Microbes and the environment, part 2:

habitats, water quality and

bioremediation

BIOS 303: Lecture 21

Lecture 21: habitats, water & bioremediation

§ Diversity of microbial habitats

§ Biofilms (Lect. 20)
§ Soil
§ The ocean, deep sea, and hydrothermal vents
§ Freshwater

§ Public health and water quality

§ Water quality monitoring/microbe detection
§ Drinking water purification
§ Wastewater treatment

§ Bioremediation and biodegradation

 Diversity of microbial habitats: soil
§ Densest and most diverse microbe communities on Earth
§ ~109 bacterial cells/g and 103-104 unique species/g!

§ Rhizosphere: soil that surrounds plant roots and

receives plant secretions; heaviest microbe
growth here/surface soil

§ Less in subsoil; even fewer in soil base: inerals/

ê organics = ê lower microbial activity
§ Also big effect of water availability
 Soil prokaryotes: who’s in there?
§ ~99% of microbes are not yet cultured
§ Metagenomics / rRNA analysis: ~80% of soil species
are unknown!
§ What medical relevance could this diversity have?

§ Most unknowns can be grouped into correct phyla:

§ 32 phyla represented
§ Majority are
Proteobacteria,
Bacteroidetes, and
Acidobacteria:
 Aquatic habitats and nutrient availability
§ Oceans and freshwater: O2 levels much lower vs. air
§ E.g., freshwater lakes: anoxic zones can form meters
below surface -> aerobic organisms consume O2
before reaches lower
§ Deep ocean: lower temp + high pressure = é [O2]

§ Primary producers: photoautotrophs use light energy

to fix CO2
§ Freshwater: plants, algae, other microbes
§ Open ocean: solely microbes
§ Ocean microbes do ~50% of all primary production
(at depths up to 200 m!)
 Diversity of microbial habitats: oceans
§ Pelagic zone = open ocean
§ More limited in nutrients vs. most
freshwater = cells smaller
§ Phototrophs esp. need limiting N,
P, and F

§ Coastal waters more nutrient-rich

= more microbes

§ ~106 microbial cells per ml seawater

Red = chlorophyll = more

phototrophs near coasts
 Ocean microbe diversity: who’s in there?
§ One µl of surface seawater:
§ Primary producers: ~150 cyanobacteria, ~10 euks
§ ~1,000 heterotrophic bacteria (eat fixed C)
§ ~10,000 bacteriophage; ≥ 10 protozoa (eat bact.!)
§ Lotta proteobacteria, cyanobacteria, bacteroidetes, etc.
§ SAR11 group: α-proteobacteria; most abundant ocean
prokaryotic group
 Ocean prokaryotes: who’s in there?
§ Prochlorococcus: most abundant O2-
producing, C-fixing ocean phototroph
§ ~Half of ocean photosynthetic biomass
Prochlorococcus:
§ Pelagic bacteria and archaea consume
dissolved, unstable organic C
§ Then available to other orgs!

§ Pelagibacter: most abundant ocean

Pelagibacter:
prokaryote (SAR11 group)
§ Oligotroph: best growth in low
nutrients
§ Smallest genome for free-living org
(1.3Mb)
 Microbes in the deep sea
§ Extremes: low temp (2-3°C), high
pressure (>200 atm), and low
nutrients

§ Cold-loving psychrophiles
Pressure tolerance:
§ Piezophiles: grow best under high
pressure (4,000-6,000 m below)
§ Piezotolerant: 0 to 3,000 m

§ Extreme piezophiles: to 10,000 m

(Moritella: 700-1,000 atm!)

Sampling the Mariana Trench,

10,897 m below the surface:
 Another extreme: hydrothermal vents
§ Hydrothermal vents: deep-sea hot
springs (volcanic origin)

§ Warm or hot: 270 to > 400°C!

