Mikrobiologi Industri

630 Microbiology for Teachers

Industrial Microbiology / Biocatalysis

Industrial Microbes

• Originally isolated from nature, but increasingly "improved" by genetic manipulation via mutagenesis and selection or recombinant DNA technology or protoplast fusion (fungi)
• To be useful in industrial microbiology, an organism must:
  • produce usable substance(s) or effect(s)
  • be available in pure culture
  • be genetically stable, but amenable to genetic manipulation
  • produce spores or other reproductive structures to allow easy inoculation
  • grow rapidly and produce product quickly in large-scale culture
  • grow in such a way that the cells are easily separated from the product
  • not be harmful to humans or agricultural plants and animals, etc.

Growth Conditions

• Composition of growth medium - generally use cheapest sources of carbon (molasses, whey, grains), nitrogen (ammonia and ammonium salts; distillers solubles), phosphorous, trace minerals and other growth factors (either of which may be used to regulate product generation)
• Other considerations - use aseptic conditions and maintain temperature, pH, oxygen concentration at optimal levels in the microenvironment in which each individual cell is growing and metabolizing
• Fermentors - many types (lift-tube, solid state, fixed bed, fluidized bed, dialysis, continuous) and sizes
  • aerobic fermentor - stainless steel cylinder with temperature control (cooling jacket vs. internal coils) and aeration system (sparger and impeller plus baffles) with process control and monitoring devices (real-time acquisition of data provides for "on-line" control of temperature, pH, pO₂, pCO₂, cell concentration, foaming, product concentration)
  • anaerobic fermentor - essentially the same as aerobic, but does not need aeration
• Scale-up of fermentation process: carried out by biochemical engineers in conjunction with microbiologists
  • steps - laboratory flask (0.1-1 L) --> laboratory fermentor (5-10 L) --> pilot plant (300-3000 L) --> commercial fermentor (10,000-400,000 L)
  • practical considerations - surface/volume ratio, uniformity of mixing (maintain appropriate conditions, especially oxygen transfer rate, at level of individual cell - consider microenvironment)

Primary vs. Secondary Metabolites

• Primary metabolite
  • metabolite formed in parallel with growth, during trophophase
  • example - alcoholic fermentation
• Secondary metabolite
  • metabolite formed after growth has occurred, during idiophase
  • characteristics:
    • formed by only a few organisms
    • not essential for growth and reproduction
    • dependent upon growth conditions - may be inducible
    • frequently produced from several intermediate products formed during trophophase; may require an inducer produced during trophophase
    • often produced as a group of related structures
    • possible to induce overproduction (not possible with primary metabolites)
  • example - antibiotic production

Biotransformation - microbial bioconversion

• Microbes can act as biocatalysts to carry out complex sequences of reactions very specifically
• Cost-effective because of high cost of chemical transformation alternatives
• Major uses include:
  • production of organic acids (chemicals), steroid hormones and antibiotics (pharmaceuticals)
  • precious metals recovery (Chlorella vulgaris)
  • bioleaching of metals - Thiobacillus ferrooxidans provides recovery of up to 70% of copper from low-grade ores

