PERFORMANCE STANDARDS FOR
IN SITU
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-* the role
CE E. RITTMAN Northwestern University Evanston, n 60208
the next. Without the activity of microorganisms in natuy, the Earth would be buried in wastes, and the nutrients necessary for the continuation of life would be locked up in detritus. The objective of in situ bioremediation is to focus that kind of natural microbial activity on lo-
FIGURE 1
Engineered bioremediation systems for cleaning up contaminated groundwater and soil (a) A system fortreating Boil above the water table (bioventing)
below
n
buccessful in situ bioremediation requires evidence correlating a decrease in contaminant concentration with microbial activity in the field, system, called bioventing, provides oxygen via vacuum pumps and nutrients via an infiltration gallery. The middle figure (b) and bottom figure (c) show systems for treating regions above and below the water table. The first system (b) distributes oxygen in the form of aqueous hydrogen peroxide through a well and an infiltration gallery and provides nutrients in aqueous solution through these same devices. The second system (c), called air sparging, provides oxygen by injecting air directly into the groundwater and distributes nutrients via an infiltration gallery. The main reason for choosing an engineered bioremediation system is to accelerate the biodegradation rate to minimize the cleanup time. On the other hand, intrinsic systems allow contaminant degradation to proceed without human enhancement, although the site must be carefully monitored to ensure t h a t t h e c o n t a m i n a t i o n is not spreading. Intrinsic bioremediation is appropriate when the intrinsic (or natural) biodegradation rate is fast enough to prevent migration of contaminants away from their source. When i n situ bioremediation works, it offers several advantages over other groundwater and soil cleanup technologies. Conventional methods for groundwater cleanup require pumping the water to the surface for treatment. Similarly, conventional methods for soil cleanup require digging up the contaminated soil and either incinerating it or transporting it to a specially designed disposal site. Pumping, excavation, and incineration require large energy inputs and are therefore costly. By minimizing pumping requirements and eliminating excavation and incineration, in situ bioremediation can reduce cleanup costs. In addition, by treating the contaminants in place, in situ bioremediation reduces human and environmental exposure. Further, for contaminated groundwater, in situ bioremediation can reduce cleanup time by destroying the contaminants close to their source instead of requiring contam-
inant dissolution and extraction before treatment. A multidisciplinary technology Despite its potential advantages, in situ bioremediation is poorly understood-and often mistrusted-by many regulators who oversee cleanups and by procurement staff in industries faced with contamination problems. As a result, some regulators and technology procurement staff approach bioremediation with skepticism, opting for more conventional technologies even when bioremediation is the most appropriate technology for a particular site. One important reason for the mistrust and misunderstanding is the lack of interdisciplinary knowledge required to fully understand bioremediation. Bioremediation requires comprehension not only of fields such as environmental engineering and hydrology, which are important in designing conventional cleanup systems, but also of microbiology. To assess the suitability of bioremediation for cleaning up a particular site, one must understand how microorganisms degrade contaminants. Both the nature of the contaminants and the environmental characteristics of the site influence the likelihood that a bioremediation project will succeed. For example, some contaminants, such as petroleum hydrocarbons, are destroyed only by organisms that require oxygen. Other contaminants, such as PCBs, are best destroyed by organisms that function when oxygen is absent. In addition, all microorganisms require elemental nutrients such as nitrogen and phosphorus in order to function. If not present naturally, the bioremediation system must supply these substances. Microbial metabolism is also sensitive to changes in water chemistry, such as increases or decreases in pH, and the bioremediation system must account for this sensitivity. Without understanding how microorganisms and contaminants interact under specific environmental conditions, one cannot evaluate a bioremediation project with any degree of certainty. For in situ ground-
1976 Environ. Sci. Technol., Vol. 27, No. 10, 1993
water and soil cleanup, the evaluation of bioremediation systems is made more difficult by the complex nature of the subsurface and the difficulty of observing it. “In Situ Bioremediation: When Does It Work?” reviews characteristics of microorganisms, contaminants, and the environment that are important in designing subsurface bioremediation systems; by explaining and connecting these characteristics, the report should increase understanding of this technology. Evaluating in situ bioremediation Commonly, bioremediation projects have been evaluated simply by determining whether (a) the contaminant levels are decreasing and (b) microorganisms from the site have the capability to metabolize the contaminant when removed to the laboratory. This type of evaluation is inadequate for two reasons. First, it fails to account for the way in which specific conditions at the site influence microbial activity. That microbes removed to the lab do not always behave the same as those in the less hospitable field conditions has been well established. Second, it fails to account for whether abiotic processes, such as volatilization or physical transport off site, may cause the reduction in contaminant concentrations. Although other processes may contribute to site cleanup during bioremediation, the microbes should be critical in meeting cleanup goals. “In Situ Bioremediation: When Does It Work?” recommends a strategy for evaluating bioremediation projects that is premised upon evidence of microbial activity at the field site itself. The strategy requires gathering the following three types of evidence: documented loss of contaminants from the site, laboratory assays showing that microorganisms in site samples have the potential to transform the contaminants under the expected site conditions, and one or more pieces of evidence showing that the biodegradation potential is realized in the field.
