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 (NH4+)
or contaminate groundwater if distributed on land (NO3-)
– 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., NH4+
• Some pollutants are terminal electron acceptors required
for growth on an energy source (organic or inorganic)
– e.g., NO3-, perchlorate (ClO4-), 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 106 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,
> 1010 bp in “metagenome” of a
one-gram soil sample
– human genome ~ 3 x 109 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., NO3-, SO42-, Fe3+)
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?
stoichiometry
how fast?
kinetics
how big?
design
how good?
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
• municipal or • bioremediation of
• septic tanks
industrial
contaminated soils
wastewater
and sediment
• decomposition
in landfills
treatment
• animal waste
“treatment”
in “lagoons”
Technological
sophistication
low
high
medium to high
Ability to
achieve
objectives
easy
easy to moderate
Science
needed
little
some to a lot
moderate to
difficult
a lot
Overview of municipal wastewater treatment
secondary treatment
primary treatment
preliminary treatment
screening
raw
wastewater
grit
removal
odor
VOCs
primary
sedimentation
VOCs
filtration
nitrification
nitrogen removal
phosphorus removal
nutrient (N&P) removal
biological
treatment
(advanced
treatment)
primary
sludge
excess biomass
disinfection
discharge
Municipal wastewater treatment:
biological processes
screening
raw
wastewater
grit
removal
odor
VOCs
primary
sedimentation
VOCs
nitrification
nitrogen removal
phosphorus removal
nutrient (N&P) removal
biological
treatment
(advanced
treatment)
primary
sludge
excess biomass
disinfection
discharge
Municipal wastewater treatment:
biological processes
“activated sludge” – an aerobic
suspended culture process
nitrification
nitrogen removal
phosphorus removal
nutrient (N&P) removal
screening
raw
wastewater
grit
removal
primary
sedimentation
biological
treatment
(advanced
treatment)
primary
sludge
“rotating biological contactor” –
an aerobic biofilm process
excess biomass
disinfection
discharge
Municipal wastewater treatment:
biological processes
screening
raw
wastewater
grit
removal
anaerobic digesters
odor
VOCs
primary
sedimentation
VOCs
nitrification
nitrogen removal
phosphorus removal
nutrient removal
biological
treatment
(advanced
treatment)
primary
sludge
excess biomass
disinfection
discharge
Municipal wastewater treatment:
biological processes
odor
VOCs
screening
raw
wastewater
bioscrubber
grit
removal
primary
sedimentation
VOCs
nitrification
nitrogen removal
phosphorus removal
nutrient removal
biological
treatment
(advanced
treatment)
primary
sludge
biofiltration of air in a soil bed
excess biomass
disinfection
discharge
Microbial groups in waste treatment
• aerobic oxidation of organic compounds: mostly
heterotrophic bacteria, some fungi
• anaerobic decomposition of organic compounds:
hydrogenotrophic
methanogens
complex
organic
substrates
aceticlastic
methanogens
fermentative bacteria
archaea
Microbial groups (continued)
• ammonia removal by nitrification (aerobic process):
ammonia-oxidizing bacteria: NH4+ + 1.5O2  NO2- + H2O + 2H+
nitrite-oxidizing bacteria: NO2- + 0.5O2  NO3Net: NH4+ + 2O2  NO3- + H2O + 2H+
• denitrification (anaerobic process): facultative
heterotrophic bacteria: organic substrates + NO3-  N2
note nitrogen removal occurs by nitrification + denitrification
• removal of ammonia and nitrogen by anaerobic
ammonia oxidation (“anammox”): anaerobic bacteria
NH4+ + NO2-  N2 + 2H2O
• 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
fraction of
chemicals
supporting
growth
xenobiotic
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 CO2,
H2O, Cl-, SO42-, etc.; e.g., aerobic metabolism of glucose:
C6H12O6 + 6O2 → 6CO2 + 6H2O
mineral (inorganic) products
– 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)
MMO
C2HCl3 + O2 + NADH + H+ → C2HCl3O + NAD+ + H2O
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