevention of product recalls

PreservativesPrevents infection, growth of and spoilage by microorganisms in products

Legal aspects

Responsibility of manufacturer to have adequate testing and preventative measures in place Responsibility of testing laboratory to testing is done properly according to regulatory bodies

Topic 2. Microbial biotechnology

Microorganisms can be used 3 ways industrially

1. Products of microbial metabolism: metabolic end product, pathway intermediates, enzymes, vitamins, products of competition

2. Bioconversion: microorganisms as biocatalysts, e.g. steroids, bioremediation3. Microbial cells or cellular components: foods or food supplements, immunising agents

against disease

Properties of an industrial microorganism

Genetically stable Suitable for scale-up Easily grown Rapid growth rate Grown on relatively cheap media or waste materials: molasses, bagasse, corn steep liquor,

whey, cassava, yam, potatoes, grapes, etc Amenable to genetic manipulation (mutation and selection)

Factors affecting growth of industrial microorganisms

Availability of carbon and energy sources Ability to replicate DNA Size and mass of cells Physical and chemical environment: media (balance between cost and maximum growth and

product formation), conditions Maintenance energy requirements: energy needed to keep the cell alive but not for growth Culture type: batch cultures (closed culture systems, nutrients are not renewed), continuous

cultures or chemostat (continuous supply of substrates gives better control over variables) Growth rates (increase in cells per unit time): cell population goes through a number of phases

when inoculated into fresh medium

Product formation

i. Primary metabolites Product formed during the primary growth (exponential) phase, e.g. ethanol Growth dependent

ii. Secondary metabolites Products formed near the end of the growth (or stationary) phase, e.g. antibiotics Not essential for growth and reproduction Highly dependent on growth conditions, repression frequently occurs

Usually produced from primary or intermediate metabolites, not the substrate

Industrial processes

Industrial processes involve taking advantage of normal cellular processes (e.g. fermentation, respiration) or genetic manipulating the microorganism yielding a useful or beneficial product

In large scale production, mass culture of microorganisms can be in a variety of reactors including: Controlled (computer) continuous culture fermenters: most common Batch fermenter Lift-tube fermenter Solid phase bioreactor Slurry phase bioreactors: a liquid-solid mixture (e.g. 10% slurry) Fixed and fluidised bed reactors: microorganisms are present as biofilms on inert surfaces Dialysis culture unit

Scale-up

Small laboratory scale, small vessel e.g. flask, bottle

Laboratory bioreactor, e.g. fermentor (1-10 L)

Pilot stage (300-3000 L)

Commercial or field stage

Downstream processing

Cell harvesting Cell disruption Product recovery

Topic 3. Industrial processes

1. Production of pharmaceutical chemicals

i. Antibiotics

Chemical substances produced by certain microorganisms which are active against other microorganisms

Produced mainly by Actinomycetes, fungi and Bacillus Produced as secondary metabolites Over 8000 antibiotic substances are known most unsuitable for large scale production: too

toxic, lack of therapeutic activity, unable to be produced commercially (e.g. growth problems, special requirements, recovery)

i. Inhibitors of cell wall synthesis Penicillins (produced by Penicillium chrysogenum and P.notatum) Cephalosporins: similar to penicillins (Cephalosporium sp.)

ii. Inhibitors of protein synthesis (produced by actinomycetes) Chloramphenicol Macrolides (erythromycin) Aminoglycosides (streptomycin, kanamycin) Tetracyclines

iii. Inhibitors of cell membrane function Nystatin Amphotericin B Polymixins

iv. Inhibitors of nucleic acid synthesis Rifampin Nalidixic acid Ciprofloxacin (ciprox)

Penicillin production

Penicillin is a -lactam antibiotic, that is the -lactam ring binds to transpeptidase enzyme in peptidoglycan synthesis. It is produced commercially by the fungus Penicillium chrysogenum

Production: an aerobic process in stirred fermenters (40-200 kL) with careful adjustment of conditions

Nutrients: the carbon source is generally lactose, ammonia or corn steep liquor is added for nitrogen and growth factors

