Site characterization and feasibility studies must determine key chemical,
physical, and microbiological properties of the site.
Main objective of site characterization is to identify the contaminants, their
concentration, and the extent of contamination.
The distribution of contaminants between soil and groundwater will largely
determine whether soil or groundwater treatment is applicable, while the extent
of contamination will largely determine the applicability of soil excavation and
Physical properties of organic contaminants
Octanol/water partition coefficient is defined as the ratio of a compound's
concentration in the octanol phase to its concentration in the aqueous phase of
a two-phase system.
Measured values for organic compounds range from 10-3
Compounds with low values (<10) are hydrophilic, with high water solubilities,
while compounds with high values ( > 104
) are very hydrophobic.
Compounds with low water solubilities and high Octanol/water coefficients will
be adsorbed more strongly to solids and are generally less biodegradable.
Highly soluble compounds tend to have low adsorption coefficients for soils
and tend to be more readily biodegradable
Vapor pressures and Henry's Law constant, HA, measure liquid—air partitioning.
Henry's Law states that the equilibrium partial pressure of a compound in the air
above the air/water interface, PA, is proportional to the concentration of that
compound in the water, usually expressed as the mole fraction, XA.
PA = HAXA
Aeration is often employed to strip volatile organic compounds from water and
is favored by large HA values.
Conversely, volatilization must be controlled or contained in many
Henry's Law constant is highly temperature sensitive, and temperature
changes of 10 °C can give threefold increases in HA.
In soil bioremediation the rate-limiting step is often the desorption of contaminants,
since sorption to soil particles and organic matter in soils can determine the
bioavailability of organic pollutants.
Bioavailability is also an important toxicity characteristic
Rates of in situ soil bioremediation are governed by mass transfer of contaminants
(desorption and diffusion), the convective—dispersive flux of oxygen and nutrients,
and the microbiological content of the soil.
On-site testing to determine the rate and extent of biodegradation can be done
immediately in consideration of ex situ process feasibility
Desorption tests measure the site-specific soil/water partition coefficients for the
contaminants of interest.
Measurements of aqueous phase and soil phase concentrations in equilibrium
may be sufficient to indicate potential problems with soil sorption
Many organic contaminants are hydrophobic, have a low water solubility, and are
tightly held to the soil phase.
Desorption of such contaminants is likely to be rate-limiting, especially (on ex situ
bioremediation where addition of nutrients and microorganisms and careful
control of environmental parameters can minimize these potential
Failure to bioremediate polyaromatic hydrocarbon (PAH) compounds has been
attributed to strong sorption to soil
Surfactants and cosolvents has been investigated in attempts to increase
bioavailability of contaminants that are strongly bound to soil.
High surfactant concentrations can be required to achieve small increases in
2% surfactant solutions are needed to remove a high percentage of compounds
such as higher-ringed PAHs, polychlorinated biphenyls (PCBs), and higher
molecular weight hydrocarbons from soils.
High surfactant concentrations can be required to achieve small increases in
Measurement of biodegradation rates by indigenous microorganisms is the first
step in microbiological characterization
These measurements can be complicated by low microbial populations or by
the absence of species capable of degrading contaminants.
Also, optimum conditions of temperature, oxygen nutrient supply, and contaminant
availability due to low solubility and sorption can limit degradation rates, especially
in early tests where these limiting factors are not well defined.
Main objective of microbial degradation tests is to determine whether the
indigenous microorganisms are capable of bioremediation when conditions are
optimized, or if inoculation by nonindigenous microorganisms will be required.
Bioremediation sites contaminated by 2-sec-buty-4,6- dinitrophenol (dinoseb),
the first site was remediated with indigenous microorganisms, second site required
inoculation with microorganisms from the first site
Chemical analyses that support process design include measurements
of pH, COD, TOC, nitrogen, phosphorus, and iron; and inhibitory, toxic,
or essential metals
Soil type, clay and organic matter content, and particle size distribution
analyses are used, for water, total suspended solids
Microbiological analyses supporting process design include BOD, plate
counts, and shake flask and/or column degradation studies with indigenous
microbes or introduced cultures
Bioremediation is usually carried out near neutral pH, although fungi
often require an acidic environment
Most microorganisms are mesophilic, requiring temperatures in the
25 to 37 °C range
More difficult to optimize are the oxygen and nutrient supply.