Techniques for demonstrating biodegradation in the fbld Techniqw
purpose
I m p l . m t m o n mahods
MeaauremenW oi tlbld samples To determine whether the number of contaminant- Standard and emerging cell-counting techniques degrading bacteria has increased over usual field from microbiology conditions Number of protozoans To determine whether the population of protozoans Standard microbiologicaltechniques for counting protozoans. including the most-probable-number that prey on bacteria has increased techniaue Rates of bacterial TO estimate the potential rates at which bacteria Determhation of Contaminant loss rate and other activity from the field can degrade the contaminant relevant markers of biodegdation in laboratory microcosmscontainina fie samDles Bacterial adaptation To assess whether bacteria from the contaminated Microcosm studies &;ore and &er tho initiation d site can metabolize the contaminant more quickly bioremediation or using samples from the than before bioremediation &an bioremediation zone and contaminated areas outside the bioremediationzone; gene probing to analyze changes in the bacteria's genetic makeup Inorganic carbon To determine whether the inorganic carbon Gas chromatography for determining gaseous concentration concantration in subsurface samples has increased, carbon dioxide; inorganic carbon anaiysis for indicating possible conversion of contaminants to determining aqueous species inorganic carbon Carbon isotope ratios To evaluate whether the inorganic carbon at the site Measurement of the '%Ii2C ratio using a mass originates tram contaminant biodegradation spectrometer Electron acceptor To determine decreases in concentrations of Oz or Standard analylical methods from wet chemistry concentration other electron acceptors used during biodegradation BYPmdUCts Of, In oxygen-depleted environments, to determine Standard chemical analylical methads anaerobic actnrlty whether byproducts of anaerobic metabolismsuch as methane, sulfides, reduced forms of metals, and nitrogen gas have accumulated Intermediary metabolite To determine the presence of intermediary Gas chromatography, high-performanceliquid formation metabolites unique to the biodegradationof a chromatography. andlor mass spectrometry particular contaminant Ratio of nondegradable To analyze whether the ratio of nonbiodegradabb standard chemical analyfical methads to degradable to degradabie components of a contaminant has substances increased Number of bacteria
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Experiments run In the Reld Comparison of amendad and unamended sukites Ta estaMish whether the contaminant 105s rate increases when growlh-stimulating materials are within the contaminated area added to the site Measuring the electron To estimate the rate of consumption of oxygen or Measurement of the rate at which oxygen (or acceptor uptake rate ather electron acceptors necessary for contaminant anather electran acceptor) is consumed once the metabolism Now of the matenal is stopped (e.g., using an oxvaen -or&\ ~---, Monitorinq To distinguish abiotic contaminant losses from Comparison of the fate of nondegradable tracer conservative tracers losses due to biodegradation compounds to the fate of the degradable contaminant cebeling contaminants To determine me fate of carbon contained in Monitoring of '%-labeled versions of the organic contaminants contaminant Stimulating bacteria within subsites
~
Modeling mass losses Direct modeling
MDdellng experlmenta To analyze whether abiotic mechanisms (e.g., dilution, transport. vdatilization)can explain all of the losses of the contaminant mass To estimate the biodegradation rate
_'he first t y p e o f evidence in the strategy-howing decreasing cont a m i n a n t concentrations-comes f r o m s t a n d a r d s a m p l i n g of t h e g o u n d w a t e r and s o i l over t i m e as c l e a n u p progresses. T h e s e c o n d type of evidence-showing the potential for microorganisms t o degrade t h e contaminants-requires taking microbes from the f i e l d and showing that they can degrade the contaminant w h e n grown in a w e l l c o n t r o l l e d l a b o r a t o r y vessel. For some cases, l a b studies m a y n o t be needed when a b o d y of peer-
Use of mathematicalmodels to represent abiotic loss mechanisms and comparison of the result with
the contaminant loss rate in the field Use of mathematicalmodels to directly estimate the biodegradationrate and compare it mth observations from the field
r e v i e w e d p u b l i s h e d studies do ments t h a t the compounds are easi l y a n d c o m m o n l y biodegraded. The third type of evidence-showing t h a t biodegradation p o t e n t i a l demonstrated in t h e laboratory i s being realized in the field-can be p r o v i d e d u s i n g a variety o f techniques, examples o f w h i c h are l i s t e d in Table 1. This third type of evidence i s the most difficult to nrovide but i s also the most esseGtia1 because it establishes t h e link between laboratorv evidence that microbes can degrade the contaminant
I
and field evidence that the contaminant i s disappearing. T h e goals o f using the techniques listed in Table 1 are, first, t o show that characteristics of t h e site's chemistry or microb i a l population change in ways that one w o u l d p r e d i c t i f bioremediat i o n were occurring and, second, to correlate these chemical and microbial changes with documented contaminant loss over time.