Since the discovery of penicillin by Flemming in 1927, production has increased over 1000-fold. This has come about because of:

i. Improvement and manipulations in media conditions

ii. Isolation of a better penicillin-producing strain: P.chrysogenum

iii. Development of submerged culture technique (instead of shallow layer culturing)

iv. Selection of mutant strains: X-rays, UV light

v. Addition of penicillin chemical building block to media: side chain precursors increase production and influence the type of penicillin produced

vi. Improvement in recovery methods

ii. Steroids

Microorganisms as biocatalysts (biotransformation) Examples: adrenal cortical steroids (to reduce inflamation: arthritis and allergies), estrogens

and androgenic steroids

iii. Vaccines

Whole microbial cells, their parts or products (e.g. antigens and antibodies) are produced in large quantities and used for the production of vaccines

New vaccines are being produced by genetic engineering techniques Example: 1986 Viral hepatitis type B: genes for the harmless protein component inserted into

S.cerevisiae

iv. Others

Human insulin Treatment of diabetes Produced from genetically engineered strains of E.coli

Interferon

Proteins produced by animal cells infected with viruses, act to inhibit cell division and activate immune system

Genes for human interferons have been cloned in bacteria and yeasts

Human growth hormone Extremely small quantities from pituitary glands Can be produced from genetically engineered E.coli and mamalian cells

2. Commercially valuable non pharmaceutical chemicals

i. Enzymes

Many microorganisms are able to produce and excrete extracellular enzymes. Most of these are useful in the degradation of insoluble polymers. They are used in industries: food, dairy, pharmaceutical and textile industries.

Amylases A series of enzymes that convert starch to dextrin and sugars. They include: -amylase: shortens the starch polysaccharide chain glucoamylase: produces glucose monomers in saccharification process Glucose isomerase: conversion of glucose to the sweeter fructose syrup Invertase: converts disaccharide sucrose to glucose and fructose

ProteasesA mixture of enzymes that break down proteins. They have uses in laundry detergents, bating of leather (finer texture and grain), manufacturing liquid glue, clarifying beer protein haze.

ExtremozymesEnzymes that operate under extreme conditions (e.g. high or low temperature and pH, high salt). Biotechnological potential of new microorganisms (extremophiles) with unique features e.g. high stability and activity, reduced risk of contamination, novel enzymes. They can be produced through recombinant DNA technology without massive culturing of the source extremophiles.

Applications of enzymes from extremophiles include: DNA polymerases (Taq polymerase) - genetic engineering, forensics Proteases - food, baking (high temp), brewing, detergents (cold water washing) -amylase and amylopullulanase - starch processing acid tolerant enzymes: animal feed additives to aid in digestion in stomach glucose isomerase - sweetener production

ii. Polysaccharides

Example: alginate is the major structural component and easily extracted from marine brown algae and produced by certain bacteria e.g. Azotobacter vinelandii, widely used in the food and pharmaceutical industry for increasing viscosity and for gelling properties

Example: agar from red algae for microbiological media

iii. Oil recovery

Microbially-enhanced oil recovery: use of microorganisms to stimulate increased production of petroleum from oil reservoirs or fields

Petroleum reservoirs Porous rock formations containing oil, gas and water generally enclosed by a impermeable shale or

clay rock boundary Conditions differ greatly: temperature, pressure, chemical composition Types: On-shore or off-shore

High temperature or lower temperatureSeawater or freshwater

Petroleum Petroleum is a fossil fuel that is decayed organic molecules of biological origin Complex mixture of alkanes, cycloalkanes and aromatics Small amounts of sulfur, nitrogen, oxygen and metal-containing compounds Viscosity is higher than water Separation: distillation Gases are natural gas containing the more volatile alkanes

Microorganisms in petroleum reservoirsAnaerobes from the following physiological groups: fermentative bacteria, sulfate reducers, sulfur reducers, metal reducers, methanogens, acetogens

Conditions affecting the presence of microorganisms Temperature Pressure Salinity and dissolved ions pH Nutrient levels Toxic compounds Anaerobic environment

Oil recovery

a. Primary recovery Recovery of oil and gas under reservoir pressure Pumping

b. Secondary recovery Waterflooding: where multiple injection bores are drilled and water is injected to displace the oil

in the reservoir to a production bore or well 30% recovery

c. Tertiary (enhanced) recovery Thermal methods: hot water, steam or gas Miscible displacement: eg CO2