Most bacteria capable of degrading organic compounds are heterotrophic and
require an organic compound as a source of carbon and energy
It may be necessary to add a readily metabolizable carbon source such as
glucose to maintain cell viability or to increase cell growth and degradation rates.
Many xenobiotic compounds can be transformed by cometabolism, in which
the transformation does not serve as an energy source for synthesis of cell biomass,
which therefore requires a separate carbon source.
Sometimes concept of cometabolism is of limited use in
bioremediation process analysis and design.
A chemical that is cometabolized at one concentration or in one environment
may be mineralized under other conditions
Cometabolism can often be accelerated by the addition of a mineralizable
compound with a structure analogous to the target compound.
This method of analog enrichment has been used to enhance the cometabolism
of PCBs by addition of biphenyl, in which the unchlorinated biphenyl serves as
a carbon source for microorganisms that cometabolize PCBs with the enzymes
induced by biphenyl
Degradation is often coupled to growth and microbial mass, so the carbon
source that best supports growth also gives the highest rate of degradation
Since nitrogen and phosphorus supplies in soil systems are inadequate to support
microbial growth and degradation of organic compounds, most bioremediation
processes supply these two compounds
Biodegradation can occur aerobically or anaerobically.
The most effective biodegradation of hydrocarbon compounds is mediated by
Oxygen supply can be a rate-limiting factor, and large-scale aerobic bioremediation
of hydrocarbons must include an aeration system as a critical design component.
Early bioremediation work focused almost exclusively on aerobic systems, but
recent improvements in anaerobic technologies have shown that a wide range of
nitroaromatic compounds, chlorinated phenols, PAHs, and PCBs can be degraded
as well or better by anaerobic processes
In the selection of a microbial system and bioremediation method, some
examination of the degradation pathway is necessary.
At a minimum, the final degradation products must be tested for toxicity and
other regulatory demands for closure.
Predicting degradation rate
Degradation rates and equilibrium properties are useful for process
During wastewater or soil treatment, target pollutants can be degraded or
mineralized, volatilized, adsorbed onto effluent solids, or discharged in the liquid
Volatilization and sorption must be minimized so that the principal fate of
contaminants in a bioremediation process is biodegradation
Table is a portion of the 196-compound data base used in a fate model to
estimate volatile emissions, concentrations of toxic compounds in sludges, and
removal of toxic compounds during activated sludge wastewater treatment
The major error in predicting the fate of toxic compounds in wastewater treatment
plants was in estimating the value of the biodegradation rate constant.
For a compound such as tetrachloroethylene, with a low biodegradation rate
constant and a high Henry's Law constant, the principal fate will be volatilization.
Published biodegradation rate constants do not provide a good comparative basis
for process design since experimental conditions vary greatly.
Many unreported factors can influence biodegradation rates, including transport effects,
Acclimation of microorganisms to toxic chemicals, inhibition, and cometabolism.
Degradation rate constants are used mainly for order-of-magnitude estimates,
and other approaches to predicting biodegradability have been developed that
are based on physical or chemical properties of compounds.
A measure of hydrophobicity can be used to estimate relative biodegradation
rates among homologous compounds.
Rates of anaerobic degradation of halogenated aromatic compounds are
correlated with the strength of the carbon halide bond that is cleaved in the
A promising method for predicting biodegradation rates is the group contribution
approach, which estimates biodegradability from the type, location, and interactions
of the substituent groups that make up a compound.
An experimental data base that assesses mixed culture biodegradation data
for 800 organic compounds is available
Good predictions of degradation rates can be obtained for mixed-culture
aerobic degradation in processes such as activated sludge treatment.
The activated sludge reactor can be optimized with respect to aeration
using a model to calculate biodegradation and volatilization rates of organics
as functions of aeration rate.
Reactor options are determined primarily by the physical properties of the waste
and the chemical and biochemical properties of the contaminants.
If the waste is found in groundwater, then a continuous supported reactor
A suspended batch reactor is preferable with contaminated soil
A polar target compound favors an aerobic reactor, while nonpolar compounds
favor anaerobic reactors
Groundwater is easily treated in a continuous process in which the microbial
biomass must be retained in the reactor by adhering to a support.
Soils are difficult to transport, so batch reactors are favored.
Agitation is a critical design parameter required for aeration, cell suspension,
and transport of contaminants and nutrients during soil remediation.
The technology for the removal of suspended and dissolved contaminants from
municipal and industrial wastewater has been well established for wastes that are
readily biodegradable under aerobic conditions.
