Microbial Biotechnology Biotechnology originated from industrial microbiology now includes all practical uses of organisms Microorganisms are

re (and will continue to be) the primary focus of biotechnology Production of natural metabolites/products on a larger scale: traditional/modern biotech Microbes engineered to make new products: modern biotech Traditional uses of microorganisms Manufacture and preservation of food and beverages using lactic acid bacteria and yeasts - still used today. Lactic acid bacteria (Lactobacillus, Leuconostoc, Streptococcus): make lots of acid convert sugars to lactic acid, inhibits or kills other microbes acid and other flavours improve taste harmless to humans (except some streps) air excluded during fermentation (anaerobes)

Processes
Fermentation
Natural fermentation *Raw materials contain desirable and associated m/o. *Incubation conditions set to favour rapid growth of desirable m/o, slow or prevent growth of others. *Metabolism of associated m/o may have some desirable result (aroma). *Problems with natural fermentation: - microbial population can vary - inconsistency of characteristics over time - risk of product failure; growth of undesirable m/o - growth of pathogens -borne illness Back slopping *Products from successful fermentation added to starting materials. *Conditions set to favour growth of m/o from previous product. *Changes in m/o may consistency of products. *High risk of product failure and foodborne illness Controlled fermentation *Starting materials (may be heat-treated) inoculated with high number population (106 cells/ml or more) of a pure culture of single or mixed strains or species = starter culture. *Incubation conditions are optimal for starter cultures. *Advantages: large volumes of product with consistent and predictable characteristics produced day to day; less chance of product failure and food-borne illness. *Disadvantage: growth of desirable secondary flora may not occur; loss of delicate flavour characteristics

Cheese and other dairy products

*Traditionally used natural fermentation (bacteria from tools and vessels) or back-slopping; today, highly specialised starter cultures used *Distinctive properties depend upon type of lactic acid bacterium used; different species or different strains

Cheese making Curdling

conversion of milk to solid mass *Protein fraction (casein) precipitated, with the fat; accomplished by lactic acid bacteria (acid denatures casein = precipitation) and addition of rennin (cleaves one form of casein =precipitation) *Whey (liquid fraction) is drained off *Making unripened cheese, e.g. cottage cheese, only involves curdling step

Ripening
development of characteristic taste and texture *Water is pressed out of curd, salt added, solid mass allowed to ripen (3-12 months) *Complex process, production of flavour-giving compounds (sulphur compounds, lactones, ketones, esters, volatile fatty acids) by microbes and hydrolysis of milk proteins =peptides, amino acids More hydrolysis, softer the cheese (e.g. camembert)

Fermented meat products

*Produced by mixing meat, fat, salt, sugar, curing agents, spices; *Semi-dry and dry sausage (salami, pepperoni)Composition: 30% added to casing, fermented naturally or with starter culture fat, 20% protein, 3% minerals (salts), 47% water; tangy taste, bacteria desirable flavour (lactate, acetate, diacetyl, breakdown products *Acids produced by starters (fermentation) and from proteolysis and lipolysis, spices); nitrite (preservative, curing agents control growth of pathogenic inhibitor of C. botulinum) contributes to pinkish colour (otherwise and spoilage m/o greyish) *Fermented products may be dried (reduce aw), smoked or heated =safety, shelf life Process: *Meat, salts, glucose, curing agent (nitrate/nitrite), spices, starter * Lactobacillus plantarum metabolises glucose to lactic acid and mixed uniformly, stuffed in casings, fermented (8-12 hrs) at 30substantial amounts of acetate, ethanol and diacetyl 43C, 80-90% RH. Incubated until pH drops to 5.2-4.6, cooked *All three can produce H2O2, oxidises myoglobin to 60C internal temp., cooled to 10C. Stored at 4-10C, 3-4 days, of product Micrococcus and S. carnosus produce catalase, break vacuum packaged down H2O2 *Starters vary depending on fermentation temp and final pH of * Micrococcus, S. carnosus and some Lactobacillus plantarum product: reduce nitrate to nitrite =pink colour (desirable) -High temp, low pH = Pedicoccus acidilactici. *Storage (curing): intracellular enzymes cause proteolysis, lipolysis -Low temp, high pH = Lactobacillus plantarum. (flavours) -P. pentosaceus can be used for both conditions *Sauerkraut (acid cabbage in German):cabbage shredded, pack * Some starters have Pedicoccus and Lactobacillus. Micrococcus sp. firmly into container, add water (to fill pockets of air), lactic acid and Staphylococcus carnosus added as secondary flora =desired bacteria naturally present: sugar =acid product colour * Spanish-type green olives *Pedicocci metabolise glucose to lactic acid (some acetate and * Animal fodder (silage): chopped plants (corn)=large air-tight silos, diacetyl) plant material fermented and preserved