*PPI Y b the evaluation strat%Y "In S i t u Bioremediation: When Does It Work?" identifies several
Envimn. Sci. Technol., Vol. 27, No. 10, 1993 1977
sites around the country where the types of data needed for the threepart evaluation have been gathered. Two of these are a Colorado industrial site (3) and a Michigan natural gas manufacturing plant ( 4 ) . At the Colorado site, a fuel holding tank under a garage leaked, creating a plume of benzene, toluene, ethylbenzene, and xylene (BTEX)in the groundwater below. An engineered bioremediation system was installed at the site in 1989.The system promoted bioremediation of the groundwater by circulating oxygen, phosphorus, and nitrogen to stimulate microbial growth. After three years of treatment, the plume of dissolved contaminants was nearly eliminated. Tests revealed that the aquifer contained a small layer that had trapped considerable quantities of BTEX. This layer is relatively impermeable and therefore had been bypassed by the fluids circulated to promote bioremediation. However, it is likely that microbes at the periphery of the remaining contamination will provide effective intrinsic bioremediation that will prevent the reemergence of a contaminant plume even though the engineered system has been shut down. Data collected during the course of the engineered bioremediation effort meet the following criteria of the three-part evaluation strategy. Documented loss of contaminants. At the monitoring well closest to the gallery used to deliver oxygen and nutrients to the site, the BTEX concentration dropped from 2030 pg/L before bioremediation to 6 pg/L after bioremediation. At other monitoring wells, the concentration dropped more than an order of magnitude, to < 46 pg/L. Laboratory assays showing that microorganisms have the potential to degrade the contaminants. Studies showed that microorganisms in the transmissive layers adjacent to the trapped contaminants could consume as much as 7 mg/L of oxygen per day, resulting in the potential destruction of as much as 2 mglL of hydrocarbons Fer day. This oxygen consumption rate was determined by placing a dewatered core from the site in a sealed glass Mason jar and measuring the amount of oxygen the microbes in the core consumed in 24 h. No direct testsother than measuring the oxygen consumption rate-of the native microbes' ability to degrade BTEX were performed. However, the ability of subsurface microorganisms to degrade BTEX is well established,
so direct lab tests were less impor-
tant for this site than for sites with contaminants for which bioremediation techniques are still emerging. Evidence that biodegradation potential is realized in the field. At this site, two types of tests provided evidence of biodegradation in the field. First, the oxygen consumption rate in microcosms constructed with cores from the site was highest when the cores came from near the layer of trapped contaminants. Thus, microbes with access to the largest supply of contaminants consumed oxygen most rapidly, supporting the expectation that bacterial growth on the hydrocarbons had been stimulated. Second, the ratio of BTEX to total petroleum hydrocarbons (TPHs) was lower in the bioremediated area than in the contaminant source. Research has shown that microorganisms prefer BTEX to other components of TPH, leaving a TPH residual that is relatively low in BTEX after a successful remediation. At the Michigan site, waste products from natural gas manufacturing leaked from a disposal pit into the surrounding groundwater. Having installed wells around the plant to prevent off-site migration of contaminated water, the company in charge of the facility chose intrinsic bioremediation to clean up the contaminants (primarily benzene, toluene, and xylene, or BTX). In monitoring this site, the company has gathered data that meet the following three criteria of the evaluation strategy. Documented loss of contaminants. The company began its extensive program in 1987 to monitor the effectiveness of intrinsic bioremediation. Since then the benzene concentration has dropped by nearly go%, and the contaminant plume has shrunk considerably. Laboratory assays showing that microorganisms have the potential to degrade the contaminants. The company performed a series of lab tests with soil cores retrieved from the field showing that the site's native microbes could degrade BTX at a high rate (5-10% per day) if supplied with adequate oxygen (12 ppm or more). Evidence showing that hiodegradation potential is realized in the field. The company used a computer-based model, BIOPLUME 11, to demonstrate that the rate of contaminant loss one would predict if bioremediation were occurring closely matched the actual contaminant loss rate in the field. In 1987
