BIORESTORATION OF AQUIFERS CONTAMINATED
WITH ORGANIC COMPOUNDS
M. D. Lee
J. M. Thomas
R. C. Borden
P. B. Bedient
J. T. Wilson
C. H. Ward
NATIONAL CENTER
FOR
GROUND WATER RESEARCH
A CONSORTIUM OF
RICE UNIVERSITY
UNIVERSITY OF OKLAHOMA
OKLAHOMA STATE UNIVERSITY
in cooperation with
R. S. Kerr Environmental Research Laboratory
U.S. Environmental Protection Agency
Ada, Oklahoma
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Volume 18, Issue 1 (1988)
29
BIORESTORATION OF AQUIFERS CONTAMINATED WITH ORGANIC
COMPOUNDS
Authors M. D. Lee
J. M. Thomas
R. C. Borden
P. B. Bedient
C. H. Ward
National Center for Ground Water
Research
Department of Environmental Science and
Engineering
Rice University
Houston. Texas
J. T. Wilson
Robert S Kerr Environmental Research
Laboratory
Subsurface Processes Branch
U S Environmental Protection Agency
Ada, Oklahoma
Referee R A Conway
Central Engineering Department
Union Carbide Corporation
South Charleston West Virginia
I INTRODUCTION
Ground water is a major source of drinking, industrial, and agricultural water in the U S
and a limited resource Therefore, contamination of ground water by anthropogenic activities
is of dire concern 1 Organic compounds can contaminate aquifers by inadvertent spills,
improper waste disposal techniques, and agricultural practices 2 The widespread distribution
of organic chemicals in ground water and the resulting adverse health effects have prompted
both the public and scientific community to examine the fate of this limited resource 1 A
1980 survey of the drinking waters from 39 communities using ground water indicated that
23% of the wells were contaminated with dichloroethylenes and other halogenated aliphatic
compounds 1 Other industrial chemicals such as toluene and phthalate esters have also been
detected in various surveys of ground water quality
There are a number of techniques available to remediate ground water contaminated with
organic compounds These include physical containment, in situ treatment with chemicals
or microbes, and withdrawal and treatment via various forms of physical, chemical, or
biological processes 4 Examples of physical containment techniques are excavation and
removal to a secure site, installation of barriers to ground water flow, and hydrodynamic
control by injection and production wells In situ chemical treatment involves inactivating
or immobilizing contaminants with chemical agents In addition to physical containment and
in situ chemical treatment, subsurface pollutants can be treated in sun by stimulating the
native microbial population Another in situ biostimulation technique which has not yet been
demonstrated is the inoculation of the subsurface with a microbial population that has
specialized metabolic capabilities Related processes such as the addition of bioemulsifiers
or surfactants to increase the availability of subsurface contaminants to the microflora can
also be used Contaminated ground water can be withdrawn and treated by physical processes
such as adsorption onto activated carbon or transfer to the gaseous phase by air stripping.
Reprinted from the CRC Critical Reweus in Environmental Control Vol 18 Issue I pages 29 89 c 1988 bs CRC Press Inc
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30 CRC Critical Reviewi in Environmental Control
chemical processes such as precipitation, oxidation, or reduction reactions, or biological
processes Combinations of these processes are often more successful than individual tech-
niques When applicable, biological treatment may offer the advantage of partial or complete
destruction of the contaminants rather than simply transferring the pollution to another phase
of the environment
II IN SITU TREATMENTS
A. Microbial Activity in Aquifers
/ Evidence for Microbial Activity in the Subsurface
Technologies for in situ biorestoration of polluted aquifers have resulted from research
indicating that subsurface microorganisms exist, are metabolically active, and often nutri-
tionally diverse Most of the research on ground water microbiology was initiated by a
review, published in 1973. by Dunlap and McNabb' of the Robert S Kerr Environmental
Research Laboratory, which addressed subsurface biological activity in relation to ground
water pollution Before publication of the review, the concept of biological activity below
the rhizosphere had not been widely received Microbiologists were skeptical about biological
activity in the subsurface because of the oligotrophy conditions that exist below the rhizosphere6
and by an early study which indicated that microbial numbers decline precipitously with
depth 7
2 Sampling Methods for Subsurface Microbes
A document that described sampling methods for subsurface microorganisms was pub-
lished in 1977 by the Environmental Protection Agency (EPA) * The method for procuring
a representative sample of unconsolidated subsurface soil has since been modified 9 A soil
sample is collected by first drilling a borehole to a desired depth with an auger and then
taking the sample with a core barrel After sample procurement, the core is extruded through
a sterile paring device that removes the outer layer of soil that has come in contact with the
core barrel The remaining soil core is thus uncontaminated by the sampling procedure and
is considered to be representative of the subsurface
Investigations of microbial activity in the subsurface conducted prior to the development
of the sampling techniques were equivocal because of the potential for contamination during
sample procurement In addition, many of the investigations were conducted using well
water instead of core material Recent evidence suggests that the majority of subsurface
microorganisms is associated with soil particles 10 In addition, well water may contain
microorganisms that are artifacts of the well because of subsurface contamination during
well installation and changes in water quality around the well
3 Microbial Numbers in the Subsurface
Methods to enumerate the subsurface microflora also have been developed Electron
microscopy, viable counts, epifluorescence microscopy, and measurements of biochemical
components have been used to estimate microbial bioniass 9 11 22 In contrast to Waksman's
study,7 which reported that microbial numbers declined with depth, uniform population
levels around I0A to 107 cclls/g dry soil, measured by epifluorescence microscopy, were
reported for profiles of uncontaminated shallow aquifers y " 13 16IK However, bacteria in a
chalk aquifer (consolidated) were sporadically distributed with depth 21 Close examination
of the subsurface strata indicated patchiness of bacterial populations, samples from the top
of the unsaturated zone of an artesian aquifer yielded the highest counts, whereas those from
bedrock and confining layers yielded the lowest total counts 24
4 Microbial Ecology of the Subsurface
Bacteria are the predominant form of microorganism observed in the subsurface, although
a few higher life forms have been detected v Some eukaryotic forms which may be
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Volume 18, Issue I (1988)
31
fungal spores or yeast cells have been observed in the upper 10 m of a soil profile 122" 22
Bacteria, protozoa, and fungi have been detected in samples of ground water collected from
I-year-old wells 2S In addition, a slow-growing amoeba has been isolated and cultured from
the ground water interface of an uncontaminated soil 24
Organic matter that enters the uncontaminated subsurface is usually the more refractory
humic substances which resist degradation while percolating through the biologically active
soil zone The organic material available for metabolism by the subsurface microflora is
likely to be present in low concentrations and difficult to degrade The majority of micro-
organisms present in such nutrient-poor environments is generally oligotrophy Character-
nation of the subsurface microflora indicates that the bacteria are usually smaller (mtrophenyl)-5-phenyl tetrazolium chloride by cytochromes lh 18 Microbial activity,
measured by the hydrolysis of fluorescein diacetate, declined with depth in the unsaturated
zone of Ultisols and Alfisols,22 however, 2-(/?-iodophenyl)-3-(/?-nitrophenyl)-5-phcnyl tet-
razolium chloride reduction varied greatly between strata of a soil profile obtained from a
shallow aquifer 24
Many subsurface microorganisms are nutritionally diverse (Table I) Simple substrates
such as glucose, glutamic acid, arginine, a mixture of amino acids, and a synthetic compound,
nitnlotnacetic acid, were mineralized in samples of uncontaminated ground water 26 Polar
solvents such as acetone, isopropanol, methanol, ethanol. and tert-butanol also have been
reported to degrade aerobically by subsurface microorganisms 27 2R More challenging con-
taminants that are aerobically degraded by subsurface microorganisms include the methylated
benzenes, chlorinated benzenes,2" chlorinated phenols,10 and methylene chloride 27 Highly
lipophilic compounds such as naphthalene, methylnapththalenes. dibenzofuran. fluorene.
and phenanthrene are also biotransformed in the subsurface 11,2
The microflora in some uncontaminated soils require little or no acclimation period to
degrade many xenobtotics For example, toluene, chlorobenzene. and bromodichloromethane
were biotransformed in uncontaminated soil, but not 1,2-dichloroethane, 1,1,2-trichloro-
ethane, tnchloroethylene, and tetrachloroethylene y Benzene, toluene, and the xylene isomers
were found to degrade in uncontaminated subsurface soils 13 In addition, methanol (80 to
100 ppm) was degraded completely after 2 months, whereas tert-butanol degraded much
slower in two uncontaminated anaerobic aquifers 14
In contrast to reports of degradation of xenobiotics added to uncontaminated soil, long
periods of acclimation to subsurface pollutants may be required before biodegradation can
occur Wilson et al 11 reported degradation of naphthalene, 1-methyl naphthalene, 2-methyl
naphthalene, dibenzofuran, and fluorene at 100 to 1000 |xg/£ in subsurface soil in the plume
of contamination from a creosote waste pit. however, degradation of these compounds was
not observed in uncontaminated soil from the same site The time and concentration required
for acclimation of the microflora to subsurface pollutants are unknown Spain and van Veld"
reported a threshold concentration of 10 ppb for adaptation to p-nitrophenol in samples of
sediment and natural water A better understanding of acclimation processes may explain
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32
CRC Critical Reviews in Environmental Control
Table 1
ORGANIC COMPOUNDS THAT HAVE BEEN
SHOWN TO BE BIODEGRADABLE IN THE
SUBSURFACE
Soil from con-
taminated
Compound
area
Aerobic
Ref
Njtural compounds
Glucose
No
Yes
26
Glutamic acid
Argininc
Solvents
Acetone
Yes
Yes
27
Elhanol
Isopropanol
(crt-Butanol
Yes
Yes
28
Methanol
Bromodichloromethane
No
Yes
9
Aromatics
Ben/ene
No
Yes
33
Xylene
Yes
Methylated ben/enes
Yes
29
Chlorinated ben/enes
Chlorinated phenols
Yes
Yes
30
Naphthalene
Yes
Yes
31 32
Dibenzofuran
Fluorene
Phenanthrene
Toluene
No
Yes
9
Chlorobenzcne
why some chemicals persist in the subsurface even though they have been reported to degrade
in laboratory cultures and samples of surface water and soil
6 Environmental Factors Which May Limit Biodegradation
Environmental factors may limit or preclude the biodegradation of subsurface organic
pollutants, even in the presence of adapted organisms The recalcitrance of compounds
thought to be biodegradable may result from a lack of an essential nutrient, substrate con-
centration, substrate inaccessibility, and the presence of toxicants 16 Transport of contami-
nants in the subsurface also affects biodegradation Transport is discussed in detail in Section
IV
Biodegradation of many organic pollutants in the subsurface may be limited by insufficient
concentrations of oxygen Alexander17 reported that even the metabolism of carbohydrates
may be inhibited in oxygen-depleted environments Lee and Ward12 found that the rate and
extent of biotransformation of naphthalene, 2-methyl naphthalene, dibenzofuran, fluorene,
and phenanthrene were greater in oxygenated ground water than in oxygen-depleted water
Contrary to general theory that complete degradation (mineralization) of hydrocarbons re-
quires molecular oxygen, more recent research suggests that alternate pathways exist under
anaerobic conditions Kuhn et al 29 reported mineralization of xylenes in samples of river
alluvium under denitrifying conditions In addition, benzene, toluene, the xylenes, and other
alkylbenzenes were metabolized in methanogenic river alluvium that had been contaminated
with landfill leachate,38 mineralization of toluene was confirmed by adding l4C-labeled
toluene and measuring the amount of |dC02 produced Grbic-Galic and VogeP" also reported
mineralization of toluene and benzene under anaerobic conditions by a methanogenic con-
sortium acclimated to ferulate Further tests indicated that water supplied the oxygen that
is first incorporated into the monoaromatic compounds 40
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Volume 18, Issue 1 (1988) 33
The presence of oxygen may inhibit the biodegradation of many halogenated aliphatic
compounds in the subsurface Degradation of trihalomethanes, trichlorocthylene. and tet-
rachloroethylene did not occur in aerobic cultures of sewage bacteria, however, the trihal-
omethanes were degraded anaerobically by mixed cultures of methanogens 41 In addition.
Bouwer and McCarty42 reported that chloroform, carbon tetrachloride, and bronnnated tri-
halomethanes, but not chlorinated benzenes, ethylbenzene. or naphthalene, were biotrans-
formed under denitrifying conditions
In addition to oxygen, other nutrients may limit the biodegradation of organic pollutants
in the subsurface Inorganic nutrients, such as nitrogen and phosphorus, may be limiting
when the ratios of carbon to nitrogen or phosphorus exceed that required for microbial
processes On the other hand, the presence of sulfate may inhibit mcthanogenic consortia
that have been reported to dehalogenate and mineralize many chlorinated aromatic
compounds 11,41
The effect of substrate concentration on biodegradation of organic compounds in suiface
soils and waters has been documented 44 Thresholds below which degradation is slow or
does not occur may exist for compounds that are readily biodegradable at higher concen-
trations Boethling and Alexander4"1 reported that 10% of 2,4-dichlorophenoxyacetate at
concentrations of 22 pg/mf and 2 2 ng/m£ was mineralized in stream water, whereas about
80% was mineralized at higher concentrations of 0 22 and 22 jjig/mf On the other hand,
microorganisms may be inhibited or killed by high concentrations of organic pollutants that
result from injection wells and hazardous waste sites Lee46 reported that glucose mineral-
ization was inhibited in subsurface soil heavily contaminated with creosote, however, glucose
was mineralized in uncontaminated and slightly contaminated core material from the same
site
Other factors such as sorption, pH, and temperature may also affect biodegradation of
pollutants in the subsurface Many of the organic compounds contaminating the subsurface
are highly lipophilic These compounds are sorbed by soil more strongly than the more
hydrophilic compounds 47 Sorption may enhance degradation by concentrating nutrients or,
conversely, may prevent degradation by rendering the substrate unavailable to the micioor-
ganism Zobell4R reported that sorption of organic material to solid surfaces in dilute nutrient
solutions increased microbial respiration In contrast. Ogram et al 4g observed that 2-4
dichlorophenoxy acetic acid sorbed to soil was completely protected from microbial deg-
radation Therefore, sorption may be important in nutrient scavenging in uncontaminated
aquifers which are generally oligotrophy, however, sorption may compete with the micro-
flora for subsurface pollutants that are relatively hydrophobic
The soil pH may affect sorption of lonizable compounds in addition to limiting the types
of microorganisms in the subsurface Methanogens, which have been implicated in miner-
alization of some aromatic hydrocarbons, are inhibited at pH values less than 6 w Nitrifi-
cation, the microbial conversion of ammonia to nitrate, is also limited at pH values below
6 and ts negligible below 5 Hambnck et al 51 also reported that mineralization of octadecane
and naphthalene in sediment was faster at a pH of 8 than 5
Temperature also influences microbial metabolism of subsurface pollutants The temper-
ature of the upper 10 m of the subsurface may vary seasonably, however, that between 9
to 18 m approximates the mean air temperature (between 3 and 25°C in the U S ) of a
particular region 52 Biodegradation of subsurface pollutants in the more northern climates
may therefore be limited by cooler temperatures Bartholomew and Pfaender" reported that
the microbial metabolism of m-cresol, nitnlotnacetic acid, and chlorinated benzenes in fresh
water and estuanne areas decreased as temperature decreased Atlas^*1 and Mulkins-Phillips
and Stewart15 also reported a direct relationship between petroleum hydrocarbon degradation
and temperature
In summary, the subsurface environment contains microorganisms that degrade many of
the organic compounds that contaminate ground water The subsurface microflora in un-
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34 CRC Critical Reviews in Environmental Contiol
contaminated aquifers is probably oligotrophy The majority of the microorganisms is as-
sociated with soil particles Even in the presence of adapted populations, environmental
factors such as temperature, pH, dissolved oxygen levels, inorganic nutrient concentrations,
and the availability and concentration of the oigumc contaminants may limit biodegradation
of subsurface pollutants
B. Biostimulation by Addition of Limiting Nutrients
/ Development of the In Sun Btostimulatton Process with Owgen Supplied b\ An Sparging
a. Application of the Degradative Activity of Subsurface Microbes
The potential for biodegradation of organic compounds in contaminated aquifers was first
recognized in 1971 Williams and Wilder"*' observed that ground water contaminated with
gasoline from a leaking pipeline in the Los Angeles-Glcndale, Calif , area contained bacteria
that degraded hydrocarbons, however, studies indicated that biodegradation of the gasoline
was limited by the availability of oxygen, mineral nutrients, and hydrocarbon surface area
These investigators suggested that the hydrocarbon-degrading bacteria could be used to clean
the aquifer of residual gasoline however, concern was expressed that bacterial growth would
plug the well and formation during the process McKce et al " conducted bacteriological
investigations using soil, water, and bacteria from the Los Angeles-Glcndale. Calif , site
and found several species of Pseudomonas and Artluohactei that could degrade gasoline
The total number of gasoline-degrading bacteria in the ground water numbered over 50.000
cells/m
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Volume 18, Issue 1 (1988) 35
table ranged from 9 2 to 30 5 m in the 46 monitoring wells installed at the site Before
biorestoration was attempted, remedial action consisted of conventional pump and treat
technologies The gasoline was contained by continuously pumping water from wells located
in the spill area About 238,000 C of the gasoline was recovered by physical methods,
however, recovery was incomplete and approximately 119,000 € of residual gasoline re-
mained The concentration of dissolved gasoline in the withdrawn ground water averaged
<5 ppm The time required for remediation of the aquifer using this pump and treat technique
was estimated to be more than 100 years
During the initial phases of remediation, problems in analyzing the concentration of
residual hydrocarbons were encountered hl These problems were later attributed to gasoline
degradation by bacteria in the ground water A program designed to investigate the potential
for biodegradation of the gasoline by these organisms was then initiated A laboratory study
indicated that supplements of air, inorganic nitrogen, and phosphate salts could increase the
numbers of hydrocarbon-degrading bacteria by 1000-fold h2 Small-scale field studies also
indicated that nutrient additions would enhance the growth of bacteria that degrade hydro-
carbons 61 A full-scale program to stimulate biodegradation of the gasoline in the aquifer
was then initiated 62 The nutrient amendment, which contained ammonium sulfate, disodium
phosphate, and monosodium phosphate, was injected into the aquifer as a 30% concentrate
by batch addition Biodegradation of I € of gasoline was estimated to require 44 g of
nitrogen, 22 g of phosphorus, and 730 g of oxygen Batch addition of the nutrients worked
as well as continuous addition and was more cost-effective, however, high concentrations
of nutrients could osmotically shock the microorganisms Oxygen was supplied by sparging
air into the wells using paint sprayer-type compressors and carborundum diffusers with a
flow rate of 0 06 mVmin As a result of the treatment, the bacterial population increased
from about I03 to 107 cells/mf High bacterial counts mirrored locations of high gasoline
concentrations at the site 61
During the biostimulation program at the Ambler, Penn , site, 32 cultures of bacteria that
actively metabolized gasoline were isolated and characterized M The isolates included species
of the genera Nocardia, Micrococcus, Acmetobacter, Flavobatterium, and Pseudomonas,
some cultures could not be identified The results of experiments that investigated the
metabolic capabilities of the isolates suggested that the Nocardia cultures were largely
responsible for the degradation of the aliphatic hydrocarbons, whereas those from the genus
Pseudomonas degraded the aromatics Gasoline was degraded by a mixed culture from
ground water However, individual components of gasoline such as branched paraffins,
olefins, or cyclic alkanes did not support the growth of any isolate
The bioreclamation program conducted by Suntech in Ambler, Penn , was reasonably
successful During the period of nutrient addition, the concentration of gasoline in the ground
water did not decline, however gasoline could not be detected in ground water 10 months
later62 A 1000-fold increase in the numbers of total and hydrocarbon-degrading bacteria
was observed in ground water from many wells 61 The waters from some wells exhibited
foaming because of high microbial numbers and associated exopolysacchandes Counts of
microorganisms determined I year after the nutrient addition was terminated indicated that
the microbial population had declined Estimates based on the amount of nitrogen and
phosphorus removed from the nutrient solution suggested that between 88,600 and 112,400
€ of gasoline were degraded However, this estimate was not particularly accurate because
some of the nutrients may have been adsorbed by the soil or lost from the biostimulation
area by dilution In addition, the estimates were based on discrete samples rather than
composited samples Large quantities of nutrients were used in this project, approximately
79 t of food-grade reagents were purchased
c. Steps in the Biostimulation Process
The basic steps involved in an in suu biorestoration program are (1) site investigation.
