Exploitation of Microorganisms

Exploitation of Microorganisms

Exploitation of Microorganisms

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Bioremediation

  • The use of microorganisms to consume and break down environmental pollutants, in order to clean a polluted site
  • Microbes can be used to tackle many contemporary environmental issues:
    • Heavy metal contamination
    • Oil spills
    • Pesticides and xenobiotics
    • Plastic waste
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Bioremediation: Heavy Metals

  • Major form of mercury in atomosphere: Hg0 - vapour
  • Oxidised to mercuric ion: Hg2+
  • Enters into aquatic environments - adsorbs to particulate matter
  • Metabolism by microorganisms results in methylation: CH3Hg+
  • Methyl-mercury binds to sulfhydryl groups (primarily cysteine amino acid residues). Acts as a molecular mimic to methionine so is distributed throughout the body. 100 times more toxic than elemental mercury
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Bioremediation: Heavy Metals

  • Several bacterial species can enzymatically biotransform methyl mercury or divalent mercury into less toxic elemental mercury
  • Enzymatic mercuric reductase 

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Bioremediation: Heavy Metals

  • Other species sequester heavy metals by secreting siderophores
  • Siderophores are biological chelators which are normally used to acquire iron from the environment. Have an extremely high affinity for metals and so can help improve removal from soil

Bioremediation: Oil Spills

  • HydrocarbonsL: rich source of organic material for microbial growth
  • Bulk storage - microbial growth is not desirable 
    • Oil-oxidising bacteria oil --> carbon dioxide
  • Oil-oxidising bacteria (Alcanivorax borkumensis) develop rapidly on slicks/films and eventually decompose the oikl
  • Bioremediation can be accelerated by the addition of nutrients (fertilisers) and oxygen. Ongoing debate on the use of dispersant agents - makes oil sink so less oxygen available to break down
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Bioremediation: Pesticides

  • One of the most widely used chemicals - 2 million tonnes estimated to be used each year
  • If they can be attacked by microorganisms:
    • eventually disappear from the soil
    • prevent toxic accumulations
    • save the bees
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Bioremediation: Polychorinated Biphenyls (PCBs)

  • Used as industrial coolants or insulators
  • Spills or leakage into the environment
  • Eventually reaches groundwater
  • Toxic, persistent and bio-accumulative
  • Reductive de-halogenation - PCBs can be decomposed by some microorganisms under anoxic conditions
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Bioremediation: Plastics

  • 40 billion kilograms produced per year
  • Half discarded in land fills - remain unaltered for decades
  • Biodegradable platics
    • Photobiodegradable
      • structure altered by sunlight (UV)
      • more amenable to microbial attack
    • Starch-based plastic
      • starch used to link short fragments of a second biodegradable polymer
  • Japanese researches have isolated species living on plastic waste (Ideonella sakaiensis) which can degrade PET into ethylene glycol and terapthalic acid - could be used in landfills to speed up decomposition of existing waste
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Antibiotics

  • Chemicals produced by microorganisms that kill or inhibit growth of other microorganisms
  • With the advent of synthetic synthesis the definition now includes substances partly or wholly produced through chemical synthesis
  • 1928 - original organism for the production of penicillin, Penicillum notatum, isolated as a chance contaminant (Alexander Fleming)
  • 1939 - Howard Florey and colleagues isolated the active ingredient from the mould, developed a powder and conducted the the first clinical trial
  • Only small quantities of the drug could be produced
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Antibiotics

  • 1941 - Andrew J. Moyer, found that culturing the organism in a specific broth, along with controlled aeration, resulted in much higher yields
  • Higher yielding strain, P.chrysogenum, isolated from an infected cantaloupe melon in U.S., could be used in a large scale fermentation process - key advance
  • Strain selection - fundamental geature of the use of microorganisms in biotechnology
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Antibiotics

  • Some antibiotics produced commercially

  • Streptomycetes produce >2/3 of clinically used antibiotics
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Toxins

  • The properties of some exotoxins allow them to be used medically
  • Botulinum Toxin
    • Clostridium botulinum - normally inhabits soil and water
    • May contaminate foods before harvest or slaughter
  • Botulism
    • Severe food poisoning caused by the consumption of food containing the exotoxin produced by C.botulinum
    • Most potent biological toxin known - attacks neural synapses
    • Causes hypotonia (muscle relaxation), paralysis, respiratory failure and death
  • Cosmetic industry: minute amounts - muscle relaxant (botox)
  • Medically: injected into patients suffering from dystonia - muscle spasms cause twisting and repetitive movements or poor posture
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Enzymes

  • Many pharmaceuticals are produced by haemolytic streptococcal strains
  • Streprokinase - Thrombolysis
    • Mammalian blood will clot if allowed to stand
    • Further standing it may dissolve as a result of an enzyme - plasmin
    • Plasmin usually present as inactive precursor - plasminogen
    • Administered intravenously to treat deep vein thrombosis or myocardial infarction
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Enzymes

