Domestication of Microbes – Applied and Industrial Microbiology
• Empirical biotechnology: microbial processes used long (>4000 years) before
development of microbiology as a science
remnants of a fermented drink in fragments of
9,000-year-old Chinese vessels
• Louis Pasteur
1857 Microbiology of lactic acids fermentations
1860 Role of yeast in ethanolic fermentation
• advances in applied microbiology led the development of microbiology
Selman Waksman (~1945)
“There is no field of human endeavor…
…where the microbe does not play an important and often dominant part.”
Microbial Applications
1. Food and beverage biotechnology
• fermented foods, alcoholic beverages (beer, wine)
• flavors
2. Enzyme technology
• production and application of enzymes
3. Metabolites from microorganisms
• amino acids
• antibiotics, vaccines, biopharmaceuticals
• bacterial polysaccharides and polyesters
• specialty chemicals for organic synthesis (chiral synthons)
4. Biological fuel generation
• ethanol or methane from biomass, single cell protein, production of biomass
• microbial recovery of petroleum
5. Environmental biotechnology
• water and wastewater treatment
• composting (and landfilling) of solid waste
• biodegradation/bioremediation of toxic chemicals and hazardous waste
6. Agricultural biotechnology
• soil fertility
• microbial insecticides, plant cloning technologies
7. Diagnostic tools
• testing & diagnosis for clinical, food, environmental, agricultural applications
• biosensors
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Control of Microorganisms
1. Preventing food spoilage
• sterilization methods, canning
• chemical & physical control of growth
• water activity, acidity, pickling, etc.
• fermented foods
2. Sanitation, prevention of waterborne disease
• disinfectants, antiseptics, etc.
• water treatment (filtration, chlorination…)
• wastewater treatment
3. Prevention of biodeterioration
[Link] cells or cell products
2. Enzymatic biotransformation products
[Link] products (de novo synthesis)
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Primary vs. Secondary metabolism
Primary metabolites:
• produced during active growth
• generally a consequence of energy metabolism and necessary for the continued
growth of the microorganism
Substrate A ➞ Product
Substrate A ➞ B ➞ C ➞ Product
• ethanol, lactic acid,…
Secondary metabolites:
• synthesized after the growth phase nears completion
• a result of complex reactions that occur during the latter stages of primary growth
Substrate A ➞ B ➞ C ➞ Primary metabolism (no product)
➘
D ➞ E ➞ Product of secondary metabolism
Substrate A ➞ B ➞ C ➞ Primary metabolism (no product)
afterwards, the product is formed by metabolism of an intermediate
C ➞ D ➞ Product
• growth phase = trophophase
• idiophase = phase involved in production of metabolites
• citric acid, antibiotics,…
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Growth in batch culture
Primary Secondary
metabolite metabolite
Fermentation
products from
pyruvate
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Ethanol
• the major microbial biotechnology: beer, wine, distilled beverages, industrial ethanol
Saccharomyces (sugar fungus)
• alcoholic (ethanolic) fermentation, principally by yeasts in the genus Saccharomyces
• Embden-Meyerhof-Parnas, glycolytic pathway
glucose + 2 ADP + 2 Pi ➞ 2 EtOH + 2 CO2 + 2 ATP
• not a facultative anaerobe, cannot grow anaerobically indefinitely (unsaturated fatty
acids and sterols can be synthesized only under aerobic conditions)
• when oxygen present glucose oxidized via the Krebs cycle to CO2 and water
(much biomass and little alcohol produced)
Zymomonas mobilis
• Alphaproteobacterium
• osmotic tolerance, relatively high alcohol tolerance
• higher specific growth rate than yeast
• anaerobic carbohydrate metabolism through the Entner-Doudoroff pathway, yielding
only 1 mol of ATP per mol of glucose ➞ more glucose converted to EtOH
• limited substrate use, only 3 carbohydrates: glucose, fructose and sucrose
• genetic engineering to expand substrate range
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Conversion of starch to fermentable sugars
1. Malting
• germination of barley to induce production of amylases
• beer
2. Amylolytic molds and yeasts
• filamentous fungus Aspergillus oryzae
• Japanese sake
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Vinegar
• sour (spoiled ) wine, vinegar (from French): vin and aigre (sour)
• production in the US about 160 million gallons per year
(2/3 used in commercial products such as sauces and dressings, production of
pickles and tomato products)
• the acetic acid bacteria divided into two genera:
Acetobacter aceti and Gluconobacter oxydans
• obligate aerobes that oxidize sugar, sugar alcohols and ethanol with the production
of acetic acid as the major end product
• ethanol oxidation occurs via two membrane-associated dehydrogenases: alcohol
dehydrogenase and acetaldehyde dehydrogenase
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Industrial Production of Acetic Acid
Trickling filter
• vinegar manufacturing industry near Orleans in 14th century
• trickling filter, wooden bioreactor (volume up to 60 m3) filled with beechwood
shavings, acetic acid bacteria grow as biofilm
• the ethanolic solution is sprayed over the surface and trickles through the
shavings into a collection basin, and recirculated
• temperature maintained at 29-35°C
• about 12% acetic acid produced in 3 days