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36 CRC Critical Reviews m Environmental Control
(2) free product recovery (3) microbial degradation enhancement study, (4) system design,
(5) operation, and (6) monitoring ¦" The first step in the process is to define the hydrogcology
and the extent of contamination of the site Important hydrogeologic characteristics include
the direction and rate of ground water flow, the depths to the water table and to the
contaminated zone, the specific yield of the aquifer and the heterogeneity of the soil In
addition, hydraulic connections between aquifers, potential recharge and discharge areas,
and fluctuations in the water table should be considered The sustainable pumping rate must
also be determined These parameters can be determined by surveying the existing data
for that site and region, reconnaissance by experienced hydrogeologists, geophysical surveys,
excavation of test pits, and installation of boreholes and monitoring wells67 Low dissolved
oxygen concentrations may indicate an active zone of hydrocarbon biodegradation The
types and concentrations of contaminants are also important The type of remedial action
chosen for a particular site depends on the time elapsed since the spill, the arcal extent of
contamination, the nature of contaminants, and whether the contamination is acute, chronic,
or periodic The urgency for action and the treatment level that must be achieved will depend
on the potential for contamination of drinking water or agricultural water wells
After defining the site hydrogeology, the next step is recovery of free product Depending
on the characteristics of the aquifer and contaminants, free product can account for as much
as 91% of the spilled hydrocarbon The remaining hydrocarbon, which is sorbed to the
soil and dissolved in the ground water, may account for 9 to 40% of the total hydrocarbon
spilled, the majority is usually sorbed, however, the dissolved phase is the most difficult to
treat The pure product can be removed using physical recovery techniques which include
(1) a single pump system that produces a mixture of hydrocarbon and water that must be
separated, but requires minimal equipment and drilling, (2) a two-pump two-well system
which utilized one well to produce a water table gradient and a second well to recover the
floating product, or (3) a single well with two pumps in which a lower pump produces a
gradient and an upper pump collects the free product 4 Physical recovery often accounts for
only 30 to 60% of the spilled hydrocarbon before yields decline "v Continued pumping of
contaminated wells may contain a spill
Prior to in situ treatment, a laboratory study is conducted to determine the nutrient
requirements that will enable the indigenous microorganisms to efficiently degrade the
contaminants 4 Kaufman70 suggested that these laboratory studies can provide a reliable basis
for field trials, however, the studies must be performed under conditions that simulate the
field For example, Kuhlmeier and Sunderland71 conducted a laboratory investigation of the
unsaturated zone using samples saturated with ground water Clearly, the results of their
study do not represent the fate of the organics in the unsaturated zone A chemical analysis
of the ground water provides little information about the nutrient requirements of the mi-
croflora 72 However, the chemistry of the site will affect the nutrient formulation Limestone
and high mineral content soils and ground waters will also affect nutrient availability by
reacting with the phosphorus 71 In addition, nutrients may sorb onto soils, especially silts
and clays, and be unavailable to the microflora
Laboratory studies conducted to determine appropriate nutrient formulations can be per-
formed using a number of techniques An increase in the number of total and hydrocarbon-
degrading bacteria has been used to identify limiting nutrients in a factorial experimental
design h2 72 However, an increase in microbial numbers does not demonstrate that the sub-
strate of interest is being metabolized Batch culture techniques designed to measure the
disappearance of the contaminant74 and electrolytic respirometer studies designed to measure
the uptake of oxygen also have been used 75 Biotransformation studies which measure the
disappearance of the contaminants or mineralization studies which indicate the complete
destruction of the compound to carbon dioxide and water will confirm that the contaminants
are being degraded Controls to detect abiotic transformation of the pollutants and tests to
detect toxic effects of the contaminants on the microflora should be included 76
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Volume 18, Issue 1 (1988)
37
NUTRIENT
ADDITION
TANK
TO SEWER OR
RECIRCULATE
4Xh
AIR
COMPRESSOR
WATER SUPPLY
COARSE
SAND
— PRODUCTION WELL
¦—INJECTION WELL
WATER TABLE
SPILLED MATERIALS
-SPARGER
CLAY
FIGURE 1 lypKal schematic tor acrobit subsurt.KC bioreMorjlion 12
l«r Tabl«*^>v
Monitoring Well
FIGURE 2 Use oI intilir.ilion gallery for reciauljlion of vy.ilcr jnd nulricnls
in in uitt bioresiorjuon
A system for injection of nutrients into the formation and circulation through the contam-
inated portion of the aquifer must be designed and constructed 4 The system usually includes
injection and production wells and equipment for the addition and mixing of the nutnent
solution 77 A typical system is shown in Figure I Placement of injection and production
wells may be restricted by the presence of physical structures In addition, wells should be
screened to accommodate seasonal fluctuations in the level of the water table Air can be
supplied with carborundum diffusers."1 by smaller diffusers constructed from a short piece
of DuPont Viaflo tubing,72 or by diffusers spaced along air lines buried in the injection
lines 7K The size of the compressor and the number of diffusers are determined by the extent
of contamination and the time allowed for treatment 77 Nutrients also can be circulated using
an infiltration gallery (Figure 2), this method provides an additional advantage of treating
the residual gasoline that may be trapped in the pore spaces of the unsaturated zone 7g
Oxygen also can be supplied using hydrogen peroxide, ozone, or soil venting (see section
on alternative oxygen sources) Well installation should be performed under the direction
of a hydrogeologist to ensure adequate circulation of the ground water4 Produced water can
be recycled to recirculate unused nutrients, avoid disposal of potentially contaminated ground
water, and avoid the need for makeup water
Inorganic nutrients can be added to the subsurface once the system is constructed Con-
tinuous injection of the nutrient solution is labor intensive but may be preferred to batch
addition in some instances Continuous addition of oxygen is recommended because the
oxygen is likely to be a limiting factor in hydrocarbon degradation
The performance of the system and proper distribution of the nutrients can be monitored
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38 CRC Critical Reviews in Environmental Control
by measuring the organic inorganic, and bacterial levels4 Carbon dioxide levels are also
an indicator of microbial activity in the formation "" Depending on the characteristics of the
nutrients and soil, nutrients can be removed from solution by sorption onto soil " For
example, about 90% of the ammonium and phosphate and 70% of the hydrogen peroxide
added to a sandy soil with low calcium, magnesium, and iron was recovered After passage
of a nutrient solution through a column packed with a clay soil that had high calcium and
magnesium but low iron and chloride levels, 100. 66, and 25% of the ammonium, phosphate,
and hydrogen peroxide were recovered, respectively However, after passage of a nutrient
solution through a column packed with a clay soil high in calcium, magnesium, and chloride,
but low in iron, 75, 100, and 15% of the ammonium, phosphate, and hydrogen peroxide,
respectively, were recovered Both soil and ground water samples should be collected and
analyzed to fully evaluate the treatment effectiveness h<1 Raymond et al 61 reported that the
most difficult problem in optimizing microbial growth in the Ambler reservoir was the
distribution of nutrients because of the heterogeneity of the dolomite formation
d. Additional Case Histories in Which Oxygen was Supplied by Air Sparging
In uru biorestoration has been largely used to treat gasoline spills and with reasonably
good success However, many of the reports on m \itu biorestoration lack sufficient data to
judge fully the overall effectiveness and costs associated with the process
In a high-pcrmeability sand aquifer contaminated with hydrocarbons in Millville, N J ,
the in Attn biorestoration program was successful in removing free product, but residual
hydrocarbons were found at the last sampling period 72 The nutrient solution was transported
through the formation at rates of 2 4 to 4 2 ft/day, aerated water was also injected, but the
dissolved oxygen was rapidly consumed and did not increase in some of the main wells
However, analysis of core material collected from the aquifer indicated that the concentration
of gasoline had not changed substantially during the biostimulation program During the
initial treatment process, inadequate dissolved oxygen levels led to the microbial formation
of phenol, however, phenol levels declined as a result of additional aeration A 10- to 1000-
fold increase in the number of gasoline-utilizing bacteria was noted in the area with the
highest gasoline levels The cleanup met the state requirement for removal of the free gasoline
and was subsequently stopped
At a gasoline spill in La Grange, Ore , 9 months of treatment by in situ biorestoration
and a vapor elimination program succeeded in removing the free product and mitigating the
vapor problems at two restaurants 78 Biodegradation of the gasoline was enhanced by cir-
culating well-aerated ground water amended with inorganic nutrients After 7 months of
treatment, the concentration of gasoline in soil ranged from 100 to 500 ppm and the average
concentration of dissolved organic carbon in the ground water was 20 ppm After an additional
3 months of treatment, the dissolved organic carbon levels in the ground water had decreased
to <5 ppm in the majority of the samples
Fumes released from a pipeline spill of gasoline temporarily closed an elementary school 81
A pumping well was used to maintain the water table below the foundation of the school
and physical recovery was used to remove two thirds of the gasoline An enhanced biodeg-
radation program was initiated by circulating nutrients and oxygen through the formation
for 6 months After the cleanup, hydrocarbons could not be detected and the fumes that had
threatened the school had been eliminated
e. Minimum Hydrocarbon Concentrations Achievable by In Situ Biostimulation
The minimum concentration of hydrocarbon that can be achieved by in situ biorestoration
is unknown and is most likely site specific A natural gradient field test in a sandy Canadian
aquifer required 434 days to reduce 1000 to 2400 ppb of benzene, toluene, and the xylene
isomers below the detection limits (1 to 2 ppb) in the absence of added nutrients and oxygen 11
The distribution of dissolved oxygen in the plume was heterogeneous and probably controlled
the biodegradation of the aromatics
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Volume 18, Issue 1 (1988) 39
Jensen et a! K2 suggested that the indigenous microflora should be able to reduce the
concentration of hydrocarbons below I ^g/f when the initial hydrocarbon concentration is
< 10 mg/f and adequate quantities of nutrients and oxygen are supplied The results of batch
experiments using ground water from hydrocarbon-contaminated aquifers showed that the
native microflora could generally reduce the concentrations of toluene, benzene, the xylenes,
trimethylbenzene, naphthalene, mcthylnaphthalene. biphenyl, ethylnaphthalene, and dime-
thylnaphthalene from a range of 400 to 1100 p,g/f to < 1 |xg/£ within 1 week in the presence
of oxygen and nutrients, however, phenanthrene and toluene persisted at higher concentra-
tions in two of the ground waters after incubation for 6 days
The concentration of trace-level orgamcs in an aquifer may be reduced by providing a
primary substrate that supports microbial growth and allows the organisms to act upon the
trace-level orgamcs as secondary substrates S1 The concentration of the trace organic or
secondary substrate is thought to be below the minimum substrate concentration (Smin)
required to support microbial growth M The Snill, concept was developed to describe limi-
tations related to transport of orgamcs into a biofilni and the subsequent kinetics of reaction
There arc several examples of Slllin A reactor-fed laboratory-grade water containing 0 59
mg/C total organic carbon (TOC) was able to reduce acetate below the Smin value (0 03 mg/
£) for acetate Shimp and Pfaendcr" demonstrated that the addition of fatty acids, carbo-
hydrates, and amino acids enhanced the ability of mixed microbial populations to degrade
substituted phenols These data suggest that the addition of naturally occurring substrates
may enhance the biodegradation potential of some xenobiotics However, the addition of a
primary substrate may not support the removal of some compounds A biofilm supported
by thymine could utilize alanine and acetate, both common metabolites, but not phenol and
galactose 1,4
f Combination of In Situ Biostimulation with Treatment Processes
In suit biorestoration has been successfully combined with other treatment processes to
reduce organic contaminants in aquifers In most cases, the contaminated ground water is
withdrawn, treated by a physical, chemical, or biological above-ground treatment technique,
and then recharged after aeration and addition of nutrients The role of biorestoration in
combination treatment schemes is often difficult to assess Yaniga et al described the
cleanup of a gasoline spill in which an air stripper was used to reduce the contaminants in
the withdrawn ground water and to supply oxygen before the water was recirculated to the
aquifer via an infiltration gallery Before recirculation, ammonium chloride, sodium mon-
ophosphate, sodium diphosphate, iron sulfate, and manganese sulfate were added in slug
batches to the treated water Additional oxygen was supplied by sparging air into the wells
As a result, the dissolved oxygen increased from a range of 0—5 to 5—10 ppm, the
hydrocarbon degrading bacteria increased from I02—101 to 101—I0J cells/mf with just
oxygen addition by air stripping and sparging and then increased to 10h cells/m£ with nutrient
addition and additional oxygen Brown et al 87 identified another gasoline-contaminated
aquifer which was treated using air sparging An estimated 95,000 to 114,000 C of gasoline
entered a 6 l-m thick coarse grain sand and fine gravel aquifer Recovery of free product
accounted for 70,000 £ of the spilled gasoline, however, an estimated 38.000 € was sorbed
to the soil at concentrations of 2000 to 3000 ppm. and 30 to 40 ppm was dissolved in the
ground water The concentration of gasoline was reduced to <50 ppm in the soil and less
than 1 ppm in the ground water by air sparging Only 1 to 2 ppm of dissolved oxygen could
be achieved in the wells by air sparging
Ground water contaminated by a spill of four solvents— methylene chloride, n-butanol,
acetone, and dimethylamline— into a glacial till aquifer was withdrawn and treated by an
activated sludge process After the sludge settled, the treated ground water was recirculated
into the subsurface through injection trenches after being aerated and amended with nu-
trients 27 The recharge water contained organisms acclimated to the solvents in addition to
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40 CRC Critical Reviews in Environmental Control
a nutrient amendment containing nitrogen, phosphate, magnesium, sulfate, carbonate, man-
ganese, and iron Additional oxygen was supplied to the aquifer using a series of injection
wells Removal efficiencies of methylene chloride, n-butanol, and acetone were >97% and
the dimethylamline levels were reduced by >93% in the above-ground treatment The
concentrations of the solvents in the resulting effluent decreased to 0 04 mg/f for n-butanol,
0 92 mg/C for methylene chloride, 0 18 mg/£ for dimethylamline, and 1 12 mg/C for acetone
from initial concentrations of 19 1, 58 5, 2 9, and 38 8 mg/
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Volume 18, Issue 1 (1988) 41
contained pockets of ethylene glycol A passive treatment system which involved adding
lime and diammonium phosphate to the soil surface continued after termination of the active
bioresloration phase By the end of the treatment program, ethylene glycol could not be
detected (detection limit, 50 ppm) in ground water collected from the production wells
A shallow basin comprised of sand and pea gravel was contaminated with isopropanol
and tetrahydrofuran ^ In addition to isopropanol and tetrahydrofuran, acetone was also
detected in the ground water and was believed to be a byproduct of isopropanol degradation
Remedial action consisted of a recovery system, treatment in an above-ground biological
reactor, and recharge of the aquifer with the effluent from the reactor which created a closed-
loop system The effluent, which contained acclimated bacteria, was also amended with
nutrients before reinjection into the subsurface The soils were flushed with the treated
ground water to remove sorbed organics and introduce acclimated organisms into the aquifer
Maximum concentrations of isopropanol (950 ppm) and acetone (190 ppm) were detected
in ground water from a centrally located well as a result of flushing pockets of contamination
from the subsurface The concentration of acetone in the ground water increased initially
until the majority of the isopropanol had been degraded, and then it declined to <0 2 ppm
Extrapolations from the data indicated that 99% of the contaminants would be removed
within 33 days Estimated cost for removal and disposal of 5700 m1 of contaminated soil
was $550,000, whereas the biological treatment program was estimated to cost one fifth as
much
Winegardner and Quince90 documented two case histories of in situ biorestoration that
involved the addition of acclimated bacteria The first case history described the cleanup of
a semi-soluble aliphatic hydrocarbon plasticizer that was spilled during a train derailment
Recovery wells were used to collect the plasticizer from the subsurface Later, surface
recharge and shallow injection were used to flush the plasticizer out of the soil, the peak
concentration of the plasticizer was >2000 ppm This treatment reduced the areal extent of
the contamination after 70 days, in addition to reducing the concentration of the plasticizer
Air stripping and carbon adsorption were used initially, however, these techniques were
replaced by biological treatment using activated sludge The water treated by the activated
sludge contained bacteria that were acclimated to degrade the plasticizer and was injected
into the subsurface to enhance in situ biorestoration The concentration of the plasticizer in
the water treated by the activated sludge was reduced from approximately 1700 to 400 ppm
after clarification, however, the contribution of the activated sludge and in sun microflora
in removal of the plasticizer in the treatment process could not be separated
The second case history involved contamination of a glacial kame deposit of sand, gravel,
silt, and clay with chloroform from a leaking pipeline Ground water was withdrawn and
treated with a mixed-media prefilter, an activated sludge bioreactor and settling vessel, and
a heated air stripper The effluent from the activated sludge bioreactor that contained bacteria
acclimated to degrade chloroform was injected into the subsurface to enhance biorestoration
The effluent from the air stripper was discharged into a process sewer or into the subsurface
A forced flushing/recovery system was used to increase the recovery of the chloroform
Biological treatment followed the physical recovery, however, treatment effectiveness was
not discussed
2 Alternate Oxygen Sources
The supply of dissolved oxygen may limit in situ biorestoration of hydrocarbons, especially
in low-permeability aquifers 72 Depending upon the temperature of the ground water, only
8 to 12 mg/
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CRC Critical Reviews 111 Environmental Control
Alternative sources of oxygen include pure oxygen hydrogen peroxide, and ozone Other
methods of supplying oxygen to the subsurface are soil venting or air flooding''2 and colloidal
dispersions of air in a surfactant matrix Concentrations of 40 to 50 mg/f of dissolved
oxygen can be achieved with pure oxygen, however, pure oxygen is somewhat expensive,
may bubble out of solution before the microflora can use it, and may be an explosion hazard
if handled improperly *"
a Hydrogen Peroxide
Hydrogen peroxide, which decomposes to form one molecule of water and one half
molecule of oxygen, also can be used as a source of oxygen (Equation 1)
H202 -» H20 + 1/2 02 (1)
Hydrogen peroxide has great potential as an alternative source of oxygen but is toxic to
microorganisms at species-specific concentrations Before application of hydrogen peroxide
to the subsurface, the tolerance range of the in utu microorganisms must be determined in
a laboratory experiment Concentrations of 3% are used as a sterilant and levels as low as
200 ppm can be toxic to microorganisms Ground water organisms inoculated into sand
columns could tolerate 0 05% hydrogen peroxide, but higher levels were toxic 44 In a study
designed to investigate the effect of increasing concentrations of hydrogen peroxide on
gasoline biodegradation, the culture acclimated to hydrogen peroxide levels that were grad-
ually increased from 0 05 to 0 2%, however, removal of gasoline was not greatly increased
in comparison to the control without hydrogen peroxide Microbial counts were higher in
columns in which hydrogen peroxide was incrementally increased than those which received
only 0 05% hydrogen peroxide These data suggest an oxygen limitation at lower concen-
trations of hydrogen peroxide Large populations of microorganisms survived high hydrogen
peroxide concentrations better than small populations ^ In a study in which the oxygen
concentration was varied from 8 to 200 ppm (using air, 60% nitrogen/40% oxygen, pure
oxygen, or a hydrogen peroxide solution), microbial growth and gasoline degradation were
greatest in columns amended with hydrogen peroxide, which provided the highest concen-
tration of available oxygen Vl At concentrations >100 ppm, hydrogen peroxide may degas
