Microbes in service of humans

Ames, IA, September, 2010

J. (Hans) van LeeuwenJ. (Hans) van LeeuwenProfessor of Environmental and

Biological Engineering &Vlasta Klima Balloun Professor

Towards a more sustainable futureTowards a more sustainable future

Small, but growing contributionSmall, but growing contribution

Historical perspectiveHistorical perspectiveAntiquity Microbial processes used long before development of microbiology as a science

remnants of a fermented drink in fragments of 9,000-year-old Chinese vessels

Antonie Philips van Leeuwenhoek (1632-1723)

The very first microbiologist made small lenses by fusion and discovered and described both bacteria and protists. Also studied sperm cells and sections of plants and muscular fibers.Later became a Fellow of the Royal Society.

The first systematic applications of The first systematic applications of microbiologymicrobiology

Louis Pasteur (1822-1895) 1857 Microbiology of lactic acid fermentation 1860 Role of yeast in ethanolic fermentation• advances in applied microbiology led to the development of microbiology

His discoveries reduced mortality from puerperal fever, and he created the first vaccine for rabies. He also made it important to make sure surgeries were more sterile. in 1888 he founded the Pasteur Institute and was named director. He is regarded as one of the main founders of modern microbiology, together with Ferdinand Cohn and Robert Koch.

Microbial applicationsMicrobial applications

1. Food and beverage biotechnology

• fermented foods, alcoholic beverages (beer, wine, kumis, sake) distilled liquors

• 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)

Microbial applications (cont’d)Microbial applications (cont’d)4. Biological fuel generation

• production of biomass, ethanol/methane/butanol, single cell protein• microbial production/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

Ethanol productionEthanol production

The major microbial biotechnology: beer, wine, distilled beverages, ethanol

Saccharomyces (brewer’s yeast)• ethanolic fermentation • Embden-Meyerhof-Parnas, glycolytic pathwayglucose + 2 ADP + 2 Pi 2 EtOH + 2 CO➞ 2 + 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

CO2Enzymes

Fermentor

Cooking

Corn

Milling

Distillation

EthanolEthanol

Typical corn dry-grind ethanol plantTypical corn dry-grind ethanol plant

Vapor

Centrifuge Dryer

Evaporator

DDGSDistillers dried grains with solubles

DDGSDistillers dried grains with solubles

Water

Thin stillage

Thin stillage backset

Thick stillage

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Syrup

DDG

Yeasts

Commercial yeast productionCommercial yeast production

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VinegarVinegarSour (spoiled ) wine, vinegar (from French): vin + aigre (sour)

• Production in the US about 160 Mgal/y; 2/3 used in commercial products such as sauces and dressings, production of pickles and tomato products

• Acetic acid bacteria are 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

Industrial production of acetic acidIndustrial 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 mixed for 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 50% higher than with trickling filter

Major organic acids from fermentationMajor organic acids from fermentation Product Microbe used Representative uses Fermentation conditions

Acetic acid Acetobacter Wide variety foods Single-step oxidation, 15%,+ ethanol 95-99% yields

Citric acid Aspergillus niger Pharmaceuticals High carbohydrate, controlled

+ molasses food additive limit trace metals; 60-80% yld

Fumaric acid Rhizopus nigricans Resin, tanning,sizing Strongly aerobic fermentation;+ sugars C:N critical; Zn limit; 60% yld

Gluconic acid Aspergillus niger Carrier of Ca and Mg Agitation; 95% yields+ glucose + salts

Itaconic acid Aspergillus terreus Polymer of esters Highly aerobic; pH <2.2; + molasses + salts 85% yield

Kojic acid Aspergillus flavus-oryzae Fungicides and Fe careful controlled to avoid

+ carbohydrate + N insectides with metals reaction with kojic acid

Lactic acid Homofermentative Carrier of Ca Purified medium used toLactobacillus and acidifier facilitate extractiondelbrueckii

Lactic acid fermentationLactic acid fermentationPyruvate is reduced to lactic acid with the coupled reoxidation of NADH to NAD+• lactic acid bacteria (e.g. Lactobacillus, Streptococcus) involved in many foodfermentations• fermented milk, cheese, fermented vegetables