Chemical flooding: solvents, surfactants, polymers Selective plugging: gels or polymers Biological treatment

Microbial treatmentsTypes: inoculation, nutrient addition, or a combinationResults: selective plugging, biosurfactant production, or biopolymer productionAdvantages: inexpensive, mobilises regions not accessible by other methods, microorganisms could spread throughout the reservoir

iv. Metals

GeomicrobiologyIndustrial processes in mining for the extraction of metals and upgrading of minerals using microorganisms as alternatives to polluting processes. Geomicrobiology can be defined as the interaction between microorganisms and metals resulting in mineral formation and decomposition, or metal solubilisation, transport and deposition.

Processesi. Mineral bioformation: "Battery active" manganese dioxidesii. Microbial release of metals (bioleaching)

Solubilisation and recovery of metals from solid minerals or ores using autotrophic or heterotrophic microorganisms

iii. Mineral beneficiation Improvement or upgrade in mineral quality, e.g. recovery of gold, silver and coal

desulfurisation

Mechanismsa. Autotrophic Oxidative process using chemolithotrophic acidophilic sulfur and Fe oxidising microbes, e.g.

Thiobacillus and SulfolobusMS + O2 MSO4 (microbial oxidation)e.g. pyrite: FeS2 + 3½O2 + H2O FeSO4 + H2SO4

b. Heterotrophic Anaerobic respiration of the metal ore (ferric oxide, MnO2)

e.g. MnO2 + 4H+ + 2e- Mn2+ + H2O

v. Methane generation (natural gas)

Methanogenic bacteria at landfills, use as power and energy source to run plant, industry or domestic purposes

vi. Solvents

Butanol, acetone, ethanol (petroleum) fermentation for the chemical industry

3. Food and food supplements

i. Alcoholic beverages and alcohol-producing microorganisms

Carbohydrate fermentation: C6H12O6 2 CH3CH2OH + 2 CO2

All products: ethanol, CO2 and biomass are used industrially Primary metabolite

Wine Grapes provide the sugar source for the yeast Different processes for red and white wines: different grapes, longer aging for red wines and

fermentation takes place with pulp/skins for reds

Brewing Production of alcohol from malted grains Malt prepared from germinated barley seeds, natural enzymes convert starch to sugars, boiled

with hops and then fermented by yeast Yeast stains: top fermenting Saccharomyces cerevisiae for ales, bottom fermenting

Saccharomyces carlsbergensis for lager

Ethanol as fuel additive Substrates can be starch wastes or cheap starchy crops (e.g. cassava or corn starch),

converted to ethanol and can be used as a fuel additive or industrial solvent Gasohol: 10% ethanol in petrol

Bakers yeast (S.cerevisiae) Bread manufacturing: aerobic production of CO2 causes dough to rise and flavour production Single cell protein: yeast can be used as a protein and mineral supplement in diets,

particularly for animals Other uses: yeast extract in media, source of vitamins, source of enzymes for food industry

Ethanol producing organisms

i. Yeasts e.g. Saccharomyces cerevisiae Alcoholic beverages (wine, beer and spirits), bread manufacturing, single cell protein and

fuel ethanol Highly tolerant of alcohol levels

ii. Bacteria e.g. Zymomonas mobilis Alcoholic beverages (spirits: Tequila and tropical spirits) and fuel ethanol Tolerant up to 10% alcohol Higher ethanol yielding than yeast

ii. Dairy products

iii. Dairy products

Cheese starter, yoghurt, buttermilk Lactic acid bacteria e.g. Lactobacillus, Streptococcus lactis, S.cremoris, utilise the sugar

lactose from milk and dairy products and carry out fermentation to lactic acid and other metabolites (organic acids, CO2)

Other microorganisms used to add a variety of flavours e.g. diacetyl or acetaldehyde

iv. Probiotics

Protect against infection from pathogens: normal metabolism creates unfavourable conditions for pathogens e.g. acid production, competition for nutrients