to form air bubbles which may block some of the pores in the aquifer Decomposition of
hydrogen peroxide may also be catalyzed by iron and fluctuations in pH 1,4 In addition to
comprising part of the nutrient formulation, certain forms of phosphate, such as potassium
monophosphate, can be used to stabilize hydrogen peroxide solutions To reduce phosphate
adsorption by the soil, a combination of simple and complex polyphosphate salts can be
used 96 Results from a field test in which hydrogen peroxide was used to increase the dissolved
oxygen content of the ground water indicated an increase from 1 to 15 ppm within 70 hr at
a monitoring well located 7 6m downgrade of the injection well
Raymond et al 97 received a patent on a process which involves stimulating biodegradation
of organic contaminants in the subsurface with hydrogen peroxide The patent described
several formulations of nutrient and hydrogen peroxide solutions and processes that can
stabilize the decomposition of hydrogen peroxide, control movement of the solution through
the aquifer, remove metal ions from the subsurface which catalyze hydrogen peroxide
decomposition, and disrupt biofilms that form at the point of injection Hydrogen peroxide
decomposition can be controlled by the addition of peroxidase, oxidase, or a transition metal
(iron, copper, manganese, chromium, or other material, including the chelated forms of
these metals) In addition, condensed phosphates can be perfused into the aquifer to deactivate
or remove substances that catalyze hydrogen peroxide decomposition
Movement of the hydrogen peroxide solution through the formation can be controlled by
hydratable polymeric materials, interface modifiers, and densifiers 97 The hydratable poly-
meric materials, such as polysaccharides, polyacrylamides, and polyacrylamide copolymers,
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Volume 18, Issue I (1988) 43
increase the viscosity of the solution An increase in viscosity reduces the rate of diffusion
and slows the movement of the solution The addition of surfactants will decrease the
interfacial tension, prevent clays from swelling, disperse materials throughout the zone of
contamination, and decrease the metal-catalyzed decomposition of hydrogen peroxide For
example, the zone of treatment can be extended into the capillary zone by adding soluble
orthophosphoric salts and condensed phosphoric acids to increase the capillary rise of the
aqueous solution Salts, such as sodium chloride, calcium chloride, and sodium bromide,
can be used to change the density of the nutrient solution Biofouling can be controlled by
adding high concentrations (0 5 to 3%) of hydrogen peroxide the effectiveness of hydrogen
peroxide in controlling biofouling may be enhanced by the addition of dilute acid
Hydrogen peroxide has been used to enhance the oxygen supply in the subsurface in many
remedial programs In most cases, the additional oxygen was required to degrade hydro-
carbons in aquifers contaminated from gasoline spills One case study involved the contam-
ination of a relatively impermeable soil (ground water movement, 0 6 to 0 9 m/year) with
gasoline 6<' About 50 to 60% of the free product was recovered, however, concentrations of
hydrocarbon in the range of 3700 to 7200 ppm remained sorbed to the soil A feasibility
study was conducted to identify an m sun microbial population capable of degrading the
hydrocarbons when supplied with nutrients and oxygen Hydrogen peroxide was used as the
source of oxygen After 2 months of operation, free product recovery reached a maximum
of 95 to 114 f/day, numbers of hydrocarbon-degrading bacteria increased one to three oiders
of magnitude, and the concentration of sorbed product declined to a range of 2300 to 2900
ppm
In another in situ biorestoration program designed to clean up gasoline from a leaking
underground storage tank, oxygen was initially supplied by air stripping and sparging and
then by hydrogen peroxide A layer of heavy silt loam which was underlaid by a layer of
fractured shale and siltstone was contaminated by the spill
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44 CRC Critical Reviews in Environmental Control
of hydrocarbons in the ground water had been reduced from 15 to 2 5 ppm Continuation
of this treatment removed the dissolved hydrocarbons in ground water from 8 of the 12
wells Between 200 and 1200 ppb remained in the other four wells96
Hydrogen peroxide-assisted biodegradation followed by granular activated carbon (GAC)
polishing was used to treat a spill of waste solvents and fuel 79 100 The source of the con-
tamination was an excavated area around several leaking tanks at a laboratory facility The
fill material and soil surrounding the storage tanks were contaminated with a mixture of
hydrocarbons composed of xylenes, benzene, toluene, ethylbenzene, and C,-Cl2 alkanes that
ranged in concentration from 1000 to 3000 ppm About 2600 £ of free product was recovered
using a sump pump, however, an estimated 1100 to 3400 £ of the hydrocarbon remained
The subsurface consisted of sand and sandy clay with fairly rapid ground water flows The
total number of bacteria in the well water ranged from 300,000 to 420,000 cells/mf. hy-
drocarbon degraders ranged from 5400 to 6100 cells/6" Carbon adsorption and enhanced
bioreclamation were considered for remedial action Carbon adsorption was estimated to
require 10 to 20 years and cost $470,000 to $850,000, whereas enhanced bioreclamation
was estimated to require 4 to 8 months and cost $180,000 to $270,000 96 Enhanced bio-
reclamation was chosen and the process design consisted of four injection wells and a
pumping well with a flow rate of 57 to 95 f/min 100 A nutrient solution consisting of
ammonium chloride and sodium phosphates was injected by batch addition Hydrogen per-
oxide was injected continuously following a short period during which only nutrients were
added, addition of nutrients without oxygen had little effect because of the initial low
dissolved oxygen content (0 8 ppm) After 72 days, 440 kg of nutrients and 945 i of the
hydrogen peroxide solution had been added The number of hydrocarbon-degrading micro-
organisms increased 130-fold and the concentration of dissolved oxygen in the ground water
increased to 10 5 ppm after the biorestoration program was initiated The concentration of
hydrocarbon decreased from 22,700 to 581 ppb in 44 days and to nondetectable levels in
one monitoring well after 72 days Elevated concentrations of contaminants detected in
another monitoring well were thought to be a result of a leaking line An estimated 570 to
1500 £ of the mixed fuels and solvents had been degraded 9h However, the formation became
partially clogged after 72 days of operation Clogging of the formation may have resulted
from the movement of silt and degradation of the cement that lined the storage tank vault 79
An activated carbon system was then used as a polishing step to reduce the hydrocarbon
concentration below 10 ppb in the tank vault and soil
The cost for a 6- to 18-month bioreclamation program at the laboratory facility was
estimated between $180,000 and $270,000 96 Estimates were $50,000 to $75,000 to start
the bioreclamation process and $130,000 to $220,000 for services and nutrients The cost
for excavation was estimated between $600,000 and $1 5 million, and the program was
projected to take less than 6 months, however, facilities on the site would restrict excavation
Withdrawal and treatment by carbon absorption was estimated to cost $470,000 to $850,000
and require 10 to 20 years because of limited extractability of the contaminants
A less successful demonstration of enhanced bioreclamation using hydrogen peroxide was
reported by Brown and Norris 101 A formation consisting of silt, sand, and gravel deposits
was contaminated by a spill of 303,000 £ of unleaded gasoline Two subsurface zones were
identified in the test area (1) a fine quartz sand with some limestone and dolomite grains
and ferromagnesium minerals with traces of limonite and pebbles of dolomite, limestone,
and granite and (2) another zone of fine quartz sand with large amounts of fines and silt
which impeded ground water flow The hydraulic conductivity ranged between 8 8 to 15 2
x I0~4 cm/sec A free-product recovery program was implemented, however, between 300
and 10,000 ppm of hydrocarbon remained in the soil and 50 to 60 ppm remained in the
ground water after 5 years The concentration of total hydrocarbons in the cores averaged
5477 ppm with a range of 4823 to 6331 ppm for several groups The highest concentrations
were detected at the water table at depths of 7 3 to 7 9 m The treatment zone was estimated
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Volume 18, Issue 1 (1988) 45
to contain 6100 ± 2500 (SD) kg of gasoline Ambient nutrient levels in the ground water
were <1 ppin and the dissolved oxygen content was <0 4 ppm Total counts and counts
of hydrocarbon-degrading bacteria grown on nutrient agar were I 2 X 10* and 2 X 102
cells/g, respectively Biostimulation was tested in a section of the plume surrounded by two
triangular patterns of monitoring wells which surrounded an inner infiltration gallery Nu-
trients were batch fed to the inner gallery and then followed by addition of hydrogen peroxide
solutions which were gradually increased from 0 to 500 ppm Phosphorus levels reached
100 to 250 ppm in the inner gallery and ranged from 1 to 10 ppm outside the gallery The
concentration of nitrogen ranged from 100 to 250 ppm in the inner gallery and from 10 to
50 ppm outside of the gallery Total bacterial counts reached I06 cells/mf in the inner gallery
and 6 X 101 cells/mf outside of the gallery, the number of gasoline-utilizers also increased
The concentration of hydrocarbon in the soil was measured after 0. 32, 91, and 164 days
during the lest and at depths of 7. 7 6, 8 2, and 8 8m below the land surface "" During
the test, the concentration of hydrocarbon was reduced from 5490 to 1874 ppm (65%)
Removal of hydrocarbons was highest (from 5643 to 1743 ppm) in the inner gallery near
the injection area, whereas a low-permeability zone was less effectively treated because of
reduced circulation of nutrients In addition, the concentration of total hydrocarbons was
reduced by 63% outside and between the galleries The hydrocarbon concentration at the
water table was reduced from 6087 to 4058 ppm and from 2946 to 1008 ppm immediately
below the water table The data indicate that substantial quantities of hydrocarbons remained
adsorbed onto the soil after m situ biostimulation, although more improvement may have
occurred with continued treatment
A field demonstration of in suit biorestoralion using hydrogen peroxide in a very gravelly
clay loam was adversely affected by the low permeability (3 9 X 10~"i to 3 3 x 10_1 cm/
sec) of the soil 102 The heterogeneity of the soil and distribution of the contaminants made
it difficult to inject nutrients and pump water The contamination resulted from a disposal
pit containing chromium sludges, electroplating wastes, chlorinated solvents, cresols. chlo-
robenzenes, and other compounds 101 ,-dichloro-
benzene Heavy metals present at concentrations > 10 mgl€ included antimony, chromium,
copper, lead, nickel, and zinc, and the concentrations of silver, cadium, and mercury were
high in some locations 101104 The formation consisted of gravel lenses and layers of fine-
grained soils with low hydraulic conductivities The water table was perched, only 1 2 to
2 4m thick, and exhibited seasonal fluctuations Direct microbial counts in soil ranged from
7 6 to 170 x I06 cells/g (wet weight), viable counts ranged from <100 to 7 x 10" cells/
g on both rich and poor media Laboratory studies conducted under aerobic conditions
indicated that the chlorobenzenes, hydrocarbons, and dramatics could be biodegraded The
total resolved hydrocarbons, i e , the organic contaminants separated by gas chromatography
and thought to represent n-alkanes, were reduced more rapidly in the aerated microcosms
than in those supplied with hydrogen peroxide, which may indicate hydrogen peroxide
toxicity Unresolved hydrocarbons representing branched alkanes were removed under aero-
bic but not anaerobic conditions The results from these microcosm studies suggested that
biological degradation was feasible, but the heterogeneity of the subsurface and the contam-
inants present seriously limited application of the biorestoration process The presence of
heavy metals was not expected to prevent biodegradation, but the treatment process could
induce metal mobilization
The treatment system design for in situ biorestoration consisted of nine extraction and
four injection wells that were connected to a central surge tank and a distribution box l0S
One upgradient and two downgradient wells were installed to monitor the influence of the
treatment in untargeted areas Nutrients were added 2 weeks before the hydrogen peroxide
After 2 months of treatment, the effectiveness of the treatment could not be determined
because of a change in analytical methods, however, a number of problems were noted with
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46 CRC Critical Reviews in Environmental Control
the field demonstration Hydrocarbon levels increased in the ground water for unknown
reasons In addition, the nutrient solution precipitated initially when added on a continuous
basis, the precipitation problem was reduced by switching to batch amendments The tem-
perature in the well water increased and fluctuated because the impermeable soil could not
sustain flow adequate to prevent the pumps from overheating Microbial numbers in the
infiltration zone remained low, perhaps due to hydrogen peroxide toxicity Antimony and
lead may have been mobilized within the aquifer and the nutrients had not reached most of
the wells at the time of this report 106
Continued treatment for 6 months resulted in decreases in the levels of chlorobenzene
and total hydrocarbons 107 The breakthrough of the nutrient solution was rapid in the highly
permeable zones, but poor in the less permeable strata Elevated concentrations of carbon
dioxide were detected in the treatment zone which suggested an increase in microbial activity
However, the concentration of many of the organic compounds did not decline and biores-
toration of the site was not successful
b. Ozone
In addition to hydrogen peroxide, ozone (O,) can be used as an alternate source of oxygen
Ozone was used in an in situ biorestoration program to remediate a hydrocarbon spill in a
railroad yard in Karlsruhe, F R G l0S The presence of organic contaminants in the drinking
water wells for the city of Karlsruhe was traced to the hydrocarbon spill in the train yard
The concentration of organics, iron, and manganese in the ground water increased but the
dissolved oxygen content decreased The water was withdrawn, treated with I g of ozone
per gram of dissolved organic carbon for 4 mm and then reinjected into the formation through
five infiltration wells at a rate of 80 to 120 mVhr Supplemental nutrients were not added
The purified water formed a barrier to prevent further contamination of the withdrawal well
The ozone treatment increased the dissolved oxygen levels in the ground water which
stimulated the microbial population and enhanced the degradation of the contaminants in
the aquifer The maximum efficiency of introducing dissolved oxygen into the ground water
was 80% of the initial concentration of ozone Oxygen consumption by the indigenous
microbes reached approximately 40 kg/day The dissolved organic carbon decreased from
a range of 2 5 to 5 5 g/m1 to a steady-state value of slightly more than I g/m\ few mineral
oil hydrocarbons remained The levels of iron and manganese were also reduced Although
the total number of bacterial cells increased, microbial counts on media which selected for
disease-causing organisms did not increase The removal of the hydrocarbons probably
resulted from both in situ microbial activity and chemical oxidation by the ozone Hydro-
carbons could not be detected in the biostimulated section of the aquifer in water collected
1 5 years after treatment
c. Soil Venting
Soil venting or air flooding can be used to supply oxygen for m situ biorestoration Organic
vapors from the unsaturated zone are removed by increasing the flow of soil gases using
vapor recovery wells and air inlet wells 109 The volatile organic contaminants partition into
the soil gas and are transported to the vapor recovery wells The increase in soil gas flow
in the unsaturated zone makes more oxygen available to reaerate the ground water
Field tests have demonstrated that soil venting is effective in removing hydrocarbons from
the unsaturated zone Following a gasoline spill in a porous and moderately permeable soil,
the concentration of vapors was reduced by 90% at a distance of 6 1 m and by 70% at 12 2
m from the vacuum source "w After reequilibration for 2 weeks, the average concentration
of the vapors was reduced to 38% of the pretest levels Soil venting has also been used to
remove from 10 to 15 kg of tnchloroethylene per day in another field trial "" A combination
of vacuum extraction and withdrawal and treatment was able to reduce the levels of carbon
tetrachloride by more than 99% in monitoring wells at a site contaminated by a storage tank
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Volume 18, Issue I (1988) 47
spill 111 After operation for 30 months, the concentration of carbon tetrachloride was reduced
by 70% in the vadose zone Levels of methane, acetone, and methylene chloride following
another tank spill were reduced below explosive limits within I week, but 6 weeks were
required to reduce the methylene chloride concentrations to drinking water standards 1,2 Soil
venting is restricted to volatile hydrocarbons in the unsaturated zone and, in some cases,
the capillary fringe In
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CRC Critical Reviews in Environmental Control
Table 2
ADVANTAGES AND DISADVANTAGES OF BIORESTORATION113114
Advaniages
Can be used to treat hydrocarbons and certain organic compounds, especially water-soluble pollutants and low
levels of other compounds that would be difficult to remove by other methods
Environmentally sound because it does not usually generate waste products and typically results in complete
degradation of the contaminants
Utilizes the indigenous microflora and does not introduce potentially harmful organisms
Fast, safe, and generally economical
Treatment moves with the ground water
Good for short-term treatment of organic contaminated ground water
Disadvantages
Can be inhibited by heavy metals and some organics
Bacteria can plug the soil and reduce circulation
Introduction of nutrients could adversely affect nearby surface waters
Residues may cause taste and odor problems
Labor and maintenance requirements may be high, especially for long-term treatment
Long-term effects are unknown
May not work for aquifers with low permeabilities that do not permit adequate circulation of nutrients
waters 2 The time required to treat subsurface pollution using in situ biorestoration can often
be faster than withdrawal and treatment procedures A gasoline spill in Ambler, Penn , was
remediated in 18 months using in situ biorestoration, whereas pump and treat techniques
were estimated to require 100 years to reduce the concentrations of gasoline to potable
levels h2 In situ biorestoration can also cost less than other remedial options Flathman and
Githens74 estimated that the cost of in situ biorestoration would be one fifth of that for
excavation and disposal of soil contaminated with isopropanol and tetrahydrofuran and in
addition would provide an ultimate disposal solution The areal zone of treatment using
biorestoration can be larger than other remedial technologies because the treatment moves
with the plume and can reach areas which would otherwise be inaccessible
There are also disadvantages to in situ biorestoration programs Many organic compounds
in the subsurface are resistant to degradation In situ biorestoration requires an acclimated
population, however, adapted populations may not develop for recent spills or recalcitrant
compounds Heavy metals and toxic concentrations of organics may inhibit microbial activity
and preclude the use of the indigenous microflora for in situ biorestoration at some sites
One option in this instance would be to remove the inhibitory substances and then seed the
subsurface with appropriately adapted microorganisms, however, the benefits to adding
microorganisms to the subsurface are still undemonstrated The formation and injection wells
may clog from profuse microbial growth which results from the addition of oxygen and
nutrients In one biostimulation project, microbial growth produced foaming in the well
casings 62 In addition, the hydrodynamics of the restoration program must be properly
managed The nutrients added must be contained within the treatment zone because the
transport of inorganics into untargeted areas can result in eutrophication High concentrations
of nitrate can render ground water unpotable Metabolites of partial degradation of organic
compounds may impart objectionable tastes and odors For example, the incomplete deg-
radation of gasoline under low dissolved oxygen conditions resulted in phenol production,
phenol was then degraded when more aerobic conditions were achieved 72 Biostimulation
projects require continuous monitoring and maintenance for successful treatment, whether
these requirements are greater than those for other remedial actions is debatable The process
results in increased microbial biomass that can exert an oxygen demand that can drive the
system anaerobic and result in the production of hydrogen sulfide or other objectionable
byproducts The long-term effects of biorestoration are unknown In situ biorestoration is
difficult to implement in low-permeability aquifers in which perfusion of nutrients and oxygen
is slow or negligible, however, many in situ physical and chemical remediation processes
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Volume 18, Issue 1 (1988) 49
Table 3
CONTAMINANTS TREATED BY IN SITU BIOSTIMULATION
Contaminants Treatment description Ref
High octane gasoline
Air sparging with nitrogen and phos-
61—64
phorus addition
Gasoline
Air sparging with complete nnx of
72
inorganics
Gasoline
Air sparging with addition of com-
78
plete inorganic nutrient solution
Gasoline
Air sparging and addition of nutrients
81
Gasoline
Dissolved oxygen supplied by an air
86 99
stripper and sparging, nutrients also
added
Gasoline
Dissolved oxygen supplied by an air
87
stripper
Gasoline
Hydrogen peroxide plus nutrients
69
Gasoline
Initial treatment utilised air stripping.