§ Can’t survive superheated areas, but
thrive in nearby gradients

§ Hot: sulfide chimneys a.k.a. “black 350°C vent emitting

smokers” sulfide- and
mineral-rich water

§ Release nutrients like sulfides and minerals

 Hydrothermal vents: who’s in there?
§ Thermophiles and hyperthermophiles!
§ Both bacteria and archaea

§ Most thermophilic: sulfur-

reducers isolated from smoker
chimney walls:
§ Pyrodictium and
Pyrolobus

§ Methanopyrus: archaeal
methanogen; oxidizes H2 ->
CH4
Diversity of microbial habitats: freshwater
§ Aquatic O2 levels much lower than in air
§ Freshwater lakes: anoxic zones just below surface
(surface microbes consume O2 before reaches lower)

§ Lotic system = running water = rivers and streams

§ Water flow = mostly sediment biofilm growth
§ Often nutrient runoff from surrounding land

§ Lentic system = free-standing water = lakes, etc.

§ Phytoplankton (e.g., diatoms) thrive
§ Organic C often produced from w/in (lotta autotrophy)
Diversity of microbial habitats: freshwater
§ Lotic system microbes are in equilibrium:
§ Chemoheterotrophs metabolize organics -> waste
§ Autotrophs use the ‘waste’ and produce O2

§ Eutrophication = response if too much nutrient-rich

organic matter (pollutants, fertilizer) enters a stream/lake
§ Hypoxia: deplete water O2 -> é chemoheterotroph
metabolism from extra organics
§ -> Algal/cyanobacterial blooms (can choke off O2
below surface)

Eutrophic Lake Mendota in

Madison, WI!
 Diversity of microbial habitats: lakes
§ Tropical lakes: warm all year = little temp change
§ Supports é productivity, but can get algae blooms
§ N2-fixing cyanobacteria can dominate = less diversity

§ Temperate lakes (around here!): nutrient locations

cycle with changes in temp

§ Summer: stratified in separate, distinct layers:

§ Epilimnion = warmer, less dense surface; microbes é
§ Hypolimnion = cooler deeper; lacks light and O2, but
organics sink from surface = nutrient-rich bottom
See text for deets!
§ Late fall/spring: turns over as cool, denser water sinks
§ Replenishes surface nutrients!
Public health and water quality/purification
§ Drinking water comes from freshwater (groundwater,
lakes, and rivers) = full of microbes, incl. pathogens!

§ Water is the most important

potential source of infectious
disease/intoxication outbreaks
§ A LOT of people commonly
use the same source

§ Water quality is the most The bubbler, a common

important single factor for local source of public
ensuring public health! drinking water
(Milw. Journal-Sentinel)
§ Monitoring quality and ensuring purification is key
 Monitoring water quality: indicator orgs.
§ We can’t screen for every possible pathogen in water
§ A few non-pathogens are fine (and unavoidable)

§ Sample for indicator microorganism = microbe whose

presence signals potential contamination with pathogens

§ What features or species of microbes can you think

of that would make good indicator organisms?
 Water quality and coliforms
§ Coliforms: facultatively anaerobic, gram-negative, non-
spore-forming, rod-shaped bacteria that ferment lactose
with gas formation within 48 hours at 35°C
§ ~ Enterics like E. coli
§ May indicate fecal contamination = water unsafe to use

§ A threshold level of
“acceptable” indicator #s
determines water quality

§ Depends on type of water

§ E.g. drinking water should
be zero/- for E. coli; higher for swimming/beach
§ Boiling advisories, beach closings (stormwater run-off)
 What makes a good indicator organism?
1. Specific to contamination source (human/animal fecal
contamination)
§ Enriching for it should not give false positives
2. Prevalent member of the contamination source

3. Survive longer in water than the hardiest pathogen

4. Suitable for all types of water sources tested

5. Not reproduce rapidly in the water source

6. Level in contaminated water should have a good

correlation to level of fecal pollution
7. Allow for easy testing
 So, a caveat on E. coli…
§ Is it specific? Nope! Non-pathogen strains/sources
§ Beaches closed more than needed due to gull poop

§ Is it a predominant member of human/animal feces?