1978 Environ. Sci. Technol., Vol. 27, No. 10, 1993
the company measured the BTX and dissolved oxygen levels at various points in the plume. These values were input into BIOPLUME I1 to predict how they should change with time if bioremediation were occurring. The field measurements of both the contaminant concentrations and the dissolved oxygen levels taken since 1967 closely match the model's predictions. In addition, the biodegradation rate predicted by the model closely matches the rate measured in the field. Monitoring at this site is ongoing to demonstrate intrinsic biorernediation's long-term effectiveness. Conclusions Whether an in situ bioremediation project is in the design or implementation phase, demonstrating its effectiveness requires more than showing that the contaminant is biodegradable under laboratory conditions. Successful in situ biore-
Jacqueline A. MacDonold directed the study on which "In Situ Bioremediation: When Does It Work?" was bosed. She is a stoff officer for the Notional Research Council's Water Science and Technology Board. She holds o B.A. degree in pure mathematics from Bryn M a w College and on M.S. degree in environmento1 science in civil engineering from the University of Illinois.
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Bruce E. Ritbnann chaired the Committee on In Situ Bioremediotion, which outhored "In Situ Bioremediotion: When Does It Work? Rittmonn is lohn Evans Professor of Environmental Engineering at Northwestern University and on octive researcher and teacher in the field of environmental biotechnology. He holds a Ph.D. in environmental engineering from Stanford University.
mediation requires evidence correlating a decrease in contaminant concentration with microbial activity in the field. The three-part strategy described in the report “In Situ Bioremediation: When Does It Work?” provides a framework for presenting such evidence to those responsible for evaluating bioremediation projects. In implementing this evaluation strategy, one must recognize that only under rare circumstances is proof of in situ bioremediation unequivocal. In the majority of cases the complexities of contaminant mixtures, their hydrogeochemical settings, and competing abiotic mechanisms of contaminant loss make it a challenge to identify biodegradation processes. Unlike controlled laboratory experiments in which measurements can usually be interpreted easily, cause-and-effect relationships are often difficult to establish at field sites. The three-part evaluation strategy relies on building a consistent, logical case from convergent lines of independent evidence. The degree of rigor with which the evidence is gathered depends on the needs of those who are evaluating the site-whether they are researchers requiring extensive data to prove their hypotheses, clients desiring the cleanup to be cost-effective, or regulators wanting to ensure protection of public health. The wider the variety of techniques used in carrying out the strategy, the stronger the case for successful bioremediation.
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Acknowledgment
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The other members of the Committee on In Situ Bioremediation are Lisa AlvarezCohen, Philip Bedient, Richard Brown, Francis Chapelle, Peter Kitanidis, Eugene Madsen, William Mahaffey, Robert Norris, Joseph Salanitro, John Shauver, James Tiedje, John Wilson, and Ralph Wolfe. Special recognition goes to Eugene Madsen, the committee’s rapporteur, who prepared the first draft of its report. Thanks also to Gregory Nyce, who efficiently managed production of the final manuscript.
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References (1) Rittmann, B. E. et al. A Critical Re-
view of In Situ Bioremediation; Gas Research Institute: Chicago, 1992. (2) National Research Council. “In Situ B i o r e m e d i a t i o n : W h e n Does It Work?”; National Academy Press: Washington, DC, 1993. (3) Nelson, C.: Hicks, R. J.; Andrews, S. D. In Bioremediation: Field Experiences: Flathman, P. E.; Jerger, D. E.; Exner, J. H., Eds.; Lewis: Chelsea, MI, in press. (4) Chiang, C. Y. et al. Groundwater 1989, 27, 823-34.
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