87, 98
99
hydrogen peroxide used later wiih
the nutrient formulation
Unleaded gasoline
Hydrogen peroxide supplied (he
101
oxygen
Mineral oil hydrocarbons
Withdrawn water treated with ozone
108
and reinfiltrated
Gasoline
Soil venting used to supply oxygen to
71
unsaturated zone
Waste solvents and alkanes
Nutrients plus hydrogen peroxide
79, 87
96 100
Melhyl chloride, n-bu-
Withdrawal and treatment by an acti-
27, 80
tanol, dimethyl aniline.
vated sludge process and recharge of
acetone
aerated nutrient-laden water
Methylene chlonde
Withdrawal and treatment with air
75, 88
stripping followed later by treatment
in an activated sludge unit and
recharge
Ethylene glycol
Treatment following withdrawal with
75. 89
ethylene-degrading bacteria and nu-
trients and then recharge
Isopropanol and
Treatment in an above-ground reactor
74
tetrahydrofuran
with addition of acclimated microbes
to the aquifer along with nutrients
Aliphatic hydrocarbon
Activated sludge and recharge of ac-
90
plasticizer
climated bacteria and nutrients
Chloroform
Activated sludge bioreactor with the
90
bacteria innoculated into the
subsurface
are subject to the same restrictions The success of in situ treatment schemes in low-
permeability aquifers depends on transporting the nutrients to the microflora or the active
agent to the contaminants The process has been used in a variety of hydrogeological
formations (Table 4)
4 Related and Innovative Processes
There are a number of innovative, generally unproven. processes that potentially can be
applied to in situ biorestoration These processes include land treatment, techniques that
decrease the surface tension to enhance the mobility and improve the biodegradability of
the contaminants, application of enzymes, and treatment beds
a. Land Treatment
Land treatment is a process in which the indigenous microflora in surface soils degrade
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Table 4
TYPES OF AQUIFERS WHERE IN SITU BIOSTIMULATION
HAS BEEN UTILIZED
Aquifer description
Flow characteristics
Ref.
High permeability dolomite
Medium to coarse sand
Alluvial fan deposit of sand,
gravel, and cobbles with
some clay and silt
Poorly sorted mixture of
boulders, pebbles, cobbles,
sand, silt, and clay
Perched water table in un-
ratified unsorted layer of
clay, silts sands gravels,
and cobbles above a clay
layer
Tank vault filled with pea
gravel surrounded by sand
and sandy clay strata
Glacial outwash composed
of silt sand, and gravel
Coarse sands and gravel
Shale and sillstone
Coarse sand with greater
than 5% gravel
Glacial till composed of
sand, gravel, and boulders
in a silty clay matrix con-
nected to a fractured
sandstone
Shallow basin containing
sand and pea gravel
Pumping rate of 265—378 f/min
Pumping rate of 65—151 f/min
Flow of 2 4 m/day
Hydraulic conductivity of 9 4 x
I0~4—I 7 x 10"'cm/sec
Pumping rate of 38—57 f/min
Flow rate in excess 100 m/yr
pumping rate of 151 C/min
Hydraulic conductivity of 8 8 x
I0~4—I 5 x 10"'cm/sec
Hydraulic conductivity of 2 1 cm/
sec
Pumping rate of 68 £/min
Gradient of 0 015—0 02 m/m. flow
of 0 61—0 91 m/yr
Flow of 27 to 38 f/min
61—64
97
78
27, 80
75, 88
79 87. 100
101
108
87 98 99
69
99
74
Table 5
FACTORS THAT CONTROL BIODEGRADATION IN LAND TREATMENT"7
Chemical structure of the waste
Presence of appropriate numbers of microorganisms capable of degrading the wastes
Concentration of the wastes
Supply of oxygen
Optimal water content of between 25 and 85% of the water-holding capacity
Optimal temperatures between 20 and 30°C
Optimal pH levels between 6 and 8
Availability of inorganic nutrients principally nitrogen and phosphorus
the organic material contained in the soil Loehr and Malina"5 suggested that land treatment
is useful for disposal of organic wastes from municipal sludge, petroleum, wood preserving,
leather tanning, coal gasification/liquefaction, food processing, and pulp and paper produc-
tion Land treatment involves the addition of the organic waste to the soil, mixing to aerate
and incorporate the organics into the soil, and, if needed, adding fertilizer to stimulate
microbial activity The process must be carefully managed to prevent overloading the as-
similative capacity of the soil and to prevent migration of the inorganic nutrients, organics,
and heavy metals 116 Major factors that control biodegradation in land treatment are listed
in Table 5 Land treatment may be advantageous in comparison to other remedial techniques
because it requires minimal operation and maintenance and is a proven technology for some
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Volume 18, Issue 1 (1988) 51
wastes 11,1 However, the process may result in incomplete destruction of the organic wastes,
the soil may be difficult to aerate effectively, the wastes must be contained within the
treatment zone, air pollution may result, and large areas of land are required Land treatment
is usually limited to the upper 1 5 m of soil,"w which restricts its use in aquifer remediation
However, land treatment may be used to treat excavated soil or wastes that are concentrated
at the water table of shallow aquifers In a survey of treatment options for ground water
contaminated with gasoline, the Law Engineering Testing Company12" recommended that
land treatment be considered when a suitable site is available In comparison to other
treatment technologies, land treatment was highly rated on the basis of effectiveness, capital
costs, reliability, and operability 121
Land treatment was used to treat a spill of 1 9 million € of kerosene 122 About 200 m'
of soil was excavated and the contaminated ground water was withdrawn and treated The
contaminated soil was treated by adding lime and fertilizer (nitrogen, phosphorus, and
potassium in the ratio of 10 1 0 85) and frequently tilling the soil to a depth of 46 cm The
fastest rate of kerosene degradation occurred during the warmer months of July and August
The concentration of kerosene was reduced from 0 87% to innocuous levels in the upper
30 cm of soil during a 21-month period However, kerosene persisted at a depth of 30 to
45 cm, perhaps a result of reduced aeration Within the first 7 months, most of the n-alkanes
and unresolved hydrocarbons were degraded A test for phytotoxicity after land treatment
indicated that the phytotoxicity of the kerosene had been reduced but not completely elim-
inated, crop yields were 20% below those in a control area 23 months after the spill
b. Techniques That Reduce the Interfacial Tension
Insoluble organic compounds that are sorbed to soils can be mobilized and made more
available for microbial attack by decreasing the interfacial tension between the compounds
and water The interfacial tension can be decreased with dispersants, surfactants, extractants,
and emulsifiers Dispersants have been used in remediation programs to control marine oil
spills with some success 121 Addition of dispersants can increase the rate of reaction, but
may not increase the extent of hydrocarbon degradation However, not all dispersants enhance
degradation of hydrocarbons and some may be toxic to microorganisms Mulkins-PhiIlips
and Stewart124 reported that only one of four dispersants stimulated biodegradation of crude
oil by marine bacteria, however, all four dispersants caused shifts in the microbial population
The addition of surfactants to mobilize organics sorbed to soils has been tested in laboratory
studies A combination of nonionic and ionic surfactants was most effective in removing
gasoline from sand columns by simple displacement and by draining the gasoline from the
capillary zone l2S Some of the surfactants identified in this study were biodegradable, whereas
others exhibited varying degrees of toxicity Ellis et al l2h demonstrated that surfactants could
remove up to 95% of the crude oil and polychlonnated biphenyls trapped in sand columns,
whereas aqueous washes failed to remove appreciable quantities of these contaminants
Surfactants may be used in combination with biorestoration to remediate aquifer contami-
nation problems A surfactant wash can mobilize the residual hydrocarbon in the unsaturated
zone and render trapped hydrocarbon in the saturated zones more available for biodegra-
dation 92 A surfactant which is biodegradable and nontoxic is required The application of
surfactants to subsurface contaminants may present additional environmental problems by
spreading contaminants to sections of the aquifer previously uncontaminated
Emulsifers can be used to increase the surface area and render the oil more degradable 127
Emulsifiers can be either chemical additives or biological agents Robichaux and Myrickl2x
reported that one chemical emulsifier increased the microbial decomposition of oil 18-fold,
however, other emulsifiers were less successful and many may have been toxic Brodenck
and Cooney129 reported that emulsifiers are produced by a variety of organisms in freshwater
environments, especially those associated with sediments Laboratory studies conducted by
Vanlooke et al 110 showed that 10 to 20% of the oil adsorbed to soil was removed after the
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52 CRC Critical Reviews in Environmental Control
addition of a nutrient solution containing ammonium nitrate and peptone, microbial metab-
olites were thought to be responsible for the enhanced desorption The ground water mi-
croflora in an aquifer contaminated with aviation fuel was reported to emulsify hydrocarbon
when supplied with dissolved oxygen, nitrogen, and phosphorus 111 Micelles and nucroe-
mulsions of the hydrocarbon are likely to be formed by bioemulsifiers which may facilitate
transport of the hydrocarbon into the cell 112 Biosurfactant-producing bacteria may be used
to remediate contaminated aquifers but their use will be controlled by (I) the physical and
chemical characteristics of the contaminant, (2) the geophysical and geochemical charac-
teristics of the formation such as pore size distribution and permeability, water quality, and
oxygen concentration, and (3) competition with indigenous microflora The hydrocarbon
that is mobilized as a result of bioemulsification may be withdrawn from the aquifer and
treated by above-ground techniques 111 The microbial conversion of the hydrocarbons to
more polar compounds such as alcohols, ketones, phenols, or organic acids will also mobilize
the contaminants
Another application of bioemulsifiers is enhanced oil recovery Clark et al 114 found several
aerobic microbial species that could be used to bioemulsify oil in situ Zajic and Akit115
found two bacterial strains that produced high concentrations of surfactants, one could remove
bitumen from tar sands when grown on hexadecane A bacterial culture supported on molasses
was able to release 19 5 to 48 7% and utilize 2 to 51% of the oil in a formation within 10
days 1,6 Field trials were successful in two of seven reservoirs, increasing yields by 10 to
200%
Extraction techniques such as steam flooding, alcohol flooding, and thermal flooding also
may be used to mobilize organic contaminants in the subsurface, however, they have not
been demonstrated in the field 117 Horizontal or vertical water sweeps can be used in perme-
able aquifers to reduce the quantity of hydrocarbons before treatment by other methods such
as in situ biorestoration
c. Enzymes as an Innovative Treatment Technique
Another innovative in situ process is the addition of enzymes to degrade specific organic
compounds In one investigation, a parathion hydrolase enyzme isolated from a mixed culture
of Pseudomonas was added to wet and dry plots of soil amended with the organic phosphorus
insecticide diazinon 118 In both wet and dry plots, removal was initially faster in the enzyme-
amended soil than in the control, however, diazinon levels in the test and control plots were
similar after 408 hr The effectiveness of an enzyme depends upon us stability in the
environment and contact with the substrate Adequate mixing to insure contact may be
difficult to achieve in an aquifer In addition, enzymes may be better substrates for microbial
metabolism than many organic pollutants The stability of an enzyme in the environment
may be adversely affected by changes in pH and solute concentrations
d. Treatment Beds
Treatment beds are another innovative process currently under development The process
consists of a trench which intercepts contaminated ground water and either a biological or
chemical treatment bed which removes the contaminants Chemical treatment beds for or-
ganic compounds include activated carbon or synthetic resins 119 Biological treatment can
be accomplished using processes similar to trickling filters in which microorganisms colo-
nizing a surface are supplied with oxygen and nutrients, if necessary, and degrade the
contaminants which enter the treatment bed Permeable treatment beds may plug or exhibit
channeling, which reduces their effectiveness Similar results could be obtained without the
treatment bed by implementing in situ biorestoration in a narrow zone that intercepts and
contains the plume
5 Potential for Anaerobic Processes
a. Anaerobic Degradation Pathways in the Subsurface
Anaerobic processes are important in the subsurface environment because oxygen may
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Volume 18, Issue 1 (1988) 53
be depleted in contaminated aquifers as a result of aerobic microbial activity However, low
levels of oxygen will support some microbial activity Once the dissolved oxygen content
in ground water declines as a result of microbial activity, replacement depends on recharge,
reaeration from soil gases, and mixture with oxygenated waters surrounding the organic
plume 140 141
Degradation of a variety of compounds under anaerobic conditions has been demonstrated
to occur in aquifers and laboratory experiments using subsurface materials However, an-
aerobiosis may retard the degradation of many compounds 47 The sequence of microbial
processes that occur as environmental conditions change from aerobic to anaerobic in the
subsurface usually follows the pattern of aerobic respiration, denitrification, manganese and
iron reduction, sulfate reduction, and finally methane formation 142 141 Net energy production
decreases as the redox potential decreases l4,i Bouwer and McCarty42 144 demonstrated dif-
ferences in the degradation of organic compounds under different redox potentials, chlo-
roform and 1,1,1-trichloroethane were degraded by methanogenic, but not denitrifying,
bacteria Ehrlich et al 21 l4,i reported the degradation of phenolics. but not polynuclcar ar-
omatics such as naphthalene, under methanogenic conditions Recently Kuhn et al 29 doc-
umented removal of tetrachloroethylene, the xylene isomers, and dichlorobenzene isomers
under denitrifying conditions Wilson and Rees18 showed that degradation of benzene, ethyl-
benzene, toluene, and o-xylene occurred in methanogenic aquifer material from a landfill,
although the process was slow compared with aerobic pathways The concentration of toluene
had been reduced by 87% after 6 weeks, however, more than 20% of the benzene, ethyl-
benzene, and o-xylene added to the microcosms persisted beyond 40 weeks In the same
study, tnchloroethylene and styrene degraded under anaerobic conditions, whereas chloro-
benzene persisted Suflita and Gibson41 reported that 13 of 19 halogenated isomers of
benzoate. phenol, and phenoxyacetate persisted at concentrations >90% of that initially
added to subsurface materials collected from a sulfate-reducing zone, however, only 3.4-
dichlorobenzene remained at concentrations >5% of that originally added to methanogenic
samples collected downgradient of the sulfate-reducing zone Maximal numbers of sulfate-
reducing and methanogenic bacteria are found at redox potentials of - 100 to - 150 and
-250 to —350 mV, respectively 146 Halogenated aliphatics such as tnchloroethylene, tet-
rachloroethylene, carbon tetrachloride, and 1,1,1-tnchloroethane can be mineralized or de-
halogenated under reducing conditions147 to potentially more toxic compounds such as vinyl
chloride 148149 Tiedje et al 150 reported the anaerobic degradation of many other compounds
by organisms from a variety of environments
b. Anaerobic Processes in In Situ Biostimulation
Anaerobic processes may be of potential use in in situ biorestoration processes The redox
potential could be selectively adjusted to favor the degradation of a particular contaminant
In addition to adjusting the redox potential, the pH of the ground water could be adjusted
to the neutral or alkaline conditions required for sulfate reduction, methanogenesis, and
usually denitrification Anaerobic degradation of organic compounds would probably require
less inorganic nutrient supplementation because less energy, and therefore biomass, is pro-
duced 113 Batterman151 added nitrate to ground water contaminated with hydrocarbons in an
attempt to promote denitrification The contaminated aquifer consisted of an 8- to 10-m
thick layer of sand which contained some silt and clay beds and a ground water flow of 4
m/day The water was withdrawn from a deeper uncontaminated aquifer, aerated, passed
through a sand filter, and amended with nitrate at 300 mg/£ before being recharged to the
shallow aquifer Phosphate was not added because it was not limiting The authors suggested
that anaerobic degradation accounted for the removal of 7 5 tons of hydrocarbon within a
period of 120 days Removal of 1 mg of the hydrocarbon required 3 3 mg of nitrate 1,2 The
concentration of aliphatics declined slowly from I 5 to about 0 7 mg/f. whereas the con-
centration of total aromatics declined from 5 5 mg/
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54 CRC Critical Reviews in Environmental Control
I year The rate of decline in the concentration of xylene was much slower than that of
benzene and toluene Water was injected during the test, which resulted in a rise in the level
of the hydrocarbons as well as the water table into the unsaturated zone There was an
overall 40% reduction in the concentration of hydrocarbon as a result of the treatment process
Insufficient information was provided to determine if anaerobic degradation was responsible
for the removal of the contaminants or if the removal was due to the oxygen introduced
when the injection water was aerated before it was recharged into the shallow aquifer
Degradation of low concentrations of organic compounds under methanogenic conditions,
with acetate added at higher concentrations as a primary substrate, has been demonstrated l4;!