After preliminary treatment such as screening, grit removal, and sedimentation,
a biological reactor degrades the organic matter in the wastewater during
The reactor is an aerobic fixed film process or a suspended-growth,
activated sludge process.
Industry relies heavily on treatment of their chemical wastes by publicly owned
wastewater treatment facilities.
Removal rates of xenobiotic compounds are not well described
Activated sludge reactors
Polychlorinated phenols and biphenyls, phenols, phthalates, and PAHs were
tested, with average removal rates of 97%. However, higher concentrations can
destabilize an activated sludge system.
The presence of cyanide, pentachlorophenol, 1,2-dichloropropane, acrylonitrile,
phenolics, and ammonia can cause instability in the operation of activated
Mobile units are often required for the treatment of groundwater pumped out of
a contaminated aquifer, or wash water used to remove chemicals from a
The advantages of an activated sludge design for treating high-organic-content
wastewaters that produce a flocculating sludge are lost in these cases.
Low microbial growth, poor flocculation, and instability problems can make
an activated sludge process very difficult to apply to groundwater treatment
Fixed film processes, similar to the trickling filter designs for wastewater
treatment, can generally be operated at lower oxygen supply costs, with
retention of bacteria in the reactor and a more stable operation.
Fixed film reactors
The BioTrol aqueous treatment system is a good example of a fixed film, continuous
reactor used successfully for the treatment of groundwater.
This reactor is packed with corrugated polyvinyl chloride media on which a
bacterial biofilm is grown
The combination of groundwater flow, air sparging, and design of the support
media facilitates the upward and lateral distribution of water and air in the reactor.
Removal of more than 95% pentachlorophenol (PCP) is achieved at a 1.8 h
Fixed film or attached growth systems require degradative microorganisms with the
ability to attach to the surfaces of an inert packing.
Fixed film reactors can treat low concentrations of organics in wastewater
because of biomass retention, and they can also treat concentrations as high
as 1000 ppm.
The high biomass loadings of fixed film reactors render them insensitive
to shock loadings, that is, to high fluctuations in organic loadings.
The outer layers of the biofilm protect the inner cells from the toxicity of high
loadings, and the adsorption of contaminants within the biofilm reduces the
soluble concentrations of contaminants
The fixed film reactor used for municipal sewage is the trickling filter reactor,
with large packing, typically 50 mm diameter stones or synthetic plastic, and
downward liquid flow, producing a liquid film over the biofilm and leaving air
voidage within the packed bed.
Liquid hold-up is low and retention times are short, so that liquid recirculation
is necessary to adequately reduce contaminant concentrations.
Recirculated effluent is usually taken from a secondary clarifier output, rather than
directly from the trickling filter effluent, to minimize the risk of clogging the
trickling filter with biomass released from the filter.
Wastewater contaminated with xenobiotics is usually treated in a fixed film
reactor with upflow through a submerged plastic packing.
Nitrogen and phosphorus may be added to the inflow so that the C: N: P
ratio is approximately 100: 5: 1.
Reactor cells may be staged, and typically a 2-hour residence time is
sufficient to give a high level of contaminant degradation
The major advantage of the upflow fixed film reactor versus the trickling bed
reactor is the longer and better control of residence time.
A good example of a successful application of a fixed film bioreactor is the
treatment of a lagoon contaminated with 36ppm pentachlorophenol (PCP),
37 ppm polynuclear aromatics (PNAs), 52ppm solvents, and a total chemical
oxygen demand (COD) of 6700 ppm
While anaerobic processes for bioremediation of groundwater are uncommon,
anaerobic treatment is used for municipal sludge processing and for agricultural
and food-processing wastewaters with a high content of biodegradable matter.
The resultant methane production provides a valuable energy source.
Since energy production is not an option in the treatment of groundwater,
anaerobic processes are considered only for compounds recalcitrant to
Anaerobic microorganisms have great potential for the reductive dehalogenation of
multihalogenated aromatic compounds. With increasing levels of halogenation,
bioremediation may be feasible only with an initial anaerobic dehalogenation.
Once lightly halogenated or nonhalogenated compounds are produced, subsequent
degradations are more rapid in an aerobic environment. This suggests an
anaerobic-aerobic process for the degradation of compounds that are highly
chlorinated or nitrated, or for PAHs.
The anaerobic bioreactor is typically an upflow packed-bed reactor. The reactor is
completely filled with liquid except for gas formed during the process.
Coarse packing (2 to 6 cm) is used since anaerobic organisms can form large
floes instead of thin attached films, causing clogging.
