Microbes in service of humans

J. (Hans) van Leeuwen

Professor of Environmental and Biological Engineering & Vlasta Klima Balloun Professor

Ames, IA, September, 2010

Towards a more sustainable future Small, but growing contribution

Historical perspective

Antiquity

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 microbiology 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 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)

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 production

T

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

Saccharomyces (brewer’s yeast)

• ethanolic fermentation • Embden-Meyerhof-Parnas, glycolytic pathway glucose + 2 ADP + 2 P i ➞ 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 CO 2 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 ➞ • limited substrate use, only 3 carbohydrates: glucose, fructose and sucrose • genetic engineering to expand substrate range more glucose converted to EtOH

Corn

Typical corn dry-grind ethanol plant

Water Enzymes

Distillation Fermentor

CO 2 Yeasts

Milling Cooking

Vapor

Evaporator

DDG

Dryer

Thick stillage

Centrifuge

Commercial yeast production

…………………………..

Vinegar

Sour (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 acid

Trickling filter

• vinegar manufacturing industry near Orleans in 14th century • trickling filter, wooden bioreactor (volume up to 60 m 3 ) 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 m 3 over 10 times higher than with surface “fermentation” and over 50% higher than with trickling filter

Major organic acids from fermentation

Product Microbe used Representative uses Fermentation conditions Acetic acid Acetobacter + ethanol Citric acid

Aspergillus niger

+ molasses Fumaric acid Rhizopus nigricans + sugars Wide variety foods Pharmaceuticals food additive Resin, tanning,sizing Gluconic acid Aspergillus niger + glucose + salts Itaconic acid Aspergillus terreus + molasses + salts Carrier of Ca and Mg Polymer of esters Kojic acid Aspergillus flavus-oryzae Fungicides and + carbohydrate + N insectides with metals Lactic acid Homofermentative

Lactobacillus delbrueckii

Carrier of Ca and acidifier Single-step oxidation, 15%, 95-99% yields High carbohydrate, controlled limit trace metals; 60-80% yld Strongly aerobic fermentation; C:N critical; Zn limit; 60% yld Agitation; 95% yields Highly aerobic; pH <2.2; 85% yield Fe careful controlled to avoid reaction with kojic acid Purified medium used to facilitate extraction

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

Lactococcal products



Nisin yield - 620 mg/L



Biomass yield - 2.3g/L



Lactic acid production

0 1 2 3 6 4 5 0 10 20 30

Lactic acid Acetic acid

0.2

0 40

Fermentation time (h)

0.6

0.4

0h 16h 24h

Milk fermentation microbes

Single cell protein

Microbial protein for use as human food/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

1. high nucleic acid content (bacteria) 2. high protein content (elevated RNA levels • treatment with RNAses – ribosomes • digestion of nucleic acids results in elevated levels of uric acid 3. sensitivity or allergic reactions

Microbes for SCP Carbon substrate Suitable microbes

Carbon dioxide

Spirulina

sp.,

Chlorella

sp.

Liquid n-alkanes Methane Methanol Ethanol

Saccharomycopsis lipolytica, Candida tropicalis Methylomonas methanica, Methylococcus capsulatus Methylophilus methylotrophus, Hyphomicrobium

sp.

Candida boidinii, Pichia angusta Candida utilis

Glucose (hydrolyzed starch)

Fusarium venetatum

Inulin (polyfructan) Spent sulfite liquor Whey Lignocellulosic wastes

Candida species, Kluyveromyces sp.

Paecilomyces variotii

(Pekilo process)

K. marxianus, K. lactis, P. cyclopium Chaetomium

sp

., Agaricus bisporus, Cellulomonas

sp.