McCarty1-" proposed a scheme to treat contaminated ground water anaerobically using the
primary substrate concept The system consists of an above-ground reactor to which substrate
and nutrients are added, a well casing bioreactor which operates anaerobically like a trickling
filter, and the aquifer The above-ground reactor is used to develop an acclimated population
The effluent from the above-ground reactor is injected into the well casing bioreactor to
introduce acclimated microbes into the aquifer or enhance adaptation of the indigenous
population to the contaminants Once the acclimated population has developed, use of the
above-ground reactor can be discontinued
A method that utilizes sequential aerobic and anaerobic conditions to degrade hazardous
wastes has been studied in soils and may be applicable to subsurface cleanup An insecticide,
methoxychlor, was slightly degraded in soil under either aerobic or anaerobic conditions
after 3 months of incubation When the samples were converted from an anaerobic to an
aerobic status, mineralization of the methoxychlor increased 10 to 70 times of that observed
in soils maintained aerobically throughout the incubation period The enhancement in
methoxychlor degradation in soils exposed to anaerobic and then aerobic conditions may be
a result of dechlorination of the insecticide under anaerobic conditions and degradation of
the dechlonnated products under aerobic conditions This anaerobic-aerobic treatment scheme
may be useful in biorestoration of aquifers contaminated with halogenated compounds The
aquifer could be managed like a sequencing batch reactor in which an acclimated population
is exposed to deoxygenated water, then to aerobic conditions, and then the treated water is
withdrawn The hydraulically managed system is then allowed to sit idle until the next cycle
is initiated
Rates of degradation under anaerobic conditions are typically slower than those under
aerobic conditions, in addition, organic compounds may not be mineralized under anaerobic
conditions even after long periods of incubation ,fi However, anaerobic treatment may be
required to degrade pollutants that are reclacitrant under aerobic conditions, also, anaerobic
treatment may require less management The application of anaerobic conditions to biores-
toration is still in the developmental stage and more research is required to demonstrate its
usefulness in the field
C. Addition of Specialized Microbial Populations to the Subsurface
In addition to stimulating the indigenous microbial population to degrade organic com-
pounds, another innovative but not yet fully demonstrated technique is to add microorganisms
with specific metabolic capabilities to the subsurface 4 Specialized organisms may be in-
oculated into the subsurface environment or the environment may be altered to favor growth
of a population with specific metabolic capacities Populations that are specialized in de-
grading target compounds are selected by enrichment cultunng or genetic manipulation
Enrichment cultunng involves exposure of microorganisms to increasing concentrations of
a contaminant or mixture of contaminants The type of microorganism that is selected, or
in essence acclimates to the contaminant, depends on the source of the inoculum, the
conditions used for the enrichment, and the substrate 127 Acclimation can result from an
increase in the number of organisms that can degrade the contaminant, new metabolic
capabilities that result from genetic changes, or an increase in the quantity of the enzymes
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Volume 18, Issue I (1988) 55
necessary for the transformation The genetic changes include overproduction of enzymes,
inactivation or alteration of regulatory gene control, or production of enzymes with altered
specificities 1,6
Genetic manipulation of microorganisms to produce specialized populations that can de-
grade target contaminants is a relatively recent development According to Kilbane,'" genetic
engineering may accelerate and focus the process of evolution Genetic manipulation can
be accomplished by two different methods In the first method, the organisms are exposed
to a mutagen such as UV light, nitrous oxide, or 8-azaquinonone. and then a population
with specialized degradative capabilities is isolated by enrichment culturing,''Sii 1,4 however,
this may produce weakened strains because the process is nonspecific and affects the entire
genome IMI In the second method, recombinant DNA technology is used to change the genetic
structure of the microorganism 1,7 The genetic structure is changed by inserting a DNA
fragment, often a plasmid that codes for a specific degradative pathway, into another or-
ganism A plasmid is a piece of DNA that exists independently from the chromosomes of
the cell lh0 The extra-chromosomal DNA can be transformed from one bacterium to another
by conjugation, transduction, or transformation Multiple degradative capabilities can be
placed on a single plasmid that will allow the organism to degrade an array of compounds
or complete the degradation of a recalcitrant molecule Genetic engineering can be used to
stabilize the degradative traits coded by the plasmid, increase the number of plasnnds in a
cell, amplify enzyme production and activity, invoke multiple degradative traits, or produce
a novel degradative pathway 161 In addition, organisms with different substrate affinities,
pH optima, or degradation rates can be fashioned 162
Genetic Engineering to Enhance Degiadalive Activity
Genetic engineering has been used to enhance the degradation of the recalcitrant pesticide.
2,4,5-trichlorophenoxyacetic acid (2,4,5-T) Biodegradation of the pesticide is usually very
slow lh1 A mixed culture of microorganisms that uses 2,4,5-T as the sole carbon and energy
source was obtained by a technique called plasmid-assisted molecular breeding IM The
technique involves inoculating a chemostat with microorganisms from a variety of hazardous
waste sites and organisms that carry an array of plasmids that code for degradation of specific
xenobiotics A pure culture that could use 2,4,5-T as the sole carbon and energy source was
isolated from the mixed population and tentatively identified as Pseitdomonas cepacia "''i
In addition, the culture, designated P cepacia ACI100, was reported to oxidize many
chlorophenols Degradation of both 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-T was
expressed in another strain of P cepacia after conjugal transfer of two plasmids from an
Alcaligenes entrophs sp that degraded some chlorinated phenoxy herbicides 1,6 An inoculum
of 2 x 107 cells/g of P cepacia ACI 100 degraded 95% of the 1000 mg/£ 2.4,5-T added
to soil at 25% moisture and incubated at 30°C l6h Less 2,4,5-T was removed with a smaller
inoculum size and different temperatures and moisture contents In addition, the 2,4,5-T-
degrading bacteria did not survive in soil without 2,4,5-T or when the concentration of the
compound had been depleted IM Field trials to determine the effectiveness of the 2,4,5-T-
degrading bacteria have not been conducted
Colaruotolo et al 167 received a patent for "microbial degradation of obnoxious organic
wastes into innocuous materials " The process involves isolation of microbial cultures from
samples of soil and leachate from a hazardous waste site by enrichment culturing and then
application of the purified strains in the field to remove the contaminants Microorganisms
capable of degrading selected isomers of chlorotoluene, dichlorotoluene, and dichloroben-
zoate were isolated Conjugation and transformation experiments were conducted to transfer
the plasmid DNA, which conferred the ability to degrade some chloroaromatics. from the
original isolates to another organism The patent claimed that the organisms could be used
to decontaminate soil, remove contaminants in the air, mineralize toxic organics in the
leachate from a chemical landfill and thereby reduce the concentrations of noxious chemicals
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56 CRC Critical Reviews in Environmental Control
Table 6
REASONS WHY INTRODUCED ORGANISMS FAIL TO FUNCTION IN THE
ENVIRONMENT171
The concentration of the compound is loo low
The environment contains some substance or organisms that inhibit growth or activity, including predators
The inoculated organism uses some other organic other than the one it was selected to metabolue
The organic is not accessible to the organism
2 Issues in Genetic Engineering of Microbes
Organisms that cannot easily exchange their genetic information with other organisms and
are restricted to growth under defined environmental conditions are preferred candidates for
genetic manipulation lhl Issues concerning the use of genetically engineered organisms in
the environment include (1) adverse effects on human health, (2) how to effectively monitor
their dispersal, (3) survival of the engineered organism in the environment, (4) regulation
of activity in nontarget areas, and (5) determination of set risk levels acceptable to the
public l6H Many scientists argue that the engineered organism is not radically different from
that which is genetically unaltered The release of genetically engineered organisms into the
environment is of great concern to some and some time may elapse before these organisms
are used The survivability of genetically altered organisms in the environment is also of
concern Surrogates of genetically engineered organisms which carried antibiotic resistance
were added to samples of sewage, lake water, and soil and survived at rates that varied with
the strain and environment tested 170 Some of the antibiotic-resistant strains reached steady-
state concentrations in lake water and sewage, however, all strains declined in the soil after
a period of 1 month Pseudomonas strains that degrade 2,4-dichlorophenol and p-nitrophenol
were isolated from soil by ennchment-culturing techniques 171 The ability of the isolates to
degrade the phenol derivatives was variable when inoculated into lake water, sewage, and
soil
Inoculation of a specialized microbial population into the environment may not produce
the desired results for many reasons (Table 6) 171 The concentration of the target compound
required to support activity of a specific degrader may be limiting Toxic or antimicrobial
substances such as antibiotics may be found in many environments High-density inoculd
may be grazed by predators and the degradative capacity severely decreased if the growth
rate of the introduced organisms is slow In addition, adequate mixing to ensure contact of
the organism with the pollutant will be difficult to achieve in the subsurface
Most hazardous waste sites involve contamination of the environment with more than one
compound Therefore a mixture of organisms may be necessary to degrade all of the com-
pounds in the waste 127 Populations that have adapted to degrade many organic contaminants
may be isolated from biological treatment processes, such as sewage treatment, which recieve
pollutants The efficacy of an inoculated population of specific degraders will depend on
environmental constraints such as temperature, pH, and the concentrations of substrate,
nutrients, and oxygen 127172 Successful results from inoculation of foreign organisms are
more likely in simple environments because the environment can be controlled more easily
An example of inoculation into a simple environment would be the introduction of bacteria
into a biological reactor, oil tanker ballast tanks, or fermentator, these also provide the
benefit of containing the microorganisms To avoid problems encountered with inoculation
of foreign organisms into the environment, samples from the contaminated environment can
be collected, microorganisms that can degrade the pollutants can be cultured by enrichment
techniques or genetically engineered, and finally the specialized population can be reintro-
duced into the environment from which they came 171 In addition, genetic manipulation of
oligotrophy bacteria with high-affinity enzyme systems may be advantageous because these
enzyme systems will allow the organism to attack low concentrations of organic pollutants 174
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Volume 18, Issue 1 (1988) 57
3 Seeding Aqueous Environments with Microorganisms
Inoculants of specialized microorganisms have been used in treatment of contaminated
water Atlas and Bartha175 tested several commercial bacterial preparations and found that
the inocula were ineffective in treating oil spills in the marine environment However, the
addition of fertilizer and a bacterial seed isolated from an estuanne environment increased
petroleum degradation in a saline but not in a freshwater pond 176 After 6 weeks, 50% of
the oil remained in the saline pond The lack of activity in the freshwater pond suggests
that the inoculum should be cultured from an environment similar to that being treated
Colwell and Walker12' suggested that seeding would be unsuccessful in environments such
as the ocean, however, contained spills and lagoons may be amenable to such treatment
Gulnick and Rosenberg177 stated that "there is no evidence to support the claim that 'seeding'
oil slicks with microorganisms reduces oil pollution by stimulating petroleum biodegradation "
4 Seeding Soil Environments with Microorganisms
The efficacy of inoculating soil with acclimated bacteria to remove selected contaminants
was tested in a series of experiments178 using experimental chambers set up in greenhouses
The contaminants, aniline and formaldehyde, were added to three types of soils (clay, sandy
loam, and organic-rich) and plants were seeded in the chambers Removal of the contaminants
by a mixed microbial population from primary sewage effluent and an acclimated population
was investigated Formaldehyde was not removed in organic soils amended with sewage
and acclimated bacteria, however, this treatment was successful in the upper and middle
zones of the sand and clay soils Aniline was removed in the organic and sandy soils after
a second application of sewage microorganisms, nutrients, and yeast extract Chemical
oxidation of the organics using hydrogen peroxide was effective in reducing aniline con-
centrations None of the treatments were successful in removing aniline from the clay soil
The removal of chlordane and 2,4-dinitrophenol by mutant-adapted microbial cultures was
also investigated The inoculum was successful in degrading 2,4-dmitrophenol from the
upper layer of the clay soil only The authors suggested that the sewage inoculum was a
low-cost, effective method for removal of aniline and formaldehyde in most soil types,
however, addition of the adapted population was not successful in these tests
Inoculation of soils to remove chlorinated organics and pesticides has been attempted
Daughton and Hsieh179 reported that inoculation of sterilized soils with a parathion-acclimated
culture reduced the concentration of the insecticide by 85%, however, the efficiency of the
inoculum in nonstenle soil was greatly reduced Focht and Brunner18" used an Acinetobacter
strain as an inoculum to degrade biphenyl and polychlonnated biphenyls (PCBs) in soils
The inoculum increased the initial and maximum mineralization rates and the disappearance
of the more heavily chlorinated biphenyls, but the overall extent of mineralization of PCBs
was not greater than that in uninoculated soil to which biphenyl had been added The process
was thought to be a cometabolic-commensal metabolism of the PCBs
Remediation of soil contaminated with hydrocarbons by inoculating with hydrocarbon-
degrading organisms has been met with varying success Schwendinger181 demonstrated that
inoculation of a hydrocarbon-degrading strain of Cellumonas in soil contaminated with
petroleum increased the rate of reclamation in comparison to soils amended with only
nutrients Jobson et al 182 reported that the application of 10" cells of oil-degrading bacteria
per cubic centimeter of soil slightly increased the degradation of the C20- to C2,-group of
n-alkanes in comparison to soils amended with fertilizer only However, Lehtomaki and
Niemela181 reported that Brewer's yeast added to soils served primarily as a fertilizer rather
than as an inoculum to actively degrade the oil Seeding boreal soil with an oil-degrading
inoculum increased microbial activity 184 In laboratory studies, the addition of 300 ppm
nitrogen and 100 ppm phosphorus, inoculation, and adjusting the pH to 7 increased microbial
activity by at least a factor of four in comparison to unamended samples after 40 days of
incubation An increase in plant growth in an oil-contaminated area in response to fertilizer
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58 CRC Critical Reviews m Environmental Control
addition was shown in field studies, however, the increased growth could have resulted from
the addition of fertilizer or enhanced removal of the petroleum In contrast, Westlake et
al IHS reported no beneficial effects from the addition of oil-degrading bacteria to boreal
soils The lack of enhancement may be a result of inadequate application of the inoculum
The type of organisms isolated from enrichment culturing depends on conditions used during
the isolation procedure For example, enrichments made at 4 and 20°C contained different
organisms, and cultures enriched on a low-quality crude were better adapted to utilize a
lower quality crude than cultures enriched on a high-quality crude IXh These data suggest
that enrichments for specialized populations should be conducted using the environmental
conditions and contaminants that are unique to the site under investigation
An inoculum of pentachlorophenol-degrading organisms has been used to decontaminate
soil, river water, ground water, and other freshwaters 1X7 A Flavobaaeuum sp that could
mineralize pentachlorophenol (PCP) was isolated from a man-made channel which was
exposed to the compound for several weeks lxx In addition to mineralizing PCP, the mi-
croorganism could attack a number of other chlorinated phenols but not all isomers IS" The
Flavobactertum sp at a cell density of I06 cells/m£ removed over 90% of the PCP added
to river water, ground water, and other fresh waters, usually within 48 hr 1X7 The organisms'
ability to degrade PCP was best between 15 and 35°C and at pH values between 7 5 and
9 Inoculum densities as low as 104 cells/mf resulted in efficient removal of PCP The time
required to remove the PCP increased with increasing concentrations of PCP When added
to uncontaminated soil, the PCP was rapidly mineralized 1X8 The highest extent of miner-
alization occurred in soils with moisture contents between 15 to 20%
Mineralization of PCP was observed at inoculum densities as low as 3 1 x 101 cells/g,
however, a slightly higher extent of mineralization was observed at a cell density of 3 1 x