Yeasts
*Most ferment sugars to ethanol and CO2 *Used to make wine, beer, whisky and bread * Saccharomyces cerevisiae or related species

Winemaking (eneology)
*Traditional crushed grapes (must) allowed to stand in container with limited contact with air, natural yeast carry out fermentation * Modern natural yeasts killed (must treated with bisulphite), pure culture of reliable strain added *Sherry -S. cerevisiae ferments all sugar, brandy added (alcohol layer of S. fermentati develops, metabolic products sherry taste. *Sweet wines (Sauternes) - Damp regions, grapes infected with Botrytis cineria, hyphae enters grapes, dries and concentrates sugars, yeast cannot ferment completely - Botrytis uses malic acid (not sugar) -Wine very sweet, less acid *Contribution of bacteria: removal of acid from premium red wines - Lactic acid bacterium, Oenococcus oeni, converts malic acid to lactic acid ess acid, more flavour - Bacteria can also spoil wine: acetic acid bacteria =vinegary taste

Other alcoholic beverages

*Whisky: distilled from malt brews *Vodka: distilled from potatoes (starch needs to saccharified) * Sake and Chinese rice wine: distilled from rice saccharified using amylase from fungi *Beer: produced from fermentation of grain (germinated barley seeds = malt) -Yeasts cannot ferment starch, must be saccharified (broken down to sugar monomers) by amylase from malt -Saccharified product (wort) fermented in vats, flavoured and preserved by hops (vine flowers)

Vinegar
S. cerevisiae (yeast) ferments sugar in fruit juice (apple or grape) =ethanol Acetobacter (bacterium) oxidises ethanol =acetic acid (vinegar)

Bread
*Bakers yeast (strain of S. cerevisiae derived from brewers yeast) added to bread mix * CO2 produced by fermentation of sugars = small bubbles which lightens the mixture and raises the bread (leaven bread)

Modern uses of microorganisms

Microbes as insecticides
*B. thuringiensis used as bioinsecticide: Bt * Bt sprayed or dusted onto plant leaves, controls caterpillars and other insect pests * Insects stop feeding one hour after consumption, die several days later * Bioinsecticides are cologically safe, specific action, no effect on plants or animals

chemical factories
*Used industrially to produce organic solvents, vitamins, amino acids, antibiotics, enzymes, pharmaceutical proteins * Rapid metabolism and growth rates * Historically, first products were the result of anaerobic fermentations *Today, processes may be aerobic or anaerobic, still termed fermentation

Anaerobic fermentations
*Cultures do not have to be aerated, rigorous aseptic technique unnecessary (contamination by aerobes is impossible, other anaerobes cannot compete against established culture) *By law, beverage alcohol and acetic acid must be made microbiologically, even if more economical chemical alternative available Ethanol production *Constituent of alcoholic beverages, used as solvent and fuel *Made by microbial fermentation before WWII, rise in petrochemical industry =inexpensive ethanol, fermentation not economical *Brazil: ethanol produced from fermentation is major source of car fuel Ethanol production *Made from sugar crops, saccharified cereal grains, straw, newsprint Acetone and butanol * Common organic solvents * Fermentation products of Clostridium acetobutylicum

