EnSoft Corp.

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Microbes in service of humans
J. (Hans) van Leeuwen Professor of Environmental
and Biological Engineering Vlasta Klima
Balloun Professor
Ames, IA, September, 2010
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Towards a more sustainable future

• Small, but growing contribution

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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.
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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.
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Microbial applications

• Food and beverage biotechnology
• fermented foods, alcoholic beverages (beer,
  wine, kumis, sake) ? distilled liquors
• flavors
• Enzyme technology
• production and application of enzymes
• Metabolites from microorganisms
• amino acids
• antibiotics, vaccines, biopharmaceuticals
• bacterial polysaccharides and polyesters
• specialty chemicals for organic synthesis
  (chiral synthons)

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Microbial applications (contd)

• Biological fuel generation
• production of biomass, ethanol/methane/butanol,
  single cell protein
• microbial production/recovery of petroleum
• Environmental biotechnology
• water and wastewater treatment
• composting (and landfilling) of solid waste
• biodegradation/bioremediation of toxic
  chemicals and hazardous waste
• Agricultural biotechnology
• soil fertility
• microbial insecticides, plant cloning
  technologies
• Diagnostic tools
• testing/diagnosis for clinical, food,
  environmental, agricultural applications

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Ethanol production
The major microbial biotechnology beer, wine,
distilled beverages, ethanol Saccharomyces
(brewers 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 Alphaproteobacteriu
m 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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Typical corn dry-grind ethanol plant
Ethanol
Yeasts
Water
Vapor
Thin stillage backset
Eliminate
Whole stillage

Evaporator
Syrup
Thin stillage
DDGS Distillers dried grains with solubles
Centrifuge
DDG
Thick stillage
Dryer
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Commercial yeast production
..
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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
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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
29-35C 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 30C 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
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Major 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 CN 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 lt2.2
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
to Lactobacillus and acidifier
facilitate extraction delbrueckii
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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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Lactococcal products

• Nisin yield - 620 mg/L
• Biomass yield - 2.3g/L
• Lactic acid production

0h
24h
16h
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Milk fermentation microbes
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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)
• high protein content (elevated RNA levels
  ribosomes
• digestion of nucleic acids results in
  elevated levels of uric acid
• treatment with RNAses

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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.
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GRAS microorganisms
Generally Regarded As Safe by the Food and Drug
Administration Normally, these organisms need no
further testing if cultivated under acceptable
conditions
Filamentous fungi Aspergillus niger
Aspergillus oryzae Mucor circinelloides
Rhizopus microsporus Penicillium roqueforti
Bacteria Bacillus subtilis Lactobacillus
bulgaricus Leuconostoc oenos Yeasts
Candida utilis Kluyveromyces lactis
Saccharomyces cerevisiae
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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
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Fungal Production and Water Reclamation Plant
Fungal inoculum
Screen
Blowers
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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,
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Growth in batch
Secondary metabolite
Primary metabolite
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Outline of fermentation design
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Amino acid production
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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 gt99 of the worlds 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
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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 Pen
icillium
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Streptomyces antibiotics
Important antibiotics produced by
Streptomyces species
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Microbial enzymes
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Microbial enzyme applications
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Enzyme applications, origins
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Mining with S and Fe bacteria
Thiobacillus, Acidothiobacillus, Beggiatoa, and
others Thiobacillus thiooxidans (Jaffe and
Waksman 1922) scattered in the
Proteobacteria a,ß, ? 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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Microbial mining with Thiobacillus
Metal recovery from low-grade ores
Slope, heap and in-situ leaching
Metal recovery from low-grade ores
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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.
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Comparison of liquid fuels
Fuel Energydensity Air-fuelratio Specificenergy Heat ofvaporization RON MON
Gasoline biogasoline 32 MJ/L 14.6 2.9 MJ/kg air 0.36 MJ/kg   9199   8189
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.
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Utilization of resources
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Algal and cyanobacterial cultivation

• High-rate photosynthesis
• J. (Hans) van Leeuwen

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Cyanobacteria
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.
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Anabaena malodorous products
Geosmin
2-Methylisoborneol
IUPAC name 4,8a-dimethyldecalin-4a-ol or, (4S,4aS,8aR)-4,8a-dimethyl-1,2,3,4,5,6,7,8-octahydronaphthalen-4a-ol
Identifiers Identifiers
CAS number 19700-21-1
PubChem 29746
SMILES CC1CCCC2(C1(CCCC2)O)C
Properties Properties
Molecular formula C12H22O
Molar mass 182.30248 g/mol
IUPAC name 1,6,7,7-Tetramethylbicyclo2.2.1 heptan-6-ol
Other names 2-Methyl-2-bornanol, MIB
Identifiers Identifiers
CAS number 2371-42-8
PubChem 16913
SMILES CC1(C2CCC1(C(C2)(C)O)C)C
Properties Properties
Molecular formula C11H20O
Molar mass 168.28 g/mol
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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/km2a) this is 7 to 30 times gt than
the next best crop, Chinese tallow
(700 US gal/acrea or 650 m3/km2a).
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Typical yield of biodiesel/ha
Some typical yields Some typical yields Some typical yields
Crop Yield Yield
Crop 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
Klass, Donald, "Biomass for Renewable Energy, Fuels,and Chemicals", page 341. Academic Press, 1998. Kitani, Osamu, "Volume V Energy and Biomass Engineering,CIGR Handbook of Agricultural Engineering", Am Society of Agricultural Engs, 1999. Biofuels some numbers Klass, Donald, "Biomass for Renewable Energy, Fuels,and Chemicals", page 341. Academic Press, 1998. Kitani, Osamu, "Volume V Energy and Biomass Engineering,CIGR Handbook of Agricultural Engineering", Am Society of Agricultural Engs, 1999. Biofuels some numbers Klass, Donald, "Biomass for Renewable Energy, Fuels,and Chemicals", page 341. Academic Press, 1998. Kitani, Osamu, "Volume V Energy and Biomass Engineering,CIGR Handbook of Agricultural Engineering", Am Society of Agricultural Engs, 1999. Biofuels some numbers
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Spirulina
Spirulina
Domain Bacteria
Phylum Cyanobacteria Chroobacteria
Order Oscillatoriales
Family Phormidiaceae
Genus Arthrospira
Species
About 35 Arthrospira maxima Arthrospira platensis About 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
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Spirulina farming
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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
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Edible algae Porphyra
Domain Eukaryota
(unranked) Archaeplastida
Phylum Rhodophyta
Class Bangiophyceae
Order Bangiales
Family Bangiaceae
Genus Porphyra
Porphyra 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
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Chondrus crispus
Kingdom 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.
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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.
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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
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Tool using sea otters
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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.
Importance to kelp protection For discussion
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.
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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)