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
The major microbial biotechnology: beer, wine, distilled beverages, ethanol
Saccharomyces (brewer’s yeast)
• ethanolic fermentation
• 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
Typical corn dry-grind ethanol plant
Distillation
Fermentor
Water Enzymes
Milling
CO2 Yeasts
Cooking
Whole stillage
Vapor
Corn
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 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 fermentation
Product
Microbe used
Representative uses
Fermentation conditions
Acetic acid
Acetobacter
+ ethanol
Wide variety foods
Single-step oxidation, 15%,
95-99% yields
Citric acid
Aspergillus niger
+ molasses
Pharmaceuticals
food additive
High carbohydrate, controlled
limit trace metals; 60-80% yld
Fumaric acid
Rhizopus nigricans
+ sugars
Resin, tanning,sizing
Strongly aerobic fermentation;
C:N critical; Zn limit; 60% yld
Gluconic acid
Aspergillus niger
+ glucose + salts
Carrier of Ca and Mg
Agitation; 95% yields
Itaconic acid
Aspergillus terreus
+ molasses + salts
Polymer of esters
Highly aerobic; pH <2.2;
85% yield
Kojic acid
Aspergillus flavus-oryzae
+ carbohydrate + N
Fungicides and
insectides with metals
Fe careful controlled to avoid
reaction with kojic acid
Lactic acid
Homofermentative
Lactobacillus
delbrueckii
Carrier of Ca
and acidifier
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
6
 Lactic acid production
5
4
0.4
3
2
Lactic acid
Acetic acid
1
0
0
0
10
20
30
Fermentation time (h)
0h
16h
0.2
24h
40
Acetic acid
(g/L)
Lactic acid
(g/L)
 Biomass yield - 2.3g/L
0.6
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 – ribosomes
• digestion of nucleic acids results in elevated levels of uric acid
• treatment with RNAses
3. sensitivity or allergic reactions
Microbes 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 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 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 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 ➞ 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,…
Secondary
metabolite
Primary
metabolite
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
Metal recovery from low-grade
ores
Slope, heap and in-situ leaching
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 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 fuels
Air-fuel Specific
ratio
energy
Heat of
vaporization
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
6.5
3.1 MJ/kg air
1.2 MJ/kg
136
104
Fuel
Energy
density
Gasoline &
32 MJ/L
biogasoline
16 MJ/L
RON*
MON*
*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 in plants and eukaryotic
algae 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
IUPAC name
1,6,7,7Tetramethylbicyclo[2.2.1]
heptan-6-ol
Other names
2-Methyl-2-bornanol, MIB
Geosmin
IUPAC name
Identifiers
4,8a-dimethyldecalin-4a-ol or,
(4S,4aS,8aR)-4,8a-dimethyl1,2,3,4,5,6,7,8octahydronaphthalen-4a-ol
Identifiers
CAS number
2371-42-8
CAS number
19700-21-1
PubChem
16913
PubChem
29746
SMILES
CC1(C2CCC1(C(C2)(C)
O)C)C
SMILES
CC1CCCC2(C1(CCCC2)O)C
Properties
Properties
Molecular formula
C11H20O
Molecular formula
C12H22O
Molar mass
168.28 g/mol
Molar mass
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 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/ha
Some typical yields
Crop
Algae
Chinese tallow[ 1, 2]
Palm oil [3]
Coconut
Rapeseed [3]
Soy (Indiana)
Peanut [3]
Sunflower [3]
Hemp
Yield
L/ha
US gal/acre
~3,000
~300
772
97
780-1490
2150
954
76-161
138
126
242
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
Phylum: Cyanobacteria =
Chroobacteria
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]
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
[5]
Domain:
Eukaryota
(unranked):
Archaeplastid
a
Phylum:
Rhodophyta
Class:
Bangiophyce
ae
Order:
Bangiales
Family:
Bangiaceae
Genus:
Porphyra
Porphyra the most domesticated of the marine algae, 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 crispus
Kingdom:
Archaeplastida
(earlier
Plantae)
Phylum:
Rhodophyta
Class:
Rhodophycea
e
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 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 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 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)