10h cells/g lxx Mineralization of PCP in one uninoculated soil began after 7 days of incubation
and mineralization proceeded to the same point as the sample inoculated with 107 cells/g
Concentrations of PCP in soil contaminated from a wood-treating landfill were reduced from
298 to 58 ppm after four applications of the inoculum in a period of 100 days In another
contaminated soil, PCP levels were reduced from 321 to 41 ppm after one application of
seed, but similar levels of removal were observed in the uninoculated control The seed
could not remove PCP from a third soil in which the concentration of PCP had been diluted
tenfold to 553 ppm and the pH adjusted to neutrality Addition of 10" cells/g soil of a culture
of PCP-degrading Arthrobacter sp reduced the half-life of PCP from 2 weeks to 15 hr 190
Edgehill and Finn191 reported that the rate of PCP disappearance was proportional to inoculum
size that ranged from 104 to 10ft cells/g soil Up to 85% of the PCP was removed within 12
days in soil in which the seed had been thoroughly mixed, however, only 50% was removed
in the unmixed soil Brown et al 192 suggested that fixed film reactors with a PCP-adapted
population may be used to treat waters contaminated with PCP at concentrations below the
threshold of toxicity A consortium that was attached to rocks from an artificial stream
amended with PCP was generally able to degrade PCP as fast as the Flavobatterium sp
described by Crawford and Mohn l8X A treatment system using two fixed film reactors in
series was then proposed, the first reactor would reduce high concentrations of PCP and the
second reactor would contain organisms that could remove PCP to low levels The consortium
was able to remove PCP to < 1 \x,g/C when the initial concentrations were < 1 gH
5 Seeding the Subsurface with Microorganisms
Inoculation of bacteria into the subsurface for biorestoration has been met with some
success, but the contribution of the introduced bacteria to the overall cleanup cannot be
readily determined In most cases, the role of the introduced bacteria in degradation of the
contaminants cannot be determined because appropriate control plots were not incorporated
into the experimental design and the results were not quantitatively measured throughout
the course of the project The biggest concern of inoculation into the subsurface is ensuring
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59
Table 7
SUMMARY OF AQUIFER REMEDIATION CASE
HISTORIES UTILIZING INTRODUCED ORGANISMS
Compound
Treatment description
Formaldehyde
Ethylene glycol and
propyl acetate
Dichlorobenzene,
dichloromethane, and
(nchloroethane
Unidentified organic
compounds
Acrylomtrile
Phenol and
chlorophenol
Mutant bacteria added after con- 199
centrations had been reduced by
air-stnpping
Initial treatment by adsorption onto 199
GAC followed by inoculation
with mutant bactena
Treatment above ground and later 200. 201
with specialized bactena
Initial treatment with air stripping 200, 201
and then inoculation with a hy-
drocarbon-degrading bactena
Hydrocarbon-degrading bacteria 202
added after levels reduced by
GAC and air stripping
Commercial degrader added to 203
above-ground treatment system
formed from rail ballast
contact between the specialized cells and the target contaminants The cells may be filtered
out of the perfusing solution or sorbed onto soil before reaching the contaminants 142 In
addition, normal die-off may control the movement and spread of bacteria in well-sorted
sand, gravels, fractured rock, and karstic limestone
Microbial movement through the subsurface depends on the characteristics of the soil and
microorganisms Only 1% of an inoculum of a Pseudomonas strain passed through a 2-in
sandstone core after washing with 123 pore volumes 193 Penetration of bactena into sand-
stone cores with hydraulic conductivities >100 mD (hydraulic conductivity = 9 6 x 10-5
cm/sec) was rapid, however, penetration in cores with hydraulic conductivities below 100
mD was slow 194 Motile bactena moved three to eight times faster than nonmotile bactena
Hagedom195 summanzed the results of selected studies on the maximum distance that mi-
croorganisms moved in vanous soils 19 8 m in 27 weeks in a fine sand, 10 7 m in a sand
and sandy clay in 8 weeks, 24 4 m in a fine and coarse sand (time of travel not reported),
30 5 m in a sand and pea gravel aquifer in 35 hr, 0 6 to 4 m in a fine sandy loam (time of
travel not reported), 457 2 m in a coarse gravel aquifer in 15 days, 28 7 m in 24 to 30 hr
in a crystalline bedrock Bactena have moved as far as 920 m in the subsurface at rates of
up to 350 m/day 196 Microbial movement through soil macropores is an important mechanism
of transport in all subsurface soils except sandy soils and those that are disturbed 197
Transport of microorganisms in the subsurface can occur However, in situ biorestoration
programs using inoculation techniques will be affected by adverse conditions that decrease
the survival of microorganisms in the environment Several factors must be considered before
an in situ biorestoration program utilizing acclimated bacteria is implemented The source,
quantity, nature and biodegradability of the contaminants, and the environmental conditions
of the site must be determined 198 In addition, laboratory tests to determine the kinetics of
degradation, the potential for inhibition under various conditions, requirements for oxygen
and nutnents, and the effects of temperature should be conducted The formation must be
permeable enough to perfuse nutnents and the inoculum through the zone of contamination
6 Aquifer Remediation Using Inoculation Techniques
Inoculation of microorganisms into the subsurface has been used in aquifer remediation
in conjunction with wastewater treatment processes These cases are summarized in Table
7 A representative system is shown in Figure 3 In one case study, 26,500 £ of acrylomtrile
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CRC Critical Reviews in Environmental Control
DIRECTION OF GROUND WATER FLOW
outside water source
INJECTION SYSTEM
treated water
clean
pool
ZONE OF CONTAMINATION
treatn ent
iment
dirty
pool
contaminati
\ water
RECOVERY SYSTEM
pneumatc recovery
pump
FIGURE 3 Combination of above-ground treatment with in %/^r/ biorcstorahon
was spilled in a metropolitan area from a leaking rail car lwThe receiving aquifer contained
significant amounts of silt and clay and hence was rather impermeable Initial treatment
involved withdrawal and treatment of the ground water by air stripping After the concen-
tration of aerylonitrile had declined to nontoxic levels, mutant bacteria were seeded into the
soil The concentration of acrylonitrile declined from 1000 ppm to nondetectablc levels (limit
of detection 200 ppb) within I month, however, the role of the bacterial seed in acrylonitrile
degradation could not be determined
Quince and Gardner2'*'2<" documented the cleanup of 378,500 € of various organic com-
pounds, including ethylene glycol and propyl acetate, over a 23.000-m2 area The soil
consisted of a thick silty clay that extended to a depth of more than 15 2 m, migration of
the organics into the main aquifer was prevented by the structure of the formation Con-
tainment and recovery of the organics were limited to the perched water table located in the
upper clay layer The contaminated ground water was withdrawn and treated by clarification,
aeration, and GAC A biostimulation program with specialized bacteria, nutrients, and air
was initiated after the levels of the contaminants had decreased from 2000 to 10,000 ppm
to <200 ppm During treatment, the concentration of ethylene glycol was reduced from
1200 to <50 mgIt, propyl acetate was reduced from 500 to <50 mg/f, and the total
concentration of spilled compounds declined from 36,000 to <100 mg/f The resulting
concentrations of contaminants were acceptable to the regulatory agencies
Quince and Gardner20"201 documented the cleanup of a number of organic chemicals
including dichlorobcnzene, methylene chloride, and trichloroethane that contaminated the
subsurface as a result of a spill from leaking tankers The treatment scheme included recovery
of product with a vacuum system, soil flushing, air stripping, and then inoculation of
commercial hydrocarbon-degrading bacteria into an above-ground reactor followed by re-
charge of the effluent into the subsurface A commercial microbial inoculum seeded into
the above-ground reactor significantly decreased the concentrations of the organic contam-
inants after 36 hr of exposure The aeration was terminated after a 95% reduction in the
organic levels was achieved The injected hydrocarbon degraders were expected to complete
the biodegradatton in situ, however, the role of the added bacteria was not demonstrated
An accidental spill of 492,000 € of organic chemicals entered a 4 6-m thick shallow
unconfined aquifer and resulted in total contaminant levels as high as 10,000 ppm 202 A
drinking water aquifer was separated from the contaminated zone by 15 2 to 18 3 m of silty
clay The contaminated ground water was withdrawn and treated by clarification. GAC
adsorption, and air stripping A program to enhance in situ biological degradation was
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Volume 18, Issue 1 (1988) 61
initiated after the concentration of the organics had declined from as high as 10.000 to 1000
ppm The results of laboratory tests indicated that the indigenous bacteria could degrade the
contaminants when supplied with nutrients Application of a commercial bacterial inoculum
did not increase the biodegradation rates of the organics, in fact, one compound (unidentified)
was degraded slower by the commercial hydrocarbon-degrading inoculum than the indigenous
population Effluent from the treatment system was amended with hydrocarbon-degrading
bacteria, air, and nutrients and injected into the vadose zone As a result, the concentrations
of the contaminants in one soil core were reduced from 800 to 150 mg/f in 2 months In
another area, the concentration of the chemicals in composited soil samples declined from
24,000 to 2000 mg/f The concentrations of the organics in the ground water were reduced
to <1 ppm, which met regulatory approval Incorporation of biological treatment into the
restoration program decreased the cost of operation and maintenance The role of the com-
merical inoculum in the removal of the contaminants could not be determined, in addition,
laboratory studies indicated that the inoculum did not enhance biodegradation
A spill of75.700 fofa 50% solution of formaldehyde from a railroad tank car contaminated
the soil and railroad bed in Ukiah, Calif 201 Contaminated surface and ground waters were
removed by a vacuum truck and 190 m1 of soil were excavated Approximately 49 million
£ of water was collected The water was initially treated with hydrogen peroxide to reduce
the concentration of formaldehyde from 30,000—50,000 to 500—1000 ppm by oxidation
The feasibility of in situ biological degradation of the remaining formaldehyde using a
commercial bacterial inoculum was then investigated A commercial inoculum that contained
specially cultured microorganisms was chosen for the project The biological treatment
system consisted of a portable aeration tank, a spray system, and a trickling filter The
ground water was heated to increase the destruction rate and the pH was adjusted as necessary
with sulfuric acid or soda ash, nitrogen and phosphorus were added as needed The inoculum
was rehydrated with chlorine-free water and added to the system at a rate of 1 4 kg/day
The concentration of formaldehyde in the treatment tank fell from >700 to about 10 mg/f
alter 24 days The oxygen uptake rate in the sump ranged from 12 to 82 mg/f hr_l and
from 29 to 51 mg/C hr"' in the ballast gravel The treatment program was temporarily
suspended for I day and the system was flushed During this period, the concentration of
formaldehyde increased greatly, however, a rapid reduction in formaldehyde levels to <1
mgIC followed The authors suggest that the removal of the formaldehyde was a result of
biological activity, however, they concede that proving the role of microorganisms in for-
maldehyde degradation would be difficult In addition, the role of indigenous and inoculated
bacteria in formaldehyde degradation could not be separated
7 Enrichment of Specific Populations
A strategy often used in industrial microbiology is to search through nature for an organism
with specialized metabolic capabilities and then culture that organism in a fermentor to
protect it from competition However, this strategy would be difficult to apply in the sub-
surface environment because the specialized population must be competitive in addition to
performing the desired transformation Enrichment cultunng techniques are often used to
isolate organisms with specialized metabolic capabilities The same concept can be used to
identify conditions that favor the colonization of that environment by organisms with special
traits
The microbial utilization of pollutants as carbon and energy sources has already been
discussed This section will emphasize the metabolism of pollutants by microorganisms
enriched on other primary substrates
Oxygenated water-table aquifers are often polluted with chlorinated organic solvents such
as trichloroethylene 21M 2(" The ubiquity of these compounds in oxygenated ground water
may result from their resistance to microbial attack under aerobic conditions in the subsurface
However, more recent work has indicated that incubation of soils or aquifer materials with
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CRC Critical Reviews in Environmental Control
methane, propane, or natural gas will enrich for microorganisms that co-oxidize trichloro-
cthylene and a variety of other halogenated organic compounds 206211 This technique may
be applicable to in situ restoration of aquifers which are contaminated with chlorinated
organic solvents
Using gaseous aliphatic hydrocarbons as the feedstock for a forced co-oxidation is ad-
vantageous because they are nontoxic, relatively inexpensive, and widely available in the
form of natural gas, liquified petroleum gas, and propane However, they do not support
anaerobic metabolism In addition, if the gases are inadvertently supplied at concentrations
that result in the microbial depletion of the available oxygen, undesirable byproducts such
as foul-smelling organics, soluble iron, or hydrogen sulfide should not be produced The
disadvantage to enriching for specialized populations using gaseous aliphatic hydrocarbons
is the explosion hazard of the hydrocarbons mixed in air at unsafe concentrations One
constraint on in situ restoration programs is that the reagents must be dissolved in the perfusion
water to reach the zone of contamination, hydrocarbons and the oxygen required for their
metabolism are not very soluble in water
Wilson and White2" developed a general relationship that may be used to predict the
extent of removal of a chlorinated organic as a function of the metabolism of a given amount
of hydrocarbon feedstock The relationship is as follows
C/C„ = e"kh (2)
where C = the final concentration of the halogenated organic to be co-oxidized, C„ = the
initial concentration of the halogenated organic, h = the amount of hydrocarbon feedstock
to be consumed, and k = a utilization constant
At present, there are limited data available to calculate utilization constants and the
generality of the relationship has not been widely tested The equation may prove to be a
powerful tool in the engineering design of in situ biorestoration programs Some utilization
constants for tnchloroethylene exist 211 As a paper exercise, the extent of removal of tri-
chloroethylene at a number of critical engineering limitations, including the solubility of
methane, propane, and pure oxygen (24, 62, and 40 mg/
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Volume 18, Issue 1 (1988) 63
Table 8
ESTIMATED QUANTITIES OF OXYGEN AND METHANE
OR PROPANE REQUIRED TO BRING THE
CONCENTRATION OF TRICHLOROETHYLENE, cis- OR
trans-l,2-DICHLOROETHYLENE OR VINYL CHLORIDE TO
5
Initial cone*
DCE
ICE or VC
((Jig/f) (p-g/f)
Methane
required
Cents/
1000 gal
mg/f (3785 f)
Propane required
Cents/
1000 gal
nig//' (3785 6)
Oxygen required
Cents/
1000 gal
mg/f (3785 f)
2 500
62
5 9
230
340
1,000
53
5 0
190
290
250
39
3 7
140
210
100
29
2 7
105
159
30
24
2 5
96
146
17
11
10
0 9
11
1 0
40
40
7
2 2
0 2
8
5 8
2 0
0 2
8
5 0
00
6 700
24
2 5
96
146
1,000
18
1 7
71
107
100
10
0 9
40
9
2
0 2
8
5 0
0 0
00
TCE = tnchloroethylcnc DCE = dichloroethylcne VC = vinyl chloride
of trichloroethylene 214 Utilization constants for these compounds are not available Wilson
and White2" estimated from the data of Fogel et al 207 that the constants are >0 3 £ water
treated per milligram methane consumed Unpublished data of Henson272 suggest that con-
stants for cis- and trans-\ ,2-dichloroethylene are >0 8 and 0 4 £ water treated per milligram
methane consumed, respectively These higher utilization constants make aquifers contam-
inated with these compounds much better candidates for in situ biorestoration (Table 8)
Hydrogen peroxide will probably not be required to achieve adequate treatment If the
contaminated water is pumped to the surface for treatment, the limited solubility of oxygen
becomes much less of a problem The water can be exposed to any desired volume of air
in a Fixed Film bioreactor211
Table 9 summarizes the prospects for biorestoration of aquifers contaminated with specific
halogenated compounds These data were compiled by comparing the relative rates of
degradation of these compounds in a variety of experimental systems to the rates of trans-
formation of trichloroethylene and cis- and trans-l ,2-dichloroethylene, then assessing the
rates in light of the relationships portrayed in Table 8 Prospects are rated "good" if hydrogen
peroxide will not be required, "fair" if hydrogen peroxide is required, and "poor" if
environmentally insigniFicant removals cannot be attained with or without hydrogen peroxide
The relationship of Wilson and White2" does not presuppose an upper limit on the
concentration of the chlorinated contaminant, however, an upper limit obviously exists and
toxicity effects have frustrated research in this area Workers at both Stanford University
and R S Kerr Environmental Research Laboratory have isolated mixed microbial popu-
lations from nature that could degrade trichloroethylene, only to lose the ability to degrade
the compound when the primary alkane oxidizer was isolated in pure culture It is tempting
to conclude that the trichloroethylene degrader is not an alkane oxidizer However, other
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CRC Critical Reviews in Environmental Control
Table 9
PROSPECTS FOR TREATMENT OF THE COMMON HALOGENATED
ORGANIC CONTAMINANTS IN AQUIFERS THROUGH CO-
OXIDATION SUPPORTED ON GASEOUS ALKANES
Pump
Treat in the
Compound
and treat
aquifer
Ref.