Homolactic fermentation• glucose degraded via EMP pathway, with lactic acid as the only end productglucose + 2 ADP + 2 Pi 2 lactic acid + 2 ATP➞• carried out by Streptococcus, Pediococcus, Lactococcus, Enterococcus andvarious 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 producedglucose + ADP + Pi lactic acid + ethanol + CO2 + ATP➞

Lactococcal products Nisin yield - 620 mg/L

Biomass yield - 2.3g/L

Lactic acid production

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Fermentation time (h)La

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Lactic acidAcetic acid

0h 24h16h

Milk fermentation microbesMilk fermentation microbes

Single cell proteinSingle cell proteinMicrobial protein for use as human food/animal feed

- source of low-cost protein?Advantages1. rapid growth rate and high productivity2. 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 cells5. little land area required6. 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 problems1. high nucleic acid content (bacteria)2.high protein content (elevated RNA levels – ribosomes • digestion of nucleic acids results in elevated levels of uric acid • treatment with RNAses3. sensitivity or allergic reactions

Microbes for SCPMicrobes for SCP

Carbon substrate Suitable microbes

Carbon dioxide Spirulina sp., Chlorella sp.

Liquid n-alkanes Saccharomycopsis lipolytica, Candida tropicalis

Methane Methylomonas methanica, Methylococcus capsulatus

Methanol Methylophilus methylotrophus, Hyphomicrobium sp.Candida boidinii, Pichia angusta

Ethanol Candida utilis

Glucose (hydrolyzed starch) Fusarium venetatum

Inulin (polyfructan) Candida species, Kluyveromyces sp.

Spent sulfite liquor Paecilomyces variotii (Pekilo process)

Whey K. marxianus, K. lactis, P. cyclopium

Lignocellulosic wastes Chaetomium sp., Agaricus bisporus, Cellulomonas sp.

GRAS microorganismsGRAS microorganismsGenerally Regarded As Safe by the

Food and Drug AdministrationNormally, these organisms need no further testing if cultivated under acceptable conditions

Bacteria Bacillus subtilis Lactobacillus bulgaricus Leuconostoc oenos

Yeasts Candida utilis Kluyveromyces lactis Saccharomyces cerevisiae

Filamentous fungi Aspergillus niger Aspergillus oryzae Mucor circinelloides Rhizopus microsporus Penicillium roqueforti

SCP examplesSCP examplesMushrooms

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 graminarium for human consumption (mycoprotein) • processed to resemble meat

MycoMax/MycoMeal

Fungal Production and Water Fungal Production and Water Reclamation PlantReclamation Plant

Fungal inoculum

Screen

Blowers

Primary and secondary metabolitesPrimary and secondary metabolitesPrimary metabolites• produced during active growth• generally a consequence of energy metabolism and necessary for the continuedgrowth of the microorganismSubstrate 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 growthSubstrate 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,…

Growth in batchGrowth in batchP

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Outline of fermentation designOutline of fermentation design

Amino acid productionAmino acid production

Citric acidCitric acidOver 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 • >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

AntibioticsAntibioticsAntibiotics 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

BacillusStreptomycesPenicillium

Streptomyces antibioticsStreptomyces antibiotics Important antibiotics produced by Streptomyces species

Microbial enzymesMicrobial enzymes

Microbial enzyme applicationsMicrobial enzyme applications

Enzyme applications, originsEnzyme applications, origins

Mining with S and Fe bacteriaMining with S and Fe bacteriaThiobacillus, Acidothiobacillus, Beggiatoa, and othersThiobacillus 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

Microbial mining with Microbial mining with ThiobacillusThiobacillus

Slope, heap and in-situ leaching Metal recovery from low-grade ores

Metal recovery from low-grade ores

BiobutanolBiobutanol

Biobutanol can be produced by fermentation of biomass by the A.B.E. process. The process uses the bacterium Clostridium acetobutylicum, also known as the Weizmann organism. It was Chaim Weizmann who first used this bacteria for the production of acetone from starch (with the main use of acetone being the making of Cordite) in 1916. The butanol was a by-product of this fermentation (twice as much butanol was produced). The process also creates a recoverable amount of H2 and a number of other by-products: acetic, lactic and propionic acids, acetone, isopropanol and ethanol.

