Microbiology of Waste Treatment/ Biodegradation of Pollutants Biology 422, Fall 2012

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Microbiology of Waste Treatment/

Biodegradation of Pollutants


Biology 422, Fall 2012

Michael D. Aitken

Department of Environmental Sciences & Engineering

Gillings School of Global Public Health

What are the pollutants of concern?


domestic wastewater or concentrated animal waste


organic
compounds that
would deplete oxygen if discharged
into surface water (river, stream, lake, estuary, ocean
); in
aggregate, referred to as “oxygen demand”


inorganic nutrients (N and P) that would stimulate excessive
algal growth (eutrophication) in a surface water body


N species that would deplete oxygen
in
surface water (NH
4
+
)
or
contaminate
groundwater if
distributed on
land

(NO
3
-
)


pathogenic microorganisms and
viruses


“emerging contaminants” (
e.g.
, pharmaceuticals, “personal
care products”, flame retardants)

Pollutants of concern (continued)


domestic solid
waste



industrial
wastewater


easily degradable organic

compounds (
e.g.
, food

production, breweries)


specific organic compounds

(
e.g.
, commercial products such as pharmaceuticals)


inorganic chemicals (
e.g.
, N, P, S, metals)


hazardous
waste


metals


specific organic compounds (
e.g.
, chlorinated solvents,

pesticides, aromatic hydrocarbons)


Summary of pollutant characteristics


Many pollutants are organic compounds that serve as
carbon and energy sources (
electron donors
) for growth
of heterotrophs


many are naturally occurring compounds


some are
xenobiotic


Some organic compounds are not known to serve as a
carbon or energy source for any microorganism


Some pollutants are inorganic compounds that serve as
an energy source for autotrophs


e.g.
, NH
4
+


Some pollutants are
terminal electron acceptors
required
for growth on an energy source (organic or inorganic)


e.g.
, NO
3
-
, perchlorate (ClO
4
-
), chlorinated hydrocarbons

Environmental applications of engineered
microbial processes


municipal wastewater treatment (ubiquitous in

developed countries)


treatment of some
industrial
wastewaters


controlled anaerobic decomposition in landfills
(“bioreactor landfills”)


composting of solid waste


bioremediation of contaminated soil or groundwater


above
-
ground (
ex situ
) or in place (
in situ
)


biofiltration

of contaminated air


common
features of all systems


open systems
(anyone can join the party!)


complex
communities
of naturally occurring microorganisms


Microbial diversity: an
under
-
explored
universe


estimated to be ~ 5 x 10
6

prokaryotic
species (bacteria
and archaea) on
Earth


we know
nothing

about most of them


for example: soil
can contain
thousands of species

of
prokaryotes
per
gram


at ~ 3
Mbp

per prokaryotic genome
,

>
10
10

bp

in “metagenome” of
a

one
-
gram
soil sample


human genome ~ 3 x 10
9

bp



one
gram of soil is genetically
more

complex
than the human genome


abundances range over several orders of magnitude

Julian Davies (2006): “once the diversity of the microbial world is
catalogued, it will make astronomy look like a pitiful science”

Underlying principles
of microbial ecology


Every organism has a unique range of capabilities, some
of which might be useful in an engineered process


Every organism has a unique range of conditions under
which it
will
grow

or at least
survive


Environmental systems are likely to be characterized by
relatively few dominant species and a large number of
low
-
abundance
species


Open

environments permit the growth of heterogeneous
communities


wastes typically are heterogeneous mixtures of organic and
inorganic compounds


therefore a diverse community of microorganisms can
be
expected in a given environmental system,
each
species with its own “niche


Factors influencing microbial communities


Environmental
conditions
govern which organisms
dominate (which organisms are
selected
)


major energy and carbon sources


dissolved oxygen concentration


aerobes


microaerophiles


anaerobes


concentration of other electron
acceptors (
e.g.
, NO
3
-
, SO
4
2
-
, Fe
3+
)


pH (
e.g.
,
acidophiles
)


temperature (
psychrophiles
,
mesophiles
, thermophiles)


salinity


availability of nutrients (e.g.,
sorbed

to a surface or within

a non
-
aqueous matrix
vs.
dissolved in water)