Nope! Bacteroides et al. are much more prevalent

§ Can it survive as long as the hardiest pathogen?

Nope! Some pathogens don’t die as quickly

§ Does its absence ensure good water quality? Nope!

Provides no info on viruses, protozoa
§ (Cryptosporidium outbreak in Milwaukee!)

§ “Good enough” for now, but working on better (e.g.,

Bacteroides, Lachnospiraceae, >1 species)
 1. Culture-dependent methods
§ E.g., most-probable-number (MPN) dilution series of
drinking water (certain [cell] = contaminated)

§ E.g., membrane filter (MF): 0.2 µM filter traps bact; put

on eosin-methylene blue (EMB): select for gram –
enterics and differential for lactose-fermenters = coliforms
Membrane filter Selective/differential
Water sample
traps bacteria media

EMB
 Membrane filter methods
§ Advantages: great selection/differentiation for coliforms
§ Indicator organisms culturable = count quite accurate
§ Technically easy

§ Disadvantage: requires ≥ 16 hours growth

§ Can be a day too late for contamination = don’t swim
right after heavy rain

§ Defined substrate tests: variation on MF; ID total

coliforms AND E. coli specifically
§ Faster and more accurate than EMB
§ Test two metabolic abilities: one common to all
coliforms, one unique to E. coli
 Defined substrate test: an example
§ IDEXX Colilert: tests coliforms for β-
galactosidase (lac enzyme) w/ colored
substrate
§ Lactose + = degrade ONPG =
yellow

§ Test E. coli for unique

β-glucuronidase
§ Degrade MUG = fluorescent
light blue w/ UV
IDEXX; you use in lab!
§ Clear = no coliforms
 2. Culture-independent qPCR method
§ Quantitative PCR on DNA can enumerate water microbes
§ BUT only quantitative if careful =
§ Determine the fraction of fecal indicators to estimate
concentration

§ Advantages:
§ More rapid ID than filtering/culturing method
§ = Can ID water quality issue before it affects people

§ Disadvantages:
§ Need training/technical expertise
§ Not all PCRs amplify DNA in proportion found in
sample, so indicator #s may not be 100% accurate
 Drinking water purification
§ Early 1900s: filtration; reduce but not eliminate
pathogens

§ 1913: chlorine gas effective and

cheap way to disinfect:

§ Primary disinfectant: sufficient

to kill existing microbes and inhibit further growth in
filtered water
§ Now hypochlorite (bleach) or chlorine gas (easy)
§ Dissipates quickly = add ammonia -> chloramine
(NH2Cl)
§ Secondary: chlorine gone when reacts with organics,
so add another dose
 Wastewater and sewage treatment
§ Sewage: liquid contaminated with human/animal fecal
materials

§ Wastewater: domestic sewage or liquid industrial waste

that cannot be discarded untreated into lakes/streams
§ Why: health, economic, environmental and aesthetic

§ Daily average of 100-200 gallons of

water per American for washing, cooking,
drinking, and sanitation

§ 16,000 treatment works in U.S.

§ Each ~1 million or less of wastewater per day
§ Total: 32 billion gallons daily!
 Wastewater and sewage treatment
§ Microbes are essential for breaking down hazardous
waste!

§ Multistep wastewater treatment before released

§ (Except storm surge = must release untreated!)
 Primary wastewater treatment
§ Wastewater has a high Biological Oxygen Demand
(BOD) = how much dissolved O2 is needed to oxidize/
break down all that waste matter

§ Primary treatment: physical separation of solid/

particulate (in)organic material from wastewater

1. Screens/grates to remove large objects

2. Settle out rest by gravity during

transfer to successively lower
levels
 Secondary wastewater treatment
§ Secondary treatment: aerobic and anaerobic microbial
metabolism to further reduce organic matter and BOD
§ From 200-1500 BOD units to < 5!
§ No removal -> lotta microbe growth where released