Comparison of liquid fuelsComparison of liquid fuels

FuelEnergydensity

Air-fuelratio

Specificenergy

Heat ofvaporization

RON* MON*

Gasoline & biogasoline

32 MJ/L 14.6 2.9 MJ/kg air 0.36 MJ/kg   91–99   81–89

Butanol fuel 29.2 MJ/L 11.2 3.2 MJ/kg air 0.43 MJ/kg   96   78

Ethanol fuel 19.6 MJ/L   9.0 3.0 MJ/kg air 0.92 MJ/kg 129   102

Methanol 16 MJ/L   6.5 3.1 MJ/kg air 1.2 MJ/kg 136 104

*Octane rating of a spark ignition engine fuel is the detonation resistance (anti-knock rating) compared to a mixture of iso-octane (2,2,4-trimethylpentane, an isomer of octane) and n-heptane. By definition, iso-octane is assigned an octane rating of 100, and heptane is assigned an octane rating of zero. An 87-octane gasoline, for example, possesses the same anti-knock rating of a mixture of 87% (by volume) iso-octane, and 13% (by volume) n-heptane.

Utilization of resourcesUtilization of resources

Algal and Algal and cyanobacterial cyanobacterial

cultivationcultivationHigh-rate photosynthesisHigh-rate photosynthesis

J. (Hans) van LeeuwenJ. (Hans) van Leeuwen

CyanobacteriaCyanobacteria

Certain cyanobacteria produce cyanotoxins including anatoxin-a, anatoxin-as, aplysiatoxin, cylindrospermopsin, domoic acid, microcystin LR, nodularin R (from Nodularia), or saxitoxin. Sometimes a mass-reproduction of cyanobacteria results in algal blooms.These toxins can be neurotoxins, hepatotoxins, cytotoxins, and endotoxins, and can be dangerous to animals and humans. Several cases of human poisoning have been documented but a lack of knowledge prevents an accurate assessment of the risks.

Chloroplasts in plants and eukaryotic algae have evolved from cyanobacteria via endosymbiosis.

Anabaena malodorous products Anabaena malodorous products

IUPAC name1,6,7,7-Tetramethylbicyclo[2.2.1] heptan-6-ol

Other names 2-Methyl-2-bornanol, MIB

Identifiers

CAS number 2371-42-8

PubChem 16913

SMILES CC1(C2CCC1(C(C2)(C)O)C)C

Properties

Molecular formula C11H20O

Molar mass 168.28 g/mol

IUPAC name4,8a-dimethyldecalin-4a-ol or, (4S,4aS,8aR)-4,8a-dimethyl-1,2,3,4,5,6,7,8-octahydronaphthalen-4a-ol

Identifiers

CAS number 19700-21-1

PubChem 29746

SMILESCC1CCCC2(C1(CCCC2)O)C

Properties

Molecular formula C12H22O

Molar mass 182.30248 g/mol

2-Methylisoborneol Geosmin

Algal oil productionAlgal oil productionMicroalgae have much faster growth-rates than terrestrial crops. The per unit area yield of oil from algae is estimated to be from between 5,000 to 20,000 US gallons per acre per year (4,700 to 18,000 m3/km2·a); this is 7 to 30 times > than the next best crop, Chinese tallow (700 US gal/acre·a or 650 m3/km2·a).

Typical yield of biodiesel/haTypical yield of biodiesel/haSome typical yields

Crop Yield

L/ha US gal/acre

Algae ~3,000 ~300

Chinese tallow[ 1, 2] 772 97

Palm oil [3] 780-1490 508

Coconut 2150 230

Rapeseed [3] 954 102

Soy (Indiana) 76-161 8-17

Peanut [3] 138 90

Sunflower [3] 126 82

Hemp 242 26

1.^ Klass, Donald, "Biomass for Renewable Energy, Fuels,and Chemicals", page 341. Academic Press, 1998. 2.^ Kitani, Osamu, "Volume V: Energy and Biomass Engineering,CIGR Handbook of Agricultural Engineering", Am Society of Agricultural Engs, 1999. 3. Biofuels: some numbers