Influencing microbial selection (continued)


Native
organisms are almost always better adapted to
the local environmental conditions than added organisms
would be


creates problems for applications of genetically engineered
“superbugs” or commercial cultures


biological process engineering involves control of the
microbial community’s immediate environment


dissolved oxygen


pH


temperature


reactor configuration (can control availability of
major

carbon
sources)


THEREFORE WE HAVE CONTROL, TO A LARGE EXTENT, OVER
MICROBIAL SELECTION.


THIS IS THE KEY TO SUCCESS IN THE APPLICATION OF
BIOLOGICAL PROCESSES TO WASTE TREATMENT

Putting microbial ecology into practice


The science: which organisms do which functions?

what conditions do they require to grow and be
competitive?



The art: providing conditions to
select for

the
microorganisms that carry out the desired function



The engineering

how much?

how fast?

how big?

how good?

stoichiometry

kinetics

design

analysis

How
important is it to know something about the
makeup of
a microbial community in either waste
treatment or bioremediation of a contaminated
environment?


depends on the desired function of the process; the more
specific the function, the more knowledge is necessary

Examples


composting


septic tanks


decomposition

in landfills


animal waste
“treatment”

in “lagoons”


municipal or

industrial
wastewater
treatment


bioremediation of
contaminated soils
and sediment

Technological

sophistication

low

high

medium to high

Ability to

achieve

objectives

easy

easy to moderate

moderate to

difficult

Science

needed

little

some to a lot

a lot

Overview of municipal wastewater treatment

biological

treatment

raw

wastewater

screening

grit

removal

primary

sedimentation

disinfection

discharge

(advanced

treatment)

primary

sludge

excess
biomass

preliminary treatment

primary treatment

filtration

nitrification

nitrogen removal

phosphorus removal

nutrient (N&P)
removal

secondary treatment

odor

VOCs

VOCs

Municipal
wastewater
treatment:

biological processes

biological

treatment

raw

wastewater

screening

grit

removal

primary

sedimentation

disinfection

discharge

(advanced

treatment)

primary

sludge

excess
biomass

nitrification

nitrogen removal

phosphorus removal

nutrient (N&P)
removal

odor

VOCs

VOCs

Municipal wastewater treatment:

biological processes

biological

treatment

raw

wastewater

screening

grit

removal

primary

sedimentation

disinfection

discharge

(advanced

treatment)

primary

sludge

excess biomass

nitrification

nitrogen removal

phosphorus removal

nutrient (N&P)
removal

“activated sludge”


an aerobic

suspended culture process

“rotating biological contactor”


an aerobic biofilm process

Municipal
wastewater
treatment:

biological processes

biological

treatment

raw

wastewater

screening

grit

removal

primary

sedimentation

disinfection

discharge

(advanced

treatment)

primary

sludge

excess
biomass

nitrification

nitrogen removal

phosphorus removal

nutrient removal

odor

VOCs

VOCs

anaerobic digesters

Municipal
wastewater
treatment:

biological processes

biological

treatment

raw

wastewater

screening

grit

removal

primary

sedimentation

disinfection

discharge

(advanced

treatment)

primary

sludge

excess
biomass

nitrification

nitrogen removal

phosphorus removal

nutrient removal

odor

VOCs

VOCs

biofiltration

of air in a soil bed

bioscrubber

Microbial groups in waste treatment


aerobic oxidation of organic compounds: mostly
heterotrophic bacteria, some fungi


anaerobic decomposition of organic compounds:

complex

organic

substrates

fermentative bacteria

archaea

hydrogenotrophic

methanogens

aceticlastic

methanogens

Microbial groups (continued)


ammonia
removal by nitrification (aerobic process):


ammonia
-
oxidizing
bacteria:
NH
4
+

+ 1.5O
2



NO
2
-

+ H
2
O + 2H
+


nitrite
-
oxidizing
bacteria: NO
2
-

+ 0.5O2


NO
3
-

Net
:
NH
4
+

+ 2O
2



NO
3
-

+ H
2
O + 2H
+


denitrification
(anaerobic process
): facultative
heterotrophic bacteria:

organic substrates
+ NO
3
-



N
2

note nitrogen
removal

occurs by nitrification + denitrification


removal
of ammonia and nitrogen by anaerobic
ammonia oxidation (“anammox”): anaerobic bacteria