Aerobic processes
*Most other industrial microbiological processes are aerobic *Supplying O2 to aerobic fermentors is an engineering challenge (not very soluble in water, rapidly used by dense cultures, inflow air needs to be filter sterilised)

Antibiotics *Success of penicillin led to search for new antibiotics after WWII. * Streptomycin - isolated from Streptomyces griseus (soil bacterium): active against TB and Gram negatives that penicillin could not control *Others are broad spectrum (active against both Gram positive and negative bacteria) *Majority of antibiotics are too chemically complex for commercial chemical synthesis *Made by aerobic fermentation

Amino acids *Several made by aerobic fermentation; used in food industry (flavouring or futritional value) * Alanine and aspartate: improve taste of fruit juice *Cysteine: enhance flavour of bread, fruit juice *Glutamate (MSG): flavour enhancer *Glycine: flavour enhancer *Histidine and tryptophan: stops food going rancid * Lysine: improves nutritional value of bread (added to bread in Japan; we obtain sufficient amounts from milk and meat)

Enzymes *Glucose isomerase (Streptomyces sp.): converts glucose in saccharified cornstarch to fructose syrups; used as sweetener in confectionery and soft drink industries * Invertase (S. cerevisiae): hydrolyses sucrose = fructose (and glucose); used in confectionery industry (sweeter than sucrose, does not crystalise as easily) * Protease (B. subtilis): hydrolyses protein; used in laundry detergents (removes stains)

Chemical reactions *Microbial chemical conversions very specific (specific actions of microbial enzymes) * Microbes used to carry out difficult chemical syntheses *Example: production of corticosteroids (antiinflammatory compounds) from progesterone involves Rhizopus nigricans (fungus) (production costs reduced from $200/gram to <$1/gram)

Genetically engineered microbes *Traditional aerobic fermentations can only make products of normal microbial metabolism *Now possible to genetically engineer microorganisms to (potentially) produce any compound normally produced by organisms * Fermentations derived from recombinant DNA technology = modern biotechnology Microbes used in traditional fermentations (e.g. lactic acid bacteria) have also be modified by recombinant DNA technology * Most products used in medicine or agriculture, some used industrially (e.g. recombinant rennin)

Other proteins *Tissue plasminogen activator, tPA (dissolves blood clots that cuts off blood supply to heart; if administered soon after heart attack, patient may recover with no serious damage) *tPA also produced by expression in mammalian cells * Human DNase (also expressed in mammalian cells): used to treat CF patients; infected with Pseudomonas aeruginosa, macrophages fight infection, die, release DNA into lung, thick mucus =breathing difficulty; DNase inhaled, breaks down mucous

Hormones *Human genes for insulin and hGH both cloned (into plasmids) and expressed in E. coli * hGH used to treat children born with pituitary gland which produces insufficient hormone (children otherwise become dwarfs) * Previously, supply was limited or obtained from cadavers (risk of prion infection) Insulin used to treat diabetes mellitus (lowers glucose levels in blood, otherwise bodys metabolism affected excessive urination, coma and death) * Previously, bovine or porcine insulin used: animal version different to human, in insulin levels in cattle) *Research into bacterial production of other human hormones: - factor VIII (treatment of haemophilia) -relaxin (makes cervix dilate during delivery)

A good host for gene expression should

1. Produce large amounts of product 2. Be easy to cultivate 3. Have a biology that is thoroughly understood 4. Produce correct form of the product *E. coli satisfies first 3 better than any other organismeasy and inexpensive to cultivate *E. coli is the best understood cellular organism: -Growth conditions can be altered in a predictable way -Fourth requirement can be a problem Glycosylation and gene expression *Human proteins are usually glycosylated: sugar molecules attached after translation; unglycosylated protein folds incorrectly * E. coli lacks glycosylation ability * Many human proteins are folded incorrectly in E. coli, protein can be purified, chemically unfolded and refolded correctly Modern uses of microorgan * Unglycosylated form may still function (less soluble) or can be chemically altered to change solubility * Alternative hosts (eukaryotic) * Mammalian cells (e.g. CHO) * Insect cells (baculovirus vectors)