Anaerobic reactors have a number of advantages, including high efficiency at
low organic loading, high loading capacity, stability with toxic substances, and
low energy requirements. COD reductions of 4 to 10kg/m3/day can be obtained
with residence times from 4 to 18 h.
A two-stage anaerobic-aerobic biofilm reactor process has been used to
metabolize hexachlorobenzene (HCB), tetrachloroethylene (PCE), and chloroform
Reductive chlorination is relatively rapid for these compounds and other highly
chlorinated compounds such as polychlorinated biphenyls, trichloroethylene,
carbon tetrachloride and 1,1,1-trichloroethane.
These are some of the most pervasive groundwater contaminants.
2,4,6-Trichlorophenol can be degraded to 4-chlorophenol in the anaerobic reactor,
and the 4-chlorophenol was mineralized in the subsequent aerobic operation.
A sequential anaerobic—aerobic process similar to the process shown in Figure
1.1 has been used to degrade nitrobenzene.
Under anaerobic conditions nitrobenzene was converted to aniline, a reaction
accelerated by the addition of glucose. Complete mineralization of aniline was
accomplished in the aerobic stage.
Anaerobic—aerobic processes have a high potential for the treatment of pulp mill
wastewater containing xenobiotic compounds.
The pulp and paper industry is under great pressure to remove chlorophenols,
chlorinated aliphatic hydrocarbons, and chlorinated dioxins and furans from
With ex situ treatment of contaminated soils, a controlled environment for soil
treatment can be maintained.
With mixing, nutrient addition, aeration, and other
environmental controls, mass transfer rates that typically limit in situ bioremediation
can be greatly increased.
The disadvantages of ex situ bioremediation are the costs of soil excavation and
reactor operation. Thus, ex situ bioremediation is favored by localized, shallow
Biological soil treatment by landfarming is a relatively simple and inexpensive
method for treating soil contaminated by compounds that are readily degraded
Contaminated soil is evacuated and usually treated in pits lined with a high-density
synthetic or clay liner (Figure 1.2). Perforated pipes can be placed in a layer of
sand between the liner and contaminated soil to collect drainage that can be
separately treated or recycled.
Alternatively, the treatment area can be graded to a sump where runoff is collected.
Aeration can be accomplished by tilling the soil or with forced aeration.
With tillage, soil is usually spread to a depth of 15 to 50 cm. For forced aeration,
soil is placed above slotted PVC pipes that are manifolded to a blower (Figure 1.2).
Nutrients may be added and pH adjusted.
Phosphorus is typically added as a salt of phosphoric acid and nitrogen as an
ammonium salt, a nitrate salt, or urea. Nutrient requirements are estimated from
contamination concentrations or laboratory treatability tests, and water is added or
sprayed onto the soil to maintain optimum moisture.
Landfarming has been widely implemented at petroleum production and storage
sites, and at sites contaminated with polynuclear aromatic residues (PNAs) or
The efficacy and design of land treatment for petroleum-contaminated soil has
been studied in controlled laboratory experiments.
The effects of soil type, fuel type, contamination level, and temperature on the
kinetics of fuel disappearance were determined for a land treatment process using
lime to raise the pH to 7.5-7.6, addition of 60 µ mol of N as NH4NO3 and 5 µ mol of
P as K2HPO4, and tilling
Disappearance of hydrocarbons was maximal at 27 °C. The C6 to C9 components
of gasoline were removed primarily by evaporation. The C10 to C 11 components
were removed by biodegradation. The medium distillates responded well to
bioremediation and increased in persistence in this order: jet fuel, heating oil,
and diesel oil.
Biodegradation can be accelerated in a prepared bed reactor with forced aeration.
These reactors are used at many Superfund sites for bioremediation of
PAHs and BTEX (benzene, toluene, ethylbenzene, and xylene).
A potential problem in soil treatment is the residual contaminant concentration
that is slowly or not noticeably degraded by soil microorganisms.
This nonbioavailable fraction is recognized by its slow transport out of soil
Slow desorption can result from entrapment in intraparticle micropores, especially
in the presence of organic matter, which can tightly bind nonionic organic
Soil leaching experiments can demonstrate whether contaminants are slowly
Released from the soil matrix, and the addition of surfactants can increase the
However, the mixing and surfactant addition that may be necessary to release
contaminants increase the cost of landfarming and favor the use of more intensive
bioremediation methods such as slurry reactors.