NH
4
+

+ NO
2
-



N
2
+ 2H
2
O


biological phosphorus removal: facultative heterotrophs


under anaerobic conditions, hydrolyze stored polyphosphate to
accumulate intracellular organic polymer (
e.g.
,
polyhydroxybutyrate
)


under aerobic conditions, oxidize stored organic polymer to accumulate
phosphate as intracellular poly
-
phosphate


Biodegradation of individual organic chemicals


What is “biodegradation”?


making a pollutant go away?



reducing the impact of the pollutant?


on the environment


on human health

General mechanisms of biodegradation


growth
-
related
metabolism


compound is
electron
donor


compound is
electron
acceptor



metabolism
not related to
growth of the organism


if
such activity is to be sustained,
then a growth substrate has to be
provided eventually

natural

xenobiotic

fraction of

chemicals

supporting

growth

fraction of

chemicals

not supporting

growth

Outcomes of biodegradation mechanisms


complete
metabolism (generally associated with growth)


mineralization

of a fraction of the initial compound mass to CO
2
,
H
2
O,
Cl
-
, SO
4
2
-
,
etc.;
e.g
.
, aerobic metabolism of glucose:

C
6
H
12
O
6

+ 6O
2

→ 6CO
2

+ 6H
2
O




assimilation

of a fraction of the initial compound mass
into

cellular biomass


incomplete metabolism (usually
fortuitous
transformation
to dead
-
end metabolites, unrelated to growth)


e.g
.
, transformation of trichloroethylene (TCE) to TCE epoxide by
methanotrophs

via methane
monooxygenase

(MMO)


C
2
HCl
3

+ O
2

+ NADH + H
+
→ C
2
HCl
3
O + NAD
+

+
H
2
O

mineral (inorganic) products

MMO

General features of metabolism


whether
growth
-
related or not, virtually all microbial
transformations of interest are catalyzed by
enzymes


if
the compound is metabolized completely, metabolism
involves one or more
pathways
, or sequences of
enzyme
-
catalyzed steps


enzymes
are coded for by
genes


synthesis
of an enzyme requires that the relevant
gene(s) be
expressed

(“turned on
”)

Problems with non
-
growth metabolism


the
necessary
enzyme(s)
for transformation of the
compound
is usually
not induced by the presence
of

the
compound


the
compound is usually not transformed extensively
(may be transformed only one step)


product(s)
of incomplete metabolism will accumulate
extracellularly


product(s)
can be just as toxic
as
parent
compound (
or more so
)


product(s)
might be consumed by other organisms; this is one
advantage of microbial communities over pure
cultures


competition
between the pollutant and the “natural”
substrate for the enzyme(s) capable of transforming the
pollutant must be considered

Postulates for biodegradation


a
biochemical mechanism for transformation or
complete metabolism of the compound must
exist


one
or more organisms possessing the relevant
gene(s) must be
present in the system


indigenous organisms


organisms inoculated into system (
bioaugmentation
)


gene(s)
coding for the relevant enzyme(s) must
be
expressed


by the compound itself


induced by some other means


mechanism
must be
manifested

Why biodegradation mechanisms might


not be manifested


limited bioavailability (generally an issue for
hydrophobic chemicals, particularly in the
subsurface)


concentration effects


substrate inhibition at high concentration


concentration too low to support
growth


inhibition
by other chemicals in the system


other conditions not favorable


pH


nutrient limitations


electron acceptor limitations