GRAS microorganisms

Generally Regarded As Safe

by the

Food and Drug Administration

Normally, 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 examples

Mushrooms Pekilo prossess

• filamentous fungus • use as animal feed

Paecilomyces variotii

• use of waste from wood processing (monosaccharides + acetate)

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 Reclamation Plant

Fungal inoculum Screen Blowers

Primary and secondary metabolites

Primary metabolites

• produced during active growth • generally a consequence of energy metabolism and necessary for the continued growth of the microorganism Substrate A ➞ Substrate A ➞ Product 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) ➘ Substrate A ➞ D ➞ B ➞ E ➞ C ➞ Product of secondary metabolism Primary metabolism (no product) afterwards, the product is formed by metabolism of an intermediate C ➞ D ➞ • citric acid, antibiotics,… Product • growth phase = trophophase • idiophase = phase involved in production of metabolites

Growth in batch

Outline of fermentation design

Amino acid production

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 • >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

Streptomyces antibiotics

Important antibiotics produced by

Streptomyces

species

Microbial enzymes

Microbial enzyme applications

Enzyme applications, origins

Mining with S and Fe bacteria

Thiobacillus, Acidothiobacillus, 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

Microbial mining with

Thiobacillus

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

Biobutanol

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 of acetone starch (with the main use 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 H 2 and a number of other by-products : acetic , lactic and propionic acids , acetone , isopropanol and ethanol.

Fuel

Comparison of liquid fuels

Energy density Air-fuel ratio Specific energy Heat of vaporization 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 Ethanol fuel 19.6 MJ/L 9.0

3.0 MJ/kg air 0.92 MJ/kg 96 129 78 102 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 resources

Algal and cyanobacterial cultivation

High-rate photosynthesis

J. (Hans) van Leeuwen

Cyanobacteria

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

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.

Anabaena malodorous products 2-Methylisoborneol Geosmin

IUPAC name Other names CAS number PubChem 1,6,7,7 Tetramethylbicyclo[2.2.1] heptan-6-ol 2-Methyl-2-bornanol, MIB Identifiers 2371-42-8 16913 SMILES Molecular formula Molar mass CC1(C2CCC1(C(C2)(C) O)C)C Properties C 11 H 20 O 168.28 g/mol IUPAC name CAS number PubChem SMILES Molecular formula Molar mass 4,8a-dimethyldecalin-4a-ol or, (4S,4aS,8aR)-4,8a-dimethyl 1,2,3,4,5,6,7,8 octahydronaphthalen-4a-ol Identifiers 19700-21-1 29746 CC1CCCC2(C1(CCCC2)O)C Properties C 12 H 22 O 182.30248 g/mol

Algal oil production

Microalgae 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 m 3 /km 2 ·a); this is 7 to 30 times > than the next best crop, Chinese tallow (700 US gal/acre·a or 650 m 3 /km 2 ·a).

Typical yield of biodiesel/ha

Some typical yields Crop

Algae Chinese tallow

[ 1, 2]

Palm oil

[3]

Coconut Rapeseed

[3]

Soy (Indiana) Peanut

[3]

Sunflower

[3]

Hemp

L/ha ~3,000 Yield US gal/acre ~300 772 780-1490 2150 954 76-161 138 126 242 97 508 230 102 8-17 90 82 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

Spirulina

Spirulina

Domain: Bacteria

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 aquaculture , feed supplement in the aquarium , and poultry industries.

[1]

Phylum: Cyanobacteria = Chroobacteria Order: Oscillatoriales Family: Phormidiaceae Genus:

Arthrospira

Species About 35

•

Arthrospira maxima

•

Arthrospira platensis

Spirulina farming

Edible 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: Porphyra

Domain: Eukaryota (unranked): Archaeplastid a Phylum: Class: Rhodophyta Order: Family: Bangiophyce ae Bangiales Bangiaceae Porphyra the most domesticated of the marine algae, [5] amanori (Japanese), [6] zakai, kim ( Korean ), [6] zicai ( known as Chinese ), Genus: [6] laver karengo ,

Porphyra

nori

( Japanese , 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 crispus

Kingdom: Phylum: Class: Order: Family: Archaeplastida (earlier Plantae) Rhodophyta Rhodophycea e Gigartinales 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 algae

Fertilizer and agar

For 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 Control

With concern over global warming , new methods for the thorough and efficient capture of CO 2 are being sought out. The carbon dioxide that a carbon-fuel burning plant produces can feed into open or closed algae systems, fixing the CO 2 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 Hg 2+ 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 kelp

Fast Facts

Type: Mammal Diet: Carnivore Average lifespan in the wild: 23 y Size: 4 ft (1.25 m) Weight: 65 lbs (30 kg) Protection status: Threatened

Tool using sea otters

SEA O TTERS ABALO N E KELP & URCHIN S

Sea otter distribution

Diet

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

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.

Importance to kelp protection

For discussion

Hypoxia

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)