Microbial Products

• Foods
  • spoilage (any condition that alters color, texture, odor, and/or taste) and putrefaction (anaerobic breakdown of proteins)
    • intrinsic factors - pH, water availability, redox potential, physical structure, nutrient availability, antimicrobial agents present (aldehydes, phenolics - consider spices)
    • extrinsic factors - temperature, relative humidity, concentrations of oxygen and carbon dioxide, types and numbers of microbes present in environment
• Food preservation (consider "margin of safety" concept)
  • filtration - removes microbes from (heat labile) fluids
  • temperature
    • cooling retards microbial growth and extends holding times
    • heating
      • pasteurization (63ûC, 30 min; 71ûC, 15 sec; or 141ûC, 2 sec) kills harmful organisms
      • canning (115ûC for 25-100 min) sterilizes
        • thermal death point - lowest temperature required to destroy a microbial suspension
        • thermal death time - time required to destroy all organisms in a population
  • dehydration (lyophilization) - limits water availability
  • chemical preservatives - organic acids (benzoate), sulfite, nitrate, nitrite, and ethyl formate are "generally recognized as safe"
  • radiation
    • ultraviolet light (UV) controls populations of microbes on food preparation surfaces
    • gamma-rays and X-rays (⁶⁰Co source) kills cells throughout foods by peroxide generation
• Food production - variations on the theme of food preservation through "controlled spoilage"
  • dairy products - fermentation of milk generates lactic acid, which precipitates milk proteins and prevents other microbial growth
    • buttermilk (from skim milk) - Streptococcus diacetylactis
    • yogurt - Streptococcus thermophilus and Lactobacillus bulgaricus
    • sour cream (from cream) - Streptococcus diacetylactis
    • acidophilus milk - Lactobacillus acidophilus
    • cheeses (lactic acid fermentations; 2000 varieties, 20 types)
      • soft - cottage (Streptococcus lactis; Leuconostoc cremoris), cream (S. cremoris, S. diacetylactis, S. thermophilus, Lactobacillus bulgaricus, Brie (S. lactis, S. cremoris; Penicillium camemberti, P. candidum, Brevibacterium linens), mozzarella (S. thermophilus, L. bulgaricus)
      • semi-soft - monterey jack (S. lactis, S. cremoris), Muenster (S. lactis, S. cremoris; Brevibacterium linens), bleu (Roquefort - S. lactis, S. cremoris; Penicillium roqueforti)
      • hard - cheddar (Streptococcus lactis, S. cremoris, S. durans; Lactobacillus casei, L. plantarum), edam (S. lactis, S. cremoris), gouda (Streptococcus lactis, S. cremoris, S. diacetylactis), swiss (S. lactis, L. helveticus, S. thermophilus; Propionibacterium shermanii, P. freudenreichii)
      • very hard - parmesan (goat milk fermented and flavored by Streptococcus lactis, S. cremoris, S. thermophilus; Lactobacillus bulgaricus), romano (cow milk fermented and flavored by Streptococcus lactis, S. cremoris, S. thermophilus; L. bulgaricus)
  • meat products (salami, summer sausage) - Pediococcus cerevisiae and Lactobacillus plantarum
  • baked goods - Baker's yeast (Saccharomyces cerevisiae) aerobically generates carbon dioxide for breads and pastries
    • "regular" bread - S. cerevisiae provides flavor and carbon dioxide for "holes" to give light texture
    • sourdough bread - S. exiguus (provides flavor and carbon dioxide ) and Lactobacillus (provides flavor)
  • d) miscellaneous foods
    • coffee (coffee beans) - Erwinia dissolvens, Saccharomyces spp.
    • kimchee (napa = Chinese cabbage) - Lactobacillus
    • olives (green olives) - Leuconostoc mesenteroides together with Lactobacillus plantarum
    • pickles (cucumbers) - Leuconostoc mesenteroides together with Lactobacillus plantarum
    • sauerkraut (cabbage) - Leuconostoc mesenteroides together with Lactobacillus plantarum
    • soy sauce (soybeans) - Aspergillus oryzae or A. soyae, Streptococcus rouxii together with Lactobacillus delbrueckii
    • tempeh (tofu = soybean curd) - Rhizopus oligosporus or Rhizopus oryzae
    • vinegar (apple juice, wine) - Acetobacter or Gluconobacter
  • e) microbes as a direct food source
    • single-cell protein - Candida grown on sulfite waste "liquors" from paper manufacturing
      • approximately 50% protein, but also 16% nucleic acid
      • taste is objectionable to many
      • may cause kidney stones or gout (high nucleic acid content) when consumed in large quantities over long periods of time
    • Spirulina (cyanobacterium - photosynthetic)
    • mushrooms - Agaricus campestris bisporus
  • food additives and supplements
    • amino acids - used as food additives or starting materials in the chemical industry
      • microbes must be modified to overproduce their products by eliminating feedback inhibition and repression mechanisms as well as inducing secretion of product
      • examples - glutamic acid (MSG), phenylalanine and aspartic acid (aspartame = Nutrasweet), lysine, tryptophan
    • vitamins - food supplements for humans and animals
      • microbial production is feasible due to complexity and expense of chemical synthesis
      • more than $700 million in market value produced annually
      • examples - vitamin B1, riboflavin (Saccharomyces); ascorbic acid (Acetobacter)
    • brewer's yeast (Saccharomyces carlsbergensis) - used as vitamin B-rich food additive (relatively high in nucleic acids, though)
    • citric acid (Aspergillus niger) - food and beverage additive
• Alcoholic beverages