Tetrachloroethylene (PCE)"
None
207, 209, 213
Trichloroethylene (TCE)
Good
Fair
206, 207, 209
cis-l 2-Dichloroeihylene
Good
207 209, 213
trans-1,2-Dichloroethy lene
Good
207, 209, 213
Vinyl chloride
Good
207, 215
Direct utilization may be possible
1, l-Dichloroethylene
Fair
207
Carbon tetrachloride"
None
209, 213
Chloroform
Poor
208. 209, 213
Methylene chloride
Fair
75, 213
Direct utilization may be possible
1,1,1-Tnchloroethane (TCA)
Poor to
Poor
209, 211, 213
good
1,1,2-Tnchloroethane (TCA)
Poor
209 213
1,1-Dichloroethane (DCA)
Poor
209 213
1,2-Dichloroethane
Poor
204 209 213,
Direct utilization may be possible
1,2-Dibromoethane (EDB)
Fair
209, 213
Direct utilization may be possible
' Removal of carbon tetrachloride and tetrachloroethylene seen in the soil exposed to natural gas is
probably an anaerobic process and not a direct result of alkane oxidation 2,2
possibilities exist In certain mixed cultures or microcosms of aquifers, trichloroethylene
started to inhibit oxidation of the hydrocarbon feedstock at a concentration of about 1000
jxg/f 270 271 This is far below concentrations that produce toxicity in ordinary heterotrophs
Perhaps the effective toxicant was trichloroethylene epoxide rather than trichloroethylene
itself, and the organism that oxidized the primary hydrocarbon feedstock was protected in
mixed populations by other organisms This toxicity threshold must be more carefully defined
to aid in identifying contaminated ground water amenable to biorestoration This work should
be done with mixed cultures or microcosms, using systems that simulate the conditions in
the subsurface environment
No technique to remediate environmental contamination is universally applicable How-
ever, there should be many contamination incidents where biorestoration through a forced
co-oxidation is the technology of choice, either alone or in conjunction with physical con-
tainment Successful application of the approach will require adequate understanding of the
physiology of the biotransformation and quantitative information on the nutritional ecology
of the active organisms
III WITHDRAWAL AND TREATMENT
A. Biological Wastewater Treatment Processes
Ground water can be withdrawn and treated by conventional biological wastewater treat-
ment processes Treatment processes used to treat contaminated ground water and leachate
from hazardous wastes include (1) suspended growth processes such as activated sludge,
lagoons, waste stabilization ponds, and fluidized bed reactors, and (2) fixed Him processes
such as trickling filters, rotating biological discs, sequencing batch reactors, and others The
wastewater can be treated on site by one of these processes or off site at a municipal or
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Volume 18, Issue 1 (1988) 65
commercial treatment plant A short discussion of each process follows with regard to
hazardous waste management More detailed discussions of these processes are presented
in J R B Associates,"1 Ehrenfeld and Bass,"9 Shuckrow et al ,217 Nyer,2IK and Canter
and Knox 219
The most commonly used municipal wastewater treatment process, activated sludge, has
a number of advantages for treatment of contaminated ground waters The units include an
aeration basin, a clarifier, and sludge recycle Following treatment in the aeration basin, a
portion of the sludge collected in the clarifier is recycled The recycling process allows an
acclimated microbial population to build up in the system, hence the name activated sludge
The settled sludge can adsorb heavy metals and some organics which may cause the sludge
to be considered a hazardous waste 217 However, some compounds are removed by volatil-
ization during the aeration step 22(1 Activated sludge treatment can reduce the soluble bio-
chemical oxygen demand (BOD) to <10 mg/£ and the total BOD, including suspended
solids, to <30 mg/£ The retention time is short and consequently the process is sensitive
to toxic and hydraulic shocks A survey of 92 industrial wastewater streams conducted by
the EPA reported mean BOD removal efficiencies of 86% 139 Specific organic compounds
can be degraded to low levels, effluent levels of phenol as low as 0 02 mg/
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66 CRC Critical Reviews in Environmental Control
In the fixed film process, wastewater is passed over a surface colonized with microor-
ganisms, the attached biofilm degrades the organic material The original fixed film process,
the trickling filter, uses a solid medium such as rock or plastic as the surface for microbial
attachment 219 Trickling filters can remove from 60 to 85% of the BOD 220 Suspended or
colloidal organics can be treated, and the process is usually limited to low organic loadings 111
Trickling filters that arc operated anaerobically are known as anaerobic filters 2211 The
anaerobic filter process can tolerate high loading rates Low pH and inorganics such as
sodium, sulfate, and heavy metals may inhibit methanogenesis and toxic organics may also
be a problem 84 The biological tower is another variation of the trickling filter The tower
is packed with a colonizable surface which may reach a height of 4 9 to 6 1 m 2|l> The
process operates in a countercurrent mode, contaminated water is sprayed on the top of the
tower as air is pulled from the bottom
A rotating biological disc is similar in concept to trickling filters Discs or drums which
are coated with a biofilm are partially submerged and rotated through the wastewater ny 222
Rotation of the discs aerates the attached biofilm The process is sensitive to shock loading
and temperature fluctuations but otherwise is moderately reliable 111 In comparison to ac-
tivated sludge, rotating biological discs require less energy and are easier to operate, but
are similar in effectiveness "(> Clarification may be required before and after treatment of
wastewater with rotating biological discs 119
Biological wastewater treatment processes may be used to treat the following classes of
organic compounds typically found in ground water alcohols, organic acids, aldehydes,
ketones, quinones, amines, amides, carbohydrates, esters, some ethers, phenolics, and some
aromatics 220 221 Compounds that may be difficult to treat with biological wastewater proc-
esses include halocarbons, high molecular weight polynuclear aromatics. pesticides, and
organometals 1,9
B. Examples of Withdrawal and Biological Treatment
Biological wastewater treatment processes have been used in remedial action at several
hazardous and nonhazardous waste sites Adequate treatment of leachates from recent mu-
nicipal refuse landfills that contained high levels of free fatty acids was achieved by biological
treatment 224 Leachates from older landfills may be more amenable to physical-chemical
treatment processes In a pilot study, Stover and Kincannon22-'1 were able to decontaminate
ground water from a hazardous waste site using activated sludge The batch-activated sludge
pilot system was seeded with organisms that were acclimated to the same compounds found
in the contaminated ground water — phenols, cresols, dichlorobenzenes, and others Fol-
lowing acclimation and stabilization of the batch-activated sludge for 3 weeks, the organisms
were able to reduce the total phenols, TOC, BOD, and COD by 80% or more within 24
hr
C. Combinations of Biological Treatment with Other Processes
Combination of conventional biological wastewater treatment with other water treatment
processes such as GAC, air stripping, and addition of acclimated bacteria have also been
successful Feasibility studies on decontamination of leachate from the Ott/Story hazardous
waste site in Muskegon, Mich , have combined activated sludge and GAC adsorption to
remove various halogenated aliphatics, benzene, and toluene 226 Initial attempts to acclimate
an activated sludge culture to the organic contaminants were minimally successful and the
addition of a commercial microbial culture was not effective, however, a combination of
GAC adsorption followed by activated sludge removed >95% of the TOC The activated
sludge organisms removed the organics that were not sorbed in the GAC treatment Treatment
efficiencies were >75% as long as the removal efficiency of the GAC was high Anaerobic
treatment combined with GAC was less effective than aerobic treatment (activated sludge)
and suffered the same decline as the aerobic process when the GAC was saturated 217 Removal
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Volume 18, Issue 1 (1988) 67
of TOC by a treatment train using GAC followed by anaerobic and then aerobic biological
treatments was also tested 227 The biological treatment steps were not necessary when the
GAC sorption sites were not significantly saturated, however, the biological treatment in-
creased the removal efficiency of the treatment train as more GAC sorption sites became
saturated The TOC removal efficiency of GAC combined with anaerobic and aerobic
treatment was less than that of GAC and aerobic treatment only
Josephsonh7 reported that a combination of powdered activated carbon and activated sludge
was used to treat ground water contaminated with hydrocarbons, pesticides, and other
organics A removal efficiency of 95 to 99% was achieved for the COD, total nitrogen, and
various organics
Air stripping followed by biological treatment was used in a pilot study to treat ground
water contaminated with trichloroethylene, freon, 1,2-dichloroethene, toluene, ethyl ben-
zene. xylenes, vinyl chloride, acetone, isopropanol, o-dichlorobenzene, 1,1-dichloroethy-
lene, and 1,2-dichlorophenol 22K Air stripping could remove all of the organics except for
the nonvolatile compounds isopropanol and acetone, however, a treatability study indicated
that the nonvolatile compounds were biodegradable Hence, a treatment system utilizing
both air stripping and biological treatment was recommended for this site
Nyer and Sauer224 described the cleanup of shallow ground water from a Gulf Coast
hazardous waste site The saline aquifer was contaminated with 400 mg/C phenol and other
organics which resulted in a TOC concentration of 1300 mgH Several options that were
considered for treatment arc reported with estimated costs per gallon in parenthesis pond
evaporation with oxygen and nutrient addition ($0 028), deep well injection ($0 183), so-
lidificationAidsorption to concentrate the liquid and then adsorb the material to trench backfill
($0 085), GAC adsorption ($0 058), biological treatment ($0 005)
A feasibility study indicated that the organics were biodegradable but carbon adsorption
would be required as a polishing technique 229 The overall treatment system would consist
of (I) pH adjustment, (2) chemical addition, (3) biological treatment with two aeration
basms, a clarifier, and a fixed activated sludge treatment system (FAST), (4) filtration through
a dual media filter, and (5) carbon adsorption The FAST system uses particles of plastic
colonized by bacteria in a well-mixed tank The system is essentially a hybrid of activated
sludge and fixed film processes The biological system was seeded with activated sludge
from a refinery that treated ballast from oil tankers Results from the pilot plant, which
included the aeration plant and clarifier only, indicated that the TOC was reduced by 70%
Addition of the FAST system, the dual media filter, and the carbon adsorption unit to the
treatment train reduced the concentration of TOC from 1300 to 18 mg/f (98%) 2ltl
Addition of mutant bacteria to a sequencing batch reactor, a process patented in 1985 by
Colaruotolo et al ,21(> was used to treat leachate collected from the Hyde Park Landfill in
Niagara Falls, N Y 221 The leachate contained chlorinated organics, phenol, and benzoic
acid A consortium of microorganisms that could degrade most of the contaminants was
isolated from the leachate, however, degradation of the pollutants by bacterial strains in the
consortium was variable 211 By genetic manipulation, organisms in the consortium that could
degrade the remaining compounds were found Tests with bench-scale sequencing batch
reactors and also pilot-plant scale units indicated that the TOC was reduced by 85% or
greater and that individual contaminants were generally reduced by 95% or greater The
biomass yield was 0 64 mg/mg feed TOC Amendments of nitrogen and phosphorus did not
improve treatment over the addition of only nitrogen Cost savings for the biological treatment
over the existing carbon adsorption system were estimated to range from $538,000 up to
5783,000
Bartha212 suggested that inoculation of microorganisms into a wastewater treatment process
should be judged with caution Inoculation may be useful for startup, disruptions, for certain
xenobiotics that cannot be degraded by the natural flora, or when the added organisms cannot
sustain themselves
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68 CRC Critical Reviews in Environmental Control
IV HYDROLOGIC CONSIDERATIONS AND MATHEMATICAL
MODELING OF BIORESTORATION
A. Hydrologic Considerations
A number of methods have been reported in the literature for the containment of contam-
inated ground water through hydraulic control or through injection-pumping networks of
wells Biorestoration of a contaminant plume may involve the addition of nutrients such as
dissolved oxygen or hydrogen peroxide or the addition of microbes capable of degrading a
particular waste In order for such additions to be successful, it may be necessary to use
hydraulic controls to minimize the migration of the plume during the in situ treatment process
Thus, hydrologic considerations cannot be neglected in the biorestoration of aquifers
Hydraulic control methods depend to a large extent on variability of aquifer hydraulic
conductivities, background velocities, and sustainable pumping rates Typical patterns of
wells which are used to provide hydraulic controls include (1) the injection-production pair,
(2) a line of downgradient pumping wells, (3) a pattern of injection-production wells around
the boundary of a plume, and (4) the "double-cell" hydraulic containment presented by
Wilson 211 Well systems can also be used to capture and withdraw entire zones of contam-
inated water for treatment above ground
Analytical equations and graphical solutions are available for estimating flow rates and
limits of hydrodynamic isolation under various boundary conditions Numerical computer
models of ground water flow and contaminant transport are required when site geology is
complex, heterogeneous, and anisotropic A simple hydrodynamic isolation system within
a uniform flow field involves the placement of a recharge well of the same strength upgradient
from a pumping well Standard equations describe the head h(x,y) as a function of pumping
rate, ambient flow rate, and transmissivity of the aquifer The region of recirculation which
connects the stagnation points can be evaluated and provides a measure of the capture zone
of contaminated ground water 234
Wilson211 presents a "double-cell" hydraulic containment system which utilizes an inner
cell and an outer recirculation cell, with four wells along a line bisecting the plume in the
direction of flow The method is more efficient in terms of flushing times and recirculation
rates than the single cell The double-cell method provides added flexibility and a back-up
system if pumps should fail in either system
Ozbilgin and Powers21"* described hydrodynamic isolation systems for several EPA haz-
ardous waste sites Pumping wells and an upgradient recharge trench were successful in
retarding the advance of a contaminated plume at the site in Nashua, N H They concluded
that hydrodynamic isolation systems are generally less costly and time consuming than
physical containment structures such as slurry walls Well systems are more flexible in that
pump rates and well locations can be altered as the system is operated over a period of time
Shafer236 indicated that pumping-injection systems can be used (1) to create stagnation
(no flow) zones at precise locations in a flow field, (2) to create gradient barriers to pollution
migration, (3) to control the trajectory of a contaminant plume, and (4) to intercept the
trajectory of a contaminant plume However, the determination of pumping rates to achieve
a pollution control objective can be difficult Thus, investigators have explored the application
of optimization theory to determine optimal pumping rates for creation of hydraulic controls
Gorelick2-17 and Atwood and Gorelick238 focus on using linear programming (LP) methods
to determine the best containment strategy in combination with a ground water flow simulator
From a specified set of potential well sites, the model approach selects well locations and
optimal pumping/recharge rate schedules to contain the contaminant plume Shafer236 ad-
vocates the use of nonlinear programming combined with a ground water flow model and
an advective transport model The optimization method is applied to examples for determining
stagnation points in a flow field and for steering the trajectory of a contaminant plume
Optimization methods offer more efficient solutions than the typical trial and error approaches
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Volume 18, Issue I (1988) 69
for exploring cleanup strategies However, nonlinear programming requires the flow and
transport models to run during each iteration in which new pumping rates are selected and
tested in the overall performance index or objective function In the present case, optimization
methods are complex and time consuming and may not offer any improvements over sim-
ulation for the complex case of biorestoration alternatives
Successful biorestoration alternatives at a particular site depend on the hydrological and
geological characteristics of the aquifer If the contaminant plume is moving rapidly through
a sandy-gravelly zone, then hydraulic controls may be required to halt the advance of the
plume and to provide injection points for added nutrients or oxygen Pumping out ground
water and surface nutrient additions prior to reinjection may provide a more controlled input
to the biorestoration process
For the case of slowly moving organic plumes in a silty sand aquifer, it may be hydro-
logically difficult to pump or inject recharge waters at rates greater than 19 or 38
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70 CRC Critical Reviews in Environmental Control
the rate of contaminant biodegradation In order to model the degradation process, rela-
tionships are needed which describe the kinetics of microbial growth and consumption of
added nutrients and electron acceptors These relationships are then combined with Equation
3 to describe the movement and consumption of the contaminant and added nutrients One
of the most popular relationships for describing the growth and decay of microorganisms
and consumption of organic substrate was originally proposed by Monod241 and modified
by Herbert et al 242 This model takes the following form
^= "XY irh ~bX <4>
and
dS „ S
— = (jiX (5)
dt K + S
where X = microbial concentration (mg/£)> H- - maximum specific utilization rate (1 /day),
Y = microbial yield coefficient (g/g), S = limiting substrate (mg/
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Volume 18, Issue 1 (1988) 71
where S, 2 = substrates 1 and 2, K, 2 = half-saturation constants for substrates 1 and 2,
M'nu mj = maximum specific utilization rates for substrates 1 and 2, and a, 2 = inhibition
constants This model can be further extended to describe growth on multiple substrates
Some contaminants will not be used as a carbon and energy source by the microorganisms
but are transformed Schmidt et al 244 have shown that transformation of these compounds
is proportional to microbial population and contaminant concentration (C) where
dC
— = - KXC (9)
dt
Schmidt et al 244 used a logistic curve to describe the change in microbial concentration in
a batch system and developed a series of equations for describing change in contaminant
concentration with time for differing initial microbial and contaminant concentrations Use
of the logistic curve greatly simplifies the mathematical computations but does not allow
simulation of the effects of changing aquifer parameters such as the addition of a second
substrate In aquifer restoration, simulation of the microbial population using Equation 4
and change in contaminant concentration by Equation 9 may provide a more useful prediction
Various workers have suggested that the kinetics of microbial growth, decay, and con-
sumption of organic contaminants in the subsurface are best described by models which
include terms for transport into attached biofilms or microcolomes It is well known that
most microorganisms in the subsurface are attached to soil particles 10 This is thought to be
due to the competitive advantage attachment gives a microorganism at low substrate
concentrations 245
Workers at Stanford University have developed a series of models for simulating deg-
radation of organics in biofilms The basic model assumes that degradation within the biofilm
can be described by Monod kinetics Mass transport into the biofilm is by diffusion alone
The diffusive flux (J) is calculated from Fick's second law
dS.