1. Anaerobic: fermentative/degradative reactions

2. Aerobic: oxidative degradation

 Secondary wastewater treatment
1. Anaerobic: fermentative/degradative reactions
§ Digests sludge = large amount insoluble organics
(fiber, cellulose)
§ Break down polymers -> monomers (soluble)
§ Then, ferment those -> acetate, H2, CO2
§ Finally, methanogenic archaea -> CO2 and CH4

§ Can burn off

CH4 or use
as fuel for
the plant!
 Secondary wastewater treatment
2. Aerobic: oxidative breakdown
§ For water with low levels of organic matter
§ Activated sludge = continuously mix wastewater to
keep aerated
§ Zoogloea ramigera et al. form flocs (aggregated
masses)
§ Other microbes bind and help oxidize organics
 Tertiary wastewater treatment
§ Any other “physiochemical or biological process” =

§ Extra steps similar to those employed for drinking water:

§ Bioreactors, precipitation, filtration, chlorination, etc.

§ Greatly reduces levels of inorganic nutrients (e.g.,

phosphate, nitrogenous)
§ Essentially no nutrients left; can’t support microbe
growth

§ Best method, but not widely used since $$$

§ Final effluent = water that is released from plant

 Biodegradation and bioremediation
§ Biodegradation: using microbes to degrade an organic
molecule (to other organic molecules or inorganics)
§ Can be aerobic or anaerobic

§ Bioremediation: using microbes to convert oil, toxic

chemicals, or another environmental pollutants into less
harmful substances
§ Most successful: crude oil/
hydrocarbons

2010 Deepwater Horizon oil

spill in the Gulf of Mexico
Pollutants are large-scale enrichment media
§ If a pollutant is food for a microbe, adding it to
environment will stimulate growth

§ Carbon source isn’t enough to thrive and survive!

§ E.g., also add essential limiting nutrients (N, P, Fe)
to stimulate metabolism
§ E.g., if aerobic, need O2

§ Not perfect: oil-eaters can degrade some but not all

§ Speed: rate of degradation not as fast as rate of
ecological damage (loss of life, water contamination)
§ Other conditions (temp, pressure, light, etc.) play a
role
 Oil spills: enhancing bioremediation
§ For the 1989 Exxon Valdez spill in Alaska, the coastline
was fertilized to enhance the growth of those microbes:

Coastline Coastline
without added with added
nutrients nutrients
E.g., oil bioremediation: 2010 Gulf oil spill
§ Oil-eating microbes played huge role in decontaminating
Deepwater Horizon oil spill

§ Hydrocarbons naturally present

at low levels in Gulf
§ Spill = é [hydrocarbon] =
enrich those species #s!

§ Metabolism = aerobic
chemoheterotrophy:
§ (CH) + O2 -> CO2 + H2O

Hydrocarbon-oxidizing bact
in oil droplets
 E.g. of bioremediation: toxic uranium
§ Uranium mining can lead to major aquifer contamination
§ Old nuclear weapons, radioactivity, other industry

§ U6+ is soluble, and bacteria can reduce U6+ to U4+

(immobile precipitate) = limit human/animal contact
§ E.g., Shewanella, Geobacter, Clostridium, etc.
 Bioremediation of synthetics
§ Xenobiotic: a synthetic chemical not produced by
organisms in nature Xenos that microbes can
break down:
§ Microbes can still break
some of ‘em down!
§ Pesticides
§ Chlorinated compounds
§ Plastics

§ Problem: “emerging
contaminants”:
pharmaceuticals, hormones
 Bioremediation of plastics
§ 311 million metric tons produced/year!
§ Most synthetic = stick around for decades

§ Biodegradable plastics: e.g., Polyhydroxyalkanoate

(PHA): common storage polymer (Ralstonia
eutropha); still expensive

§ 2016: discovery of PET-degrading Ideonella

sakaiensis
§ One of toughest and most abundant plastics
§ Isolated from plastic bottle recycling plant!
§ Next step: scale up! (Lect. 22)