  • brews (beer, ale)
    • germinate grains (barley, wheat, rice) to allow amylase activity to release fermentable sugars (malting)
    • dry and crush malted grains, then rehydrate and allow further enzymatic activity (mashing)
    • add hops (dried flowers of the female vine Humulus lupulis), heat in brew kettle
    • remove hops, add Saccharomyces carlsbergensis for beer or S. cerevisiae for ale (pitching), then ferment 7-12 days (---> 2-5% ethanol)
    • "age" (lagering)
    • pasteurize or filter, then package
  • wines, champagnes
    • press grapes (must)
    • add Saccharomyces ellipsoideus, then ferment 3-5 days (---> 8-14% ethanol)
    • settle
    • "age"
    • bottle (champagne has "extra" sugar and is fermented after bottling to generate carbon dioxide for bubbles)
    • "age" some more...how long depends on the wine
  • distilled alcoholic beverages (whiskey, brandy, rum, etc.)
    • generate brew (Saccharomyces spp.)
    • distill to increase alcohol content to 25-100% ethanol
    • add flavoring, "age," then package
• Pharmaceuticals
  • products of genetically engineered microbes include:
    • insulin - treatment of diabetes
    • growth hormones - human (treat dwarfism), epidermal (promotes wound healing), bone (treat osteoporosis), animal (promotes livestock growth)
    • tissue plasminogen activator (TPA) - dissolves blood clots
    • blood clotting factors (VII, VIII, IX) - restore clotting mechanisms in hemophiliacs without chance of transmitting AIDS or hepatitis
    • erythropoietin - treatment of certain forms of anemia
    • cytokines - interferons (IFN), interleukins (IL), and other cytokines that act as anticancer agents or immune modulators
      • IFN-gamma stimulates cancer cells to produce tumor-associated antigens so they can be detected and eliminated by the immune system
      • IL-2 stimulates T cells to promote immune responses
      • tumor necrosis factor alpha (TNFa) and granulocyte-macrophage colony stimulating factor (GM-CSF) work together with IL-2 in cancer therapy
    • vaccine antigens - prevention of bacterial, fungal, metazoan, viral diseases (e.g., recombinant Hepatitis B vaccine now in use)
    • monoclonal antibodies (mAb)
      • diagnostic applications - determine ovulation, pregnancy; identification of infectious agents
      • therapeutic applications - specific drug delivery in cancer therapy; destruction of platelet-catalyzed blood clots in heart disease therapy
  • chemotherapeutic agents -
    • antibiotics are secondary metabolites produced by bacteria (Bacillus, Nocardia, Streptomyces) or fungi (Aspergillus, Cephalosporium, Penicillium)
    • more than 8000 known, several hundred discovered per year (but most are unusable)
    • more than 100 tons produced annually, worth more than $5 billion
    • cheaper to produce by fermentation than by chemical synthesis, but their structures (and thus their activities) may be modified by subsequent chemical steps (semisynthetic antibiotics)
    • steps toward commercial production include:
      • isolation - usually by screening (cross-streak method)
      • testing for toxicity and efficacy
      • optimization and purity of yield - gene amplification, other genetic engineering of microbes
      • developing extraction and purification steps - organic chemistry applications
    • examples:
      • ß-lactams
        • because they contain the ß-lactam group, they inhibit cell wall synthesis by blocking the transpeptidase that catalyzes peptide cross-linking
        • examples - penicillin, ampicillin, cephalosporins
      • aminoglycosides
        • these molecules containing amino sugars bonded by glycosidic linkage inhibit protein synthesis in Gram (-) bacteria by binding to 30S ribosomal subunits
        • examples - streptomycin, kanamycin, gentamicin
      • macrolides
        • these lactone rings connected to sugar moieties inhibit protein synthesis in Gram (+) bacteria by binding to 50S ribosomal subunits
        • (examples - erythromycin, oleandomycin, spiramycin, tylosin
      • tetracyclines
        • these naphthacene ring systems inhibit protein synthesis by binding to 30S ribosomal subunits
        • examples - chlortetracycline, oxytetracycline
• Commodity chemicals - organic compounds
  • acetic acid and other organic acids
  • acetone, butanol (Clostridium acetobutylicum), ethanol - solvents
  • ethanol - energy production (fuel component)
• Enzymes
  • secondary metabolites produced by bacteria or fungi during idiophase
  • useful (due to specificity of reaction) in food processing (especially dairy products), pharmaceutical, and textile industries
  • examples: proteases (detergent additives); amylase, glucoamylase and glucose isomerase (polysaccharide digestion to help start fermentations)
• Biopolymers
  • exopolysaccharides can be used as stabilizers, etc.
  • microbial plastics:
    • poly-ß-hydroxybutyrate (PHB), which is commonly used by some bacteria as a lipid storage material, can be used as a raw material for plastic based packaging materials
    • the nature of the raw material (and thus the plastic that can be synthesized from it) can be selected by varying the carbon source used to grow the bacteria - using acetate (C2) and butyrate (C4) yields PHB; caproate (C6) yields poly-ß-hydroxycaproate PHC; valerate (C5) yields poly-ß-hydroxyvalerate PHV; mixtures yield co-polymers
• Biosensors - bioelectronics utilize the abilities of microbes to measure pollutants and contaminants

