Beruflich Dokumente
Kultur Dokumente
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
Detrimental
• Causative agents of diseases
• Causing food poisoning and spoilage
• Affecting water quality
• Deterioration of materials and products
• Biological warfare
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:
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
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.
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.
Others include:
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
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.
Sampling
Sampling 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 processing
Laboratory 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
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
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.
Environmental monitoring
Air 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 collection
Ensures 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.
Media
Eliminate 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.
Methods
All methods must be validated and documented in full.
Equipment
Should be tested and calibrated.
Staff
Proficient in the testing carried out.
Validation
Methods
Ensures that a method is giving the correct answer with respect to sensitivity, reproducibility and
repeatability
Products
Ensures that compounds are not interfering with the testing procedure and the ability to detect
microorganisms in test methods.
Sterilisation
Impossible to sterilise all products due to the heat lability of ingredients, it is time consuming,
and can be expensive.
Disinfection
Restricted to production facility and equipment because disinfectants are often toxic chemicals
that cannot be added to products.
Monitoring programs
Preventative measures, that show if quality control process are working correctly and used in the
prevention of product recalls
Preservatives
Prevents infection, growth of and spoilage by microorganisms in products
Legal aspects
• 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
Product formation
i. Primary metabolites
• Product formed during the primary growth (exponential) phase, e.g. ethanol
• Growth dependent
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
Downstream processing
• Cell harvesting
• Cell disruption
• Product recovery
Topic 3. Industrial processes
• 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)
Penicillin production
Penicillin is a β -lactam antibiotic, that is the β -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:
v. Addition of penicillin chemical building block to media: side chain precursors increase
production and influence the type of penicillin produced
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
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:
• α -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
Proteases
A 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.
Extremozymes
Enzymes 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.
• 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
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 temperature
Seawater 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
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
Microbial treatments
Types: inoculation, nutrient addition, or a combination
Results: selective plugging, biosurfactant production, or biopolymer production
Advantages: inexpensive, mobilises regions not accessible by other methods, microorganisms
could spread throughout the reservoir
iv. Metals
Geomicrobiology
Industrial 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.
Processes
i. Mineral bioformation: "Battery active" manganese dioxides
ii. 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
Mechanisms
a. Autotrophic
• Oxidative process using chemolithotrophic acidophilic sulfur and Fe oxidising microbes, e.g.
Thiobacillus and Sulfolobus
MS + 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
• Methanogenic bacteria at landfills, use as power and energy source to run plant, industry or
domestic purposes
vi. Solvents
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
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)
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
Mushrooms
• fruiting body of Agaricus spp., can be edible and poisonous
4. Pollution control
i. Bioremediation
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
Oily sludges
• Common contaminants: oily wastes from machinary and lubrication
Bioprocesses
1. Bioreactors: slurry or solid phase
2. Composting
3. Land farming: specialised form of composting for large scale organic contamination
4. 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
• 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
• 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
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
Note: illustrations in these notes are from Brock Biology of Microorganisms, Madigan, et al.
(2000)