L.acidophilus: probiotic (restores balance in the microbial community) available as powder, in yogurts

v. Vinegar

Acetic acid bacteria (Acetobacter and Gluconobacter) convert ethanol to acetic acid Can be produced from any alcoholic substance, common starting material is pure alcohol,

wine or apple cider (i.e. distilled, wine or cider vinegar)

vi. Fermented fruits and vegetables

Cucumber (pickles), cabbage (sauerkraut), soy sauce (fermented soy beans) Lactic acid, acetic acid or propionic acid bacteria grow and reduce the pH

vii. Others

Amino acids Lysine, glutamate (MSG) Carried out by regulatory mutants where the amino acids are over produced as metabolites,

e.g. Corynebacterium glutamicum

Organic acids citric acid (Aspergillus niger) lactic acid

Vitamins

Single cell protein

Mushrooms fruiting body of Agaricus spp., can be edible and poisonous

4. Pollution control

i. Bioremediation

Bioremediation is a process of biodegrading or removing environmental contaminants using microorganisms, that is stimulating microorganisms to remove toxic pollutants from a soil or water environment. The contaminant range can be easily biodegradable through to recalcitrant compounds including petroleum hydrocarbons, PAHs, solvents, xenobiotics.

Types of materials treated (areas contaminated)

Soils Surface or subsurface

Groundwaters Contaminated from subsurface leaks (tanks, pipes, etc) and spills around oil refineries,

service stations by mobile contaminants Particularly important in rural areas which draw water from aquifers, and run-offs into lakes

and rivers

Marine oil spills Environmental concern: mostly shoreline and sediments Surface slicks or open ocean spills are not such a problem: natural dispersion, biodegradation

(lighter easily biodegradable) and evaporation

Wastewaters and wastestreams Most common contaminants: emulsions of oils and hydrocarbons and other organic wastes

mixed with water from industrial processes

Oily sludges Common contaminants: oily wastes from machinary and lubrication

Bioprocesses1. Bioreactors: slurry or solid phase2. Composting3. Land farming: specialised form of composting for large scale organic contamination4. In-situ treatment: underground treatment e.g. groundwater contamination

Microorganisms

Widely distributed: more than 100 species (30 genera) known to oxidise or transform organic contaminants, a lot less can mineralise them e.g. Pseudomonas, Mycobacterium, Nocardia, Alcaligenes, White-rot fungi

Types: Naturally occurring Introduced Genetically engineered

Possible outcomes: Mineralisation: complete breakdown to cell biomass, CO2 and water Transformation: changing the pollutant to another chemical form

ii. Sewage treatment

Application of microorganisms for treatment of chemical and biological pollutants from the used water supply of a community

Wastewater: dissolved and solid matter in the water system from domestic and industrial activities and stormwater drains

Pollutants: faecal material, oils and greases, detergents and soaps, animal and vegetable matter, pesticides and fertilisers

Wastewater treatment process

1. Primary treatment (non biological) Removal of coarse solids by a physical process

2. Secondary treatment (microbial)Decomposition of organic matter by microorganismsSteps include: trickling filters: decomposition by Zoogloea activated sludge: aerobic decomposition by mixed bacteria oxidation ponds: Chlorella utilise inorganic nutrients sludge digestion: anaerobic decomposition

3. Tertiary treatment (advanced) (chemical and/or biological) Removes any additional pollutants and nutrients to yield a high quality wastewater Includes: chemical flocculation, final filtration, phosphate and nitrate removal (microbial or

chemical process), chlorination

iii. Control of greenhouse gases

Using microorganisms to remove carbon dioxide and methane from the atmosphere Example: farming of cyanobacteria and algae to remove CO2 from the atmosphere Example: the use of methylotrophic bacteria to use methane in the atmosphere

5. Agriculture

i. Biological control and bioinsecticides

The control of an agricultural pest (insect) or disease causing organism by a microorganism or microbial toxin to prevent infection of a plant

ii. Vaccines

Example: foot and mouth disease virus, gene for the protein coat of the virus inserted into E.coli plasmid, E.coli produce the protein

iii. Fertility of soil and symbiotic associations with plants

Example: Mycorrhizaea, a fungus-plant root association Example: Rhizobium (nitrogen fixation) form nodule on certain crop plants giving them a

have a selective advantage in low nitrogen soils

Note: illustrations in these notes are from Brock Biology of Microorganisms, Madigan, et al. (2000)