J = -D—! (10)
dz
where Sf = concentration of rate-limiting substrate, z = coordinate orthogonal to biofilm,
and D = diffusivity Different dtffusivities are assigned to the biofilm and an effective
diffusion layer adjacent to the biofilm Williamson and McCarty246 originally developed this
model to simulate either substrate or oxygen limited biodegradation in wastewater treatment
biofilms Rittman and McCarty247 modified this approach to describe the steady-state biofilm
surrounding an injection well receiving tertiary-treated wastewater Bouwer and McCarty24"
have further expanded the approach to allow simulation of secondary utilization of trace
organics while the biofilm is supported by an undifferentiated COD Most recently, Kissel
et al 249 have employed the biofilm concept to model carbonaceous oxidation, nitrification,
and denitnfication within a mixed culture biofilm
Much of the work on biofilms at Stanford occurred as an outgrowth of field studies on
organic degradation near an injection well In this region, substrate fluxes and ground water
velocities will be high and a biofilm can be expected to develop Actual biofilms are rare
in most aquifers and the majority of the microorganisms are present as microcolomes Molz
et al 250 have recently modified the biofilm concept to describe the growth and decay of
microorganisms present in microcolomes An average colony radius and thickness is used
to describe the microcolomes Growth ard decay within the colony is simulated by Monod
kinetics and includes both oxygen and substrate limitation Transport to the microcolony is
limited by a diffusion layer at the colony surface
D. Modeling Subsurface Transport
Keely et al 251 present a concise overview of evolving concepts of subsurface contaminant
transport They argue that state-of-the-science methods may cost more at the outset but may
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CRC Critical Reviews in Environmental Control
yield overall benefits in the form of reduced cleanup costs compared with conventional
methods The authors make the point that ground water processes are difficult to understand
and to model due to interactions which may not be simple to describe Biotransformation
in the presence of dissolved oxygen in an aquifer represents an example where research
results may pave the way for reduced cleanup costs at many sites If more detailed data can
be obtained about potential pathways and mechanisms of transport, the state of the art will
be advanced along with the potential for less costly sue restoration
The major physical processes of importance in ground water transport are advection and
dispersion 2,2 Advection is the transport of a contaminant by the bulk ground water flow
Dispersion is the spreading of a contaminant front due to molecular diffusion and small-
scale variations in fluid velocity throughout the aquifer
The major chemical processes of interest are adsorption, ion exchange, hydrolysis, and
oxidation-reduction reactions Adsorption is "the process in which matter is extracted from
the solution phase and concentrated on the surface of the solid material "251
I Dispersion
Dispersion, the spreading of a contaminant front as it moves in the ground water, is an
area of particular controversy at this time The dispersion process can be most easily described
as consisting of three components (1) molecular diffusion resulting from Brownian motion
of individual molecules, (2) hydrodynamic dispersion resulting from variations in interstitial
pore velocities, and (3) macrodispersion resulting from structural variations in hydraulic
conductivity and, consequently, velocity Differences in permeability between layers can
result in different ground water velocities and large variations in solute concentration When
an aquifer is monitored using a fully screened well, ground water from different layers is
mixed, resulting in a smoothing of the apparent solute breakthrough curve This smoothing
can result in very large apparent dispersion when matched against simple two-dimensional
solute transport models
The effects of molecular diffusion, hydrodynamic dispersion, and macrodispersion are
frequently combined to form a dispersivity tensor which in some cases can be reduced to
three main components longitudinal dispersivity (a,), transverse dispersivity (a,), and vertical
dispersivity (av)
Dispersion coefficients (D) used in the advection dispersion equation are found by
D — avm (11)
where v is the resultant velocity scalar and m is assumed equal to 1
The physics and mathematics necessary to describe molecular diffusion and hydrodynamic
dispersion are well established Bear2M and Fried254 provide comprehensive experimental
and theoretical reviews of these processes The significance of the third component,
macrodispersion, is the subject of much debate Anderson255 summarizes much of the current
research on the nature and significance of macrodispersion At present, there appear to be
two dominant approaches
1 Macrodispersion occurs due to random variations in permeability which can never be
adequately characterized, consequently, the only reasonable method is to employ a
stochastic procedure for describing the average movement of a solute
2 The apparent spreading of many solute fronts is due to variations in permeability which
are complex but measureable, consequently, more effort should be expended towards
measuring the actual permeability distributions and using these as input for determin-
istic simulations
No work has yet been focused on the effect of varying aquifer parameters on solute
transport as it relates to biorestoration At present, the predictive accuracy of biorestoration
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Volume 18, Issue 1 (1988) 73
modeling is severely limited by uncertainties in solute transport simulations When simulating
the transport of a contaminant and oxygen or other nutrient, the most commonly used
numerical models will predict significant mixing between the contaminants and oxygen and
high rates of biodegradation In real aquifers, contaminants may be trapped in a few areas
of low permeability while the oxygen or other nutrients are forced through the high-permc-
ability zones In this situation, little mixing of the contaminants and oxygen will occur, and
consequently, little biodegradation Until solute transport models are developed which can
adequately describe the complexities of subsurface flow, the accuracy in which biorestoration
can be simulated will be limited
2 Chemical Processes
The major chemical processes which affect the transport of organic contaminants in ground
water are adsorption and hydrolysis
Adsorption is a surface process in which a compound "sticks" to the solid aquifer material
In the case of neutral, nonpolar organics, this stickiness is due to the much higher affinity
of the compound for other organics attached to the soil than for the polar water phase In
the case of polar molecules, adsorption may be due to dipole dipole forces The attraction
due to this mechanism is typically much weaker than that for hydrophobic compounds
Naturally occurring organic material in aquifers is commonly present as a humic-kerogen
film over the clay particles This organic material may originate from humic or fulvic acids
deposited with the original sediment or from infiltrating rain water Organic material is
effectively preserved in tight clays where diffusion of oxygen is limited and the redox
potential is low
Hydrolysis reactions can have a major impact on the mobility of organic compounds in
aquifers These reactions are typically pH dependent and catalyzed by metal ions The pH
of most solutions will approach equilibrium shortly after entering an aquifer In this case,
hydrolysis reactions can be modeled as a simple first-order decay process
When attempting to develop models for simulating the adsorption of contaminants in
ground water, many early investigators assumed that at low concentrations contaminants
move independently of other solutes, the reaction kinetics are fast relative to ground water
flow, and the natural reactants are uniformly distributed throughout the aquifer These
assumptions allow the reactions to be analyzed using the equilibrium isotherm approach
Under this approach, the variation in adsorbed contaminant concentration is described by
an adsorption isotherm
S - f(C) (12)
where C = concentration of the contaminant in solution and S = concentration in the
nonmobile solid phase This relationship can be incorporated into the advection dispersion
equation by considering the loss of solute to adsorption
If S is a linear function of C, then the effect of adsorption can be replaced by a constant
retardation factor (R) In this special case, the adsorbed contaminant will move according
to Equation 2 with an effective velocity, v', where v' = v/R and an effective dispersion
coefficient, D' = D/R A common method of calculating R is by the relation2"
R = 1 + p Kd/n (13)
where n is the aquifer porosity, p the bulk density, and Kd the partition coefficient in grams
of contaminant adsorbed per gram aquifer
The use of a retardation factor depends on the following assumptions
1 Adsorption can be described by a linear relationship between solute and solid phase
concentration
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74 CRC Critical Reviews in Environmental Control
2 The reaction kinetics are fast relative to ground water flow
3 Natural reactants attached to the aquifer material can be assumed uniformly distributed
in space
4 Contaminant transport is independent of other liquid phase organics
E. Mathematical Models of Subsurface Biorestoration
Equation 3 with terms included for biodegradation can be solved to obtain the concentration
of the contaminant in space and time by both analytical and numerical methods Analytical
solutions generally require less effort for the model user to employ, but may also require
simplification of the aquifer conditions and biodegradation processes For uniform flow in
an aquifer in which biodegradation may be approximated as a first-order decay, solute
concentrations in space and time can be calculated using the one-dimensional solution of
Ogata and Banks256 or the two-dimensional solution of Wilson and Miller 257 Approximating
biodegradation as a first-order decay would be appropriate when the microbial concentration
is constant, growth is only dependent on the contaminant concentration, and the contaminant
concentration is significantly less than the half-saturation constant (K) Simkins and
Alexander258 provide useful guidelines for determining when consumption of substrate may
be approximated as a first-order decay
Numerical solution of Equation 3 allows the user much more flexibility in specifying
aquifer geometry and biodegradation kinetics The most common mathematical formulations
for approximating the solute transport equation are finite differences, finite elements, and
the methods of characteristics
Finite difference models have been developed for a variety of field situations including
saturated and unsaturated flow and for transient and constant pollutant sources Finite dif-
ference methods operate by dividing space into rectilinear cells along the coordinate axes
Homogeneous values within each cell are represented by values at a single node Partial
differentials can then be approximated by finite differences and the resulting set of equations
solved by iteration 259 260 Approximating the differentials by a difference requires that the
remaining terms of the Taylor's expansion be dropped, resulting in a truncation error and
significant spreading of the simulated contaminant front This spreading has been termed
numerical dispersion and can often mask the actual physical dispersion process 219
The finite element method also operates by breaking the flow field into elements, but in
this case the elements may vary in size and shape In the case of a triangular element, the
geometry would be described by the three corner nodes where heads and concentrations are
computed The head or concentration within an element is allowed to vary in proportion to
the distance to these nodes Complex interpolating schemes are sometimes used to predict
accurately parameter values within an element and thereby reduce the truncation errors
common in finite difference procedures Some numerical dispersion may still occur but is
usually much less significant The use of variable size and shape elements also allows greater
flexibility in the analysis of moving boundary problems which occur when there is a moving
water table or when contaminant and flow transport must be analyzed as a coupled problem
A disadvantage of the finite element method is the greater mathematical complexity and
generally higher computing costs 261262
The method of characteristics (MOC) is most useful where solute transport is dominated
by convective transport One of the most commonly used models employs a procedure where
idealized particles are tracked through the flow field 261 In step one, a particle and associated
mass of contaminant is translated a certain distance according to the flow velocity The
second step adds on the effect of longitudinal and transverse dispersion and sources and
sinks for the contaminant
All of these techniques can be used to simulate in situ biorestoration under certain cir-
cumstances, although no single procedure will be applicable to every situation Only very
limited work has been done on simulating the simultaneous effects of advection, dispersion,
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Volume 18, Issue 1 (1988) 75
and chemical and biological processes In the following section, the few studies that have
been performed are reviewed and the potential weakness of each technique discussed
Kosson et al 264 ay a simple one-dimensional finite difference solution to simulate
the movement of h ous industrial wastewaters through an acclimated soil column Ad-
sorption is assumei e linear and is described by a retardation factor A portion of the
influent wastewater is assumed nondegradable Biodegradation of the remainder is simulated
as a first-order decay Experimental data are also provided by a field scale column used to
study the degradation process The model adequately matches experimental data from the
later portion of the column biodegradation test when an acclimated microbial population
has developed Agreement between model and experimental results was not as good during
the earlier part of the test before the microbial population had reached steady slate
Angelakis and Rolston265 present a mathematical model for simulating the movement of
insoluble (particulate) and soluble organic carbon through the unsaturated soil profile Trans-
formation from insoluble to soluble and finally to carbon dioxide is assumed to follow first-
order kinetics Transport of carbon dioxide is by gaseous diffusion Simulation results are
obtained from analytical and numerical solutions These results compare favorably with
experimental data from a series of column tests performed using primary wastewater effluent
Insoluble and soluble organic carbon distributions were adequately matched A variable
gaseous diffusion coefficient was required to match the observed carbon dioxide distribution
Baehr and Corapcioglu26" present a one-dimensional model for simulating gasoline trans-
port in the unsaturated zone which includes transport by air, water, and free hydrocarbon
phases The hydrocarbon is assumed to be composed of "n" components of differing
solubility and volatility Exchange between the air, water, hydrocarbon, and adsorbed phases
is assumed to be rapid and described by equilibrium partition coefficients Biodegradation
of the hydrocarbon is limited by the availability of oxygen which can enter the soil dissolved
in the water phase or by gaseous diffusion Microorganism growth was not simulated directly
since biodegradation was assumed to be rapid relative to mass transport and to be limited
by the availability of oxygen The equations are solved numerically using a finite difference
procedure Model simulations indicated that the rate of biodegradation was very sensitive
to (he diffusive properties of the soil No experimental data are presented to test the model
predictions
Sykes et al 2b7 simulate the anaerobic degradation of a landfill leachate plume in the
saturated zone at the Canadian Forces Base in Borden, Ontario Microbial growth, decay,
and substrate utilization are simulated using Monod kinetics When substrate concentrations
are significantly below the half-saturation constant and microbial populations are close to
steady state, the biodegradation kinetics are reduced to a first-order decay The nonlinear
equations are generated using a Galerkin finite element approximation and solved using a
Newton Raphson iteration procedure Model simulations indicate that the majority of the
degradable organics can be expected to be removed within a few meters of the landfill This
finding was confirmed in field studies at the site Sensitivity analyses performed using a
one-dimensional solution indicated that, under certain circumstances, pulses of organics
could escape from the landfill before a significant microbial population had developed
Molz et al 250 present a numerical model for simulating substrate and oxygen transport
and use by attached microorganisms The microbial population is assumed to be immobile
and present in microcolonies of an average thickness and radius Transport into the micro-
colonies of oxygen and substrate is limited by diffusion through a stagnant layer adjacent
to the microcolony Microbial growth and consumption of oxygen and substrate within the
microcolony is described by Monod kinetics A one-dimensional solution is obtained nu-
merically using an Eulenan-Lagrangian finite element solution The numerical model will
then be used to simulate the transport and biodegradation of substrate and oxygen in a
laboratory column The simulation results indicate that degradation is most rapid near the
column inlet The initial microbial population has a significant effect on the simulated
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76 CRC Critical Reviews in Environmental Control
breakthrough at the beginning of the simulation but has little effect on the steady-state
substrate distribution Large substrate loadings at the column inlet quickly exceed the avail-
able oxygen supply, resulting in anaerobic conditions throughout the majority of the column
Laboratory testing of the simulation model is planned
Borden and Bedient140 present a numerical model of oxygen-limited biodegradation of
hydrocarbons in the saturated zone Numerical solutions are obtained by approximating one-
dimensional flow as a series of completely mixed reactors and two-dimensional flow using
an explicit finite difference solution corrected for numerical dispersion One-dimensional
model simulations indicate that biodegradation will be very rapid near the contaminant source
when oxygen is present When no oxygen is present at the source, biodegradation will be
slow and limited by the transport of oxygen into the contaminant plume Two-dimensional
simulations indicate that horizontal and vertical mixing are the major sources of oxygen to
the contaminant plume and control the biodegradation process When adsorption of the
hydrocarbon to the aquifer is significant, advective fluxes of oxygen into the plume and
resulting biodegradation is also significant Sensitivity analyses with the model suggest that
for many aquifers the reaction between oxygen and hydrocarbon may be approximated as
an instantaneous reaction since oxygen transport is rate limiting Borden et al 141 employ
these results to modify the U S G S Solute Transport Model261 to simulate oxygen-linnted
biodegradation of creosote wastes at a Superfund site The model gave an adequate description
of the observed oxygen and hydrocarbon distributions in the shallow aquifer at the site and
was used to study various remedial actions, including no action and removal of the contam-
inant source
Dawson et al 26K modify a petroleum reservoir code to simulate enhanced in situ biores-
toration using the equations presented by Borden and Bedient 140 Advective and dispersive
transport is calculated using a finite element-modified MOC solution which allows a large
time step and strongly advection-dominated flow Because the rates of biodegradation can
be very high relative to transport, a time-splitting scheme is employed where the microbial
kinetic terms are solved separately using an implicit solution with a much smaller time step
This model is then employed to simulate enhanced in situ biorestoration by the injection of
oxygen and production of contaminated water by a five-spot pattern Simulations are per-
formed for a variety of conditions including uniform and random permeabilities and variable
adsorption
F. Model Use and Limitations
The current technology for simulating subsurface biorestoration is still in its infancy Some
progress has been made in developing kinetic descriptions of the biodegradation process and
combining these with available solute transport models Unfortunately, little reliable field
data have been available to rigorously test these models Considerable uncertainty exists
over the importance of simulating transport into biofilms or microcolonies Also, the effects
of variations in aquifer parameters on the efficiency of biorestoration are unknown At
present, the technology is not available to quantitatively predict the efficiency of enhanced
biorestoration, but significant advances are being made in our ability to describe the process
V CONCLUSIONS
Of the available biological aquifer remediation techniques, the most effective demonstrated
methods are enhancement of the native population and withdrawal and treatment by various
wastewater treatment processes 269 Before any aquifer remediation technique can be imple-
mented, a thorough understanding of the hydrogeology and contamination problems of the
site must be obtained and used to design the treatment system 4 When successful, costs for
in situ biorestoration are generally less than physical or chemical remediation techniques
In situ biorestoration compares favorably with other common remedial actions such as
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Volume 18, Issue 1 (1988) 77
excavation and withdrawal and treatment by carbon adsorption or air stripping 87 Excavation
is generally limited to surface soils above the water table and can be restricted by the physical
construction on the site Excavated soils must be transported to a secure landfill with the
potential for liability from an accident during transport or a leak from the receiving landfill
The contaminated ground water must be withdrawn before air stripping and carbon adsorp-
tion The ability to withdraw organic contaminants is a function of the solubility of contam-
inants and the permeability of the soil Highly soluble organics in high-permeability soils
can be effectively withdrawn Compounds which are hydrophobic can be effectively treated
with carbon adsorption, but because of their hydrophobicity are also more likely to be
adsorbed to the soil and not removed by withdrawal systems Carbon adsorption generates
spent carbon that must be treated or sent to proper disposal facilities Air stripping is effective
for compounds with high volatilities, but not those with high boiling points or high solu-
bilities Air stripping simply transfers the contaminants to another phase of the environment
unless expensive carbon adsorption or incineration is used to treat the off gases
Addition of oxygen, nitrogen, phosphorus, and trace minerals stimulates the acclimated
indigenous microbial population to aerobically degrade many subsurface contaminants In
suit biorestoration has been chiefly used to treat gasoline-contaminated aquifers, but also
has been employed with ethylene glycol and solvents including acetone, tetrahydrofuran,
methylene chloride, n-butanol, dimethyl aniline, and isopropanol Biorestoration effective-
ness will be affected by toxic levels of organics and heavy metals In general, in situ
bioreclamation has been effective in reducing the quantity of the contaminants but not in
completely eliminating them The treatment moves with the plume, allowing treatment of
trapped or sorbed contaminants, or by using soil flushing or an infiltration gallery, m situ
microbial treatment can reach areas that are not accessible by other techniques Biorestoration
has been used in a number of aquifers, but may be of limited usefulness in those with low
permeabilities Undesirable metabolic and inorganic nutrients may escape from the treatment
zone and affect ground water or surface water quality Alternative oxygen sources such as
ozone, hydrogen peroxide, pure oxygen, and air flooding or soil venting may speed the
removal of the organic contaminants, but their impact on the microbial population and the
geochemistry of the site is not fully understood Innovative processes such as treatment beds
or land treatment can be used in some situations In the presence of an acclimated microbial
population, many aquifers will be anaerobic because the microorganisms will have depleted
the dissolved oxygen It will be possible to use anaerobic degradation to remove contami-
nants, although the technology for this treatment has not yet been developed Reducing the
interfacial tension between the hydrocarbon and ground water with surfactants, dispersants,
or emulsifiers will mobilize the contaminants and may make them available for microbial
degradation Combinations of in situ biorestoration treatment with other chemical, physical,
or biological treatment processes have been successfully utilized in aquifer remediation
Treatment at the surface by biological wastewater processes is a proven technology The
biological processes include activated sludge, lagoons, waste stabilization ponds, fluidized
bed reactors, trickling filters, rotating biological discs, and sequencing batch reactors All
of these processes are dependent upon extraction of the contaminated ground water from
the subsurface Combinations of conventional wastewater treatment processes and other
water treatment processes have also been successful
Alteration of the subsurface microbial community has a great deal of potential to allow
degradation of recalcitrant compounds in the subsurface The organisms are selected by
enrichment cultunng or genetic manipulation However, introduction of non-native micro-
organisms may be limited by movement of the organisms through the subsurface, survival
of the organisms, and accessibility of the organic contaminants Addition of an acclimated
population may be more successful when combined with wastewater treatment processes
where the environment can be more closely regulated Although the aquifer remedial actions
that have used a microbial seed have not conclusively shown that the added organisms were
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CRC Critical Reviews in Environmental Control
responsible for removal of the contaminant, the concentrations of the contaminants were
reduced Alteration of the environment to promote the activity of a particular component of
the microbial community is another promising technology Field tests and further research
are currently underway for this technology The environment is altered to promote the growth
of organisms that co-oxidize halogenated aliphatics when supported on gaseous hydrocarbons
such as methane, propane, or natural gas
Techniques for simulating the subsurface biorestoration process are under development,
but little reliable field data have been generated that can be applied to these models Some
of the major considerations in simulating transport and biodcgradation of organic contam-
inants in the subsurface are poorly understood, these include the importance of transport of
organics to the bacteria and the variability in aquifer parameters
ACKNOWLEDGMENT
Although the research described in this article has been supported by the U S Environ-
mental Protection Agency through Assistance Agreement No CR-812808 to Rice University,
it has not been subjected to Agency review and therefore does not necessarily reflect the
views of the Agency and no official endorsement should be inferred
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14 Stetzenbach, L. D , Kelly, L M., and Sinclair, N A , Isolation, identification, and growth of well water
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15 Smith, G A , Nickels, J S , Daws, J D , Findlay, R H , Yashio, P. S , Wilson, J T., and White,
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20 Hoos, E. and Schweisfurth, R , Investigations about the distribution of bacteria between 10 and 90 meters
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265 Angelakis, A N and Rolston, D. E., Transient movement and transformation of carbon species in soil
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