Agricultural Uses of Microbes

• Rhizobium - facilitates nitrogen fixation in symbiotic legumes
• Agrobacterium tumefaciens - Ti plasmid is used as a gene vector for transferring genes coding for important functions to plants
• Bacillus thuringiensis spores contain protein crystals toxic for tomato worms
• polyhedrosis virus is used to control pine caterpillars and cotton bollworms

Biodeterioration Management

• Jet fuel - growth of Cladosporium resinal at water/fuel interface is controlled with fungicides
• Paper production - microbes and slime produced by them controlled with biocides (environmental problems)
• Electronics - microbes damage computer chips by growing on trace contaminants at junctions (must use ultrapure water - no microbes, no organics - to prevent this)
• Paints - growth of fungi, etc. in paints (esp. latex-based) is controlled by use of quaternary ammonium salts, barium salts and chlorinated phenolics (mercury compounds no longer used due to toxicity for humans)
• Textiles and leather goods - fungal growth is controlled with phenolics in textiles and copper compounds in leathers
• Metals and concrete - microbial metabolites (e.g., sulfuric acid produced by Thiobacilli) are frequently responsible for corrosion of concrete sidewalks, highways, bridges and building as well as sewer pipes

Biodegradation and Bioremediation

Bioremediation is also called Biodegradation Enhancement and includes any purposful use of microbes to degrade unwanted substances in the environment

• natural products
  • petroleum - certain bacteria (some cyanobacteria, pseudomonads, corynebacteria, mycobacteria), green algae and fungi (several molds and yeasts) oxidize hydrocarbons at aerobic water/oil interfaces (with optimal conditions, up to 80% removal within 1 year after a spill)
  • biodegradable plastics
    • photobiodegradable - structure of polymer altered by UV light in sunlight so that it is now amenable to biodegradation
    • biochemically biodegradable starch-linked polymers - starch-digesting bacteria in soil attack the starch, releasing polymer fragments which are degraded by other microbes
• xenobiotics - chemically synthesized compounds not found in nature (pesticides, synthetic polymers, etc.) and thus would seem unlikely to be degradable by naturally existing microorganisms; these products tend to be persistent in nature, and many nations are working to ban the use of many of them; microbes that can degrade xenobiotics are rather diverse and typically include both bacteria and fungi
  • PCBs - certain Pseudomonas species have been engineered to accelerate breakdown of polychlorinated biphenyls (formerly used by electric industry as transformer insulation)
  • PAHs (poly aromatic hydrocarbons) can be difficult to degrade, but there are microbes in the environment that can accomplish this task, especially when working together
  • pesticides - herbicides, insecticides and fungicides
    • these are typically rather complex molecules
    • some xenobiotics are good carbon sources and electron donors for soil microbes, so they are more readily degraded than others
    • other xenobiotics, such as chlorinated insecticides, are recalcitrant to degradation; thus they have rather long persistence times in the environment
      • lindane - 3 years for 75-100% disappearance
      • DDT - 4 years for 75-100% disappearance
      • chlordane - 5 years for 75-100% disappearance
    • to degrade these xenobiotics, microbes may employ co-metabolism, in which an organic material other than the xenobiotic is used as the primary energy source and the xenobiotic is degraded as a secondary process
• Genetically engineered microbes in bioremediation
  • Microbes can be "engineered" to carry out the biochemical processes needed for bioremediation
    • concerns about long-term the effects of genetically engineered microbes on the environment are manifold
    • fate of genetically engineered microbes is similar to that of other allochthonous organisms, but even more extreme because they are typically engineered to require nutrients, etc. not naturally present in the environments into which they may be introduced ... so they will die out when the material they were engineered to degrade has been removed from the area
  • Naturally-occurring microbes bioremediate just as well as engineered microbes in many cases ... it is important to adjust environmental conditions to favor their growth, however
  
  from: http://www.cas.muohio.edu/~stevenjr/mbi630/industrialmicro630.html