• the life of a well-packed and maintained generator is about 20 years
Submerged, batch process (Frings acetator)
• stainless steel tank with a high-speed mixer
microbes, air, ethanol and nutrients are mixed to provide a favorable environment
for microbial growth
• 30°C maintained by circulation of cooling water
• 12% acetic acid in about 35 h
• production rate per m3 over 10 times higher than with surface “fermentation” and
over 5% higher than with trickling filter
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Lactic acid fermentation
• pyruvate is reduced to lactic acid with the coupled reoxidation of NADH to NAD+
• lactic acid bacteria (e.g. Lactobacillus, Streptococcus) involved in many food
fermentations
• fermented milk, cheese, fermented vegetables
Homolactic fermentation
• glucose degraded via EMP pathway, with lactic acid as the only end product
glucose + 2 ADP + 2 Pi ➞ 2 lactic acid + 2 ATP
• carried out by Streptococcus, Pediococcus, Lactococcus, Enterococcus and
various Lactobacillus species
• important in dairy industry (yogurt, cheese)
Heterolactic fermentation
• glucose degraded via pentose phosphate pathway
• in addition to lactic acid, also ethanol and CO2 produced
glucose + ADP + Pi ➞ lactic acid + ethanol + CO2 + ATP
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Net Gain Net Gain
2 ATP 1 ATP
2 lactate per glucose 1 lactate + 1 ethanol + 1 CO2
per glucose
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Cheese making
Pre-ripening
• inoculation with starter culture of lactic acid bacteria
• acidification by fermentation of lactose to lactic acid
Coagulation of milk proteins (casein)
• addition of chymosin (rennin), an acid protease from calves’ stomach -
renneting
since 1980s also recombinant enzyme produced in yeast
• catalyzes specific hydrolysis of κ-casein Æ leads to coagulation in the
presence of Ca2+ to form a gel (curd)
• also traps bacteria that continue lactic acid fermentation
• removal of liquid whey from the curd
• addition of secondary microbiota
• treatments vary for different cheese varieties
Separation of curd and whey
• accelerated by decrease in pH
• salting
• curd pressed and placed into mold
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Ripening
• storage at controlled humidity
at ~9°C for up to a year
• modification of proteins and
fats by proteases and lipases
• complex development of flavor
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Amino acids
• annual worldwide production of over 400,000 tons
• uses as food additives, medicines, starting material in chemical synthesis
glutamic acid (80% of total), lysine (10%)
• production of glutamic acid (MSG) for use in foods; other amino acids (L-lysine)
• Corynebacterium glutamicum
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Citric acid
• over 130,000 tons produced worldwide each year
• used in foods and beverages
• iron citrate as a source of iron
preservative for stored blood, tablets, ointments,…
in detergents as a replacement for polyphosphates
• a microbial fermentation for production of citric acid developed in 1923
• today >99% of the world’s output produced microbially
Aspergillus niger
• submerged fermentation in large fermenters
• sucrose as substrate, and citric acid produced during idiophase
• during trophophase mycelium produced and
CO2 released
• during idiophase glucose and fructose are
metabolized directly to citric acid
Antibiotics
• Antibiotics are small molecular weight compounds that inhibit or kill microorganisms
at low concentrations
• often products of secondary metabolism
• the significance of antibiotic production is unclear, may be of ecological significance
for the organism in nature
• antibiotics produced by various bacteria, actinomycetes & fungi
Bacillus
Streptomyces
Penicillium
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Enzymes
Commercially produced enzymes:
• enzymes used in industry, such as amylases, proteases, catalases, isomerases
• enzymes used for analytical purposes, such as glucose oxidase, alcohol
dehydrogenase, hexokinase, cholesterol oxidase
• enzymes used in medicine, such as asparaginase, proteases, lipases
• different levels of quantity and quality
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Amylases
• hydrolysis of starch (glucose polymer), one of the most readily available plant
polysaccharides
• amylases are enzymes that hydrolyse starch
production of sweeteners from starch: maltose or glucose syrups
(further transformation to high fructose syrup with glucose isomerase)
• starch hydrolyates used as additives in the manufacture of candies, baked goods,
canned goods, and frozen foods
Glucose Isomerase
• D-glucose ketoisomerase: causes the isomerization of glucose to fructose
• since reaction is reversible the ration of glucose and fructose depends on the
enzyme and reaction conditions
• high fructose corn syrup
fructose 2x sweeter than sucrose
Chymosin
• site-specific proteolysis by chymosin detaches hydrophilic “tails” of κ-casein
resulting in coagulation (curlding)
• calf chymosin (prochymosin) cloned and expressed in E. coli (first genetically
engineered protein approved for human consumption, 1990)
Proteases
• used in laundry detergents
Industrial enzymes produced by Bacillus species
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Single Cell Protein Production
• microbial protein for use as human food or animal feed
• source of low-cost protein?