  • Streptodornase
    • Liquefies puss - breaks down deoxyribonucleoprotein and DNA
    • Streptodornase and Streptokinase together used topically to liquefy blood clots and puss in wounds (Varidase)
  • L-asparaginase
    • Produced by many bacteria, e.g. Escherichia coli
    • Employed in cancer chemotherapy
    • L-asparagine is an essential requirement of some tumours
    • L-asparagine - selective, targeted treatment
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Dextrans

  • Polysaccharides produced commercially used as a plasma substitute
  • Lactic acid bacteria (Leuconostoc) can produce dextrans (from sucrose)
  • Glucose polymer (1,6alpha-linkages)
  • High/variable molecular weight
  • Dextran produced depends on the strain used for production

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Amino Acids

  • Pharmaceutical use: ingredients of infusion solutions for parenteral nutritional food supplements
  • Commercial use: food supplements
    • Produced commercially in fermentation processes
  • Brevibacterium flavum
  • Corynebacterium glutamicum
  • Both produce glutamic acid by a fermentation process
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Vitamins

  • Most made commercially through chemical synthesis
  • A few are too complex to be manufactured cheaply
  • Riboflavin (vitamin B2)
    • Deficiency characterised by enflamed tongue, dermatitis, 'burning feet'
    • Produced commercially in high yields by the mould Ashbya gossipii and the bacterium Bacillus subtilis
  • Vitamin B12
    • Synthesis in nature exclusively by microorgansisms, dietary needs satisified by food intake or absorption of the vitamin produced in the gut by microbes - Propionibacterium, Pseudomonas
    • Deficiency - low red blood cell, nervous system, dementia
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Vaccines

  • Live vaccines (attenuated)
    • symptomless infection - involve immune response and infection
    • Bacille Calmette-Guerin (BCG) strain causes tuberculosis in cattle not humans
    • Protection against human strain - cross protection
  • Killed vaccines
    • do not replicate/cause infection
    • Typhoid, cholera, influenza
  • Component vaccines
    • Hepatitis B (HBsAg)
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Insecticides

  • Bacillus thuringiensis
    • Crystalline inclusion - parasporal body
    • Endotoxin
    • Active against insect larvae (paralysis and death)
    • Spores produced as dusting powder)
    • GM crops contain cry genes - produce endotoxin
  • Bacillus sphaericus
    • Two proteins (51 and 42kDa) toxic to mosquito larvae
    • Microbe introduced into waters where larvae develop
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Brewing

  • Fermentable sugars and nutrients extracted from malt/sugar/hops
  • Placed into a fermentation vessel and brewing yeasts added
  • Ferment sugars and produces alcohol and carbon dioxide
  • Top fermenting yeasts - Saccharomyces cerevisiae
  • Production of ales
  • Bottom fermenting yeasts - Saccharomyces carlsbergensis
  • Production of lager
  • Yeasts broken down - full of B vitamins and proteins
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Probiotics

  • Live microorganisms which, when administered in adequate amounts, confer a health benefit on the host
  • Human intestine
    • Human intestine - contains 1.2kg of bacteria
    • Stable ecosystem: first few months of life to old age
  • Probiotics - evidence?
    • The general gut microflora prevent growth of pathogenic bacteria
    • Probiotics do not generally change the gut flora - must be taken daily to have any noticeable effect
    • Commercial strains: Lactobacillus and Bifidobacterium
    • Very profitable
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Antibiotic-Associated Diarrhoea

  • Systemic use of antibiotics disrupts normal gut flora
  • Clostridium difficile becomes a major pathogen
  • Produces two toxins - A and B, which cause psuedomembranous colitis, toxic megacolon and mortality
  • Vacomycin/metronidazole administered in combination with probiotics
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Genetic Engineering

  • Ralstonia metallidurans
    • Resistant to an array of heavy metals (zinc, cadmium, lead copper)
    • Able to breakdown a variety of pollutants
  • Recombinant strain (adding genes) used to decontaminate soils allowing re-vegetation
  • Resistance is plasmid-borne and can be exported to other organisms
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Genetic Engineering

  • Pharmaceuticals
    • Higher yielding strains
  • Probiotics
    • Probiotic E.coli strain engineered to secrete an anti-HIV peptide
    • Prevents HIV from entering cells
  • Insecticides
    • Expressing bacterial genes in plants
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Vaccines

  • Live vaccines (first generation)
    • Delete genes involved in disease
  • Component vaccines (second generation)
    • Relevant antigens expressed in bacteria, purified and administered
  • DNA vaccines (third generation)
    • Defined fragments of the pathogen's DNA which encode an antigen
    • Cloned into a plasmid and injected
    • DNA taken up by animal cell, transcribed and translated
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Mammalian Genes

  • Human proteins have high pharmaceutical value
  • Present in small amounts in normal tissue - costly to purify
  • Genetically engineered micoorganisms to produce these proteins
  • Insulin --> diabetes
  • Blood clotting factors --> haemophilia
  • Interferons --> anti-cancer agent
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