SpirulinaSpirulinaSpirulinaDomain: Bacteria

Phylum: Cyanobacteria = Chroobacteria

Order: Oscillatoriales

Family: Phormidiaceae

Genus: Arthrospira

SpeciesAbout 35•Arthrospira maxima •Arthrospira platensis

Spirulina common name for food supplements from two species of cyanobacteria: Arthrospira platensis, and Arthrospira maxima. These and other Arthrospira species were once classified in the genus Spirulina. There is now agreement that they are a distinct genus, and that the food species belong to Arthrospira; nonetheless, the older term Spirulina remains the popular name. Spirulina is cultivated around the world, and is used as a human dietary supplement as well as a whole food and is available in tablet, flake, and powder form. It is also used as a feed supplement in the aquaculture, aquarium, and poultry industries.[1]

Spirulina farmingSpirulina farming

Edible algaeEdible algae

Dulse (‘’Palmaria palmata’’) is a red species sold in Ireland and Atlantic Canada. It is eaten raw, fresh, dried, or cooked like spinach

Edible algae: PorphyraEdible algae: PorphyraDomain: Eukaryota

(unranked): Archaeplastida

Phylum: Rhodophyta

Class: Bangiophyceae

Order: Bangiales

Family: Bangiaceae

Genus: PorphyraPorphyra the most domesticated of the marine algae, [5] known as laver, nori (Japanese), amanori (Japanese),[6] zakai, kim (Korean),[6] zicai (Chinese),[6] karengo, sloke or slukos.[2] The marine red alga has been cultivated extensively in Asian countries as edible seaweed to wrap rice and fish that compose the Japanese food sushi, and the Korean food gimbap. Japanese annual production of Porphyra spp. is valued at 100 billion yen (US$ 1 billion).[7]

Chondrus crispusChondrus crispusKingdom: Archaeplastida

(earlier Plantae)

Phylum: Rhodophyta

Class: Rhodophyceae

Order: Gigartinales

Family: Gigartinaceae

Genus: Chondrus

Species: C. crispus

Irish moss (Chondrus crispus), often confused with Mastocarpus stellatus, is the source of carrageenan, which is used as a stiffening agent in instant puddings, sauces, and dairy products such as ice cream. Irish moss is also used by beer brewers as a fining agent.

Other uses of algaeOther uses of algaeFertilizer and agarFor centuries seaweed has been used as fertilizer. It is also an excellent source of potassium for manufacture of potash and potassium nitrate.Both microalgae and macroalgae are used to make agar.

Pollution ControlWith concern over global warming, new methods for the thorough and efficient capture of CO2 are being sought out. The carbon dioxide that a carbon-fuel burning plant produces can feed into open or closed algae systems, fixing the CO2 and accelerating algae growth. Untreated wastewater can supply additional nutrients, thus turning two pollutants into valuable commodities. Algae cultivation is under study for uranium/plutonium sequestration and purifying fertilizer runoff.

Chlorella, particularly a transgenic strain which carries an extra mercury reductase gene, has been studied as an agent for environmental remediation due to its ability to reduce Hg2+ to the less toxic elemental mercury. Cultivated algae serve many other purposes, including bioplastic production, dyes and colorant production, chemical feedstock production, and pharmaceutical ingredients.

Sea otters and kelpSea otters and kelpFast FactsType: MammalDiet: CarnivoreAverage lifespan in the wild: 23 ySize: 4 ft (1.25 m)Weight: 65 lbs (30 kg)Protection status: Threatened

Tool using sea ottersTool using sea otters

SEA O TTERSABALO NE KELP &URCHINS

Sea otter distributionSea otter distribution

Sea otters were hunted for their fur to the point of near extinction. Early in the 20th century only 1,000 to 2,000 animals remained. Today, 100,000 to 150,000 sea otters are protected by law.

DietSea urchins, abalone, mussels, clams, crabs, snails and about 40 other marine species. Sea otters eat approximately 25% of their weight in food each day.

Importance to kelp protectionFor discussion

HypoxiaHypoxia

Gulf of Mexico "Dead Zone" due to excessive algal growth supported by fertilizer runoff in the Mississippi Low-oxygen areas appear in red.(NASA and NOAA)