Advantages:
1. rapid growth rate and high productivity
2. high protein content (30-80% of dw)
3. ability to utilize a wide range of cheap carbon sources
methane, methanol, molasses, whey, lignocellulose waste, etc.
4. relatively easy selection of cells
5. little land area required
6. production independent of season and climate
• protein content and quality largely dependent on the specific microbe utilized and
on the fermentation process
• fast growing aerobic microorganisms
Some problems:
• high nucleic acid content
• high protein content (elevated RNA levels - ribosomes)
- digestion of nucleic acids results in elevated levels of uric acid
- treatment with RNAses
• sensitivity or allergic reactions
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Single Cell Protein
Mushrooms
Pekilo prossess
• filamentous fungus Paecilomyces variotii
• use of waste from wood processing (monosaccharides + acetate)
• use as animal feed
Pruteen
• methanol (from methane - natural gas) as C1 carbon source
• methylotrophic bacteria (Methylophilus methylotrophus)
• feed protein
Quorn
• fungal mycelium, Fusarium (mycoprotein) for human consumption
• processing to resemble meat
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Microbial polymers
• e.g. xanthan gum from Xanthomonas campestris
Production of solvents: Acetone-butanol fermentation
Clostridium acetobutylicum
• utilizes EMP pathway for glucose
catabolism with the formation of C3
and C4 products
• biphasic fermentation: during growth
acetate and butyrate are formed
(acidogenic phase)
• as pH drops the culture enters
stationary phase and there is a
metabolic shift to solvent phase
(solvetogenic phase)
• acetyl-CoA as central intermediate,
which can be:
a) reduced to butyrate and butanol
b) cleaved via decarboxylation to
acetone
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Waste-water and sewage treatment
Objectives of wastewater treatment:
• reduce the organic content of wastewater
• BOD
• trace (toxic) organics that are recalcitrant to biodegradation
• removal / reduction of nutrients to reduce pollution of receiving waters
• nitrogen, phosphorous
• removal or inactivation of pathogenic microorganisms and parasites
Treatment methods:
• various types of bioreactors to produce effluent that can be discharged into the
natural environment without adverse effects
• combinations of physical & chemical treatment and aerobic / anaerobic
biological biodegradation
Activated Sludge Process
1. Preliminary treatment
• removal of debris and coarse materials that may clog equipment
2. Primary treatment
• screens, settling tanks and skimmers to remove suspended solids
• physical separation
3. Secondary treatment
• aerobic microbiological process: trickling filters, activated sludge
• effluent from primary treatment aerated, aerobic bacteria growing in flocs degrade
organic material
• an important characteristic of the process is the recycling of a large proportion of the
biomass
• removal of BOD and nutrients:
1. oxidation of biodegradable organic matter
(soluble organic matter converted to new cell mass)
2. flocculation, separation of newly formed biomass from effluent
4. Tertiary or advanced treatment
• removal of ammonia and phosphate
• oxidation of ammonia to nitrate followed by denitrification
• accumulation of polyphosphate granules
• chemical treatments
5. Disinfection (when needed)
• chlorination
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Microbiological mining - Sulfur and Iron-Oxidizing Bacteria
• Thiobacillus, Acidithiobacillus, Beggiatoa, and others
Thiobacillus thiooxidans (Jaffe and Waksman 1922)
• scattered in the Proteobacteria: α,β, γ subdivisions
• acidophiles
• chemolithotrophs: energy from oxidation of reduced sulfur compounds or iron
• used in bioleaching of ores
• problems with acid mine drainage
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Microbiological mining - Leaching of metals with Thiobacillus
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