1662 Environ. Sci. Technol., Vol. 25, No. 10. 1991
0013-936W91/0925-1662$02.50/0@ 1991 American Chemical Society
Determinimi in situ FACTS AND CHALLENGES Contamination of water, soil, and sediment by toxic organic chemicals is widespread and frequent. Incidents range from industrial chemical waste contamination at Love Canal, NY, ( 1 ) to halogenated hydrocarbons and pesticides in groundwater (2, 3 ) to oil spills in Prince William Sound, AK, ( 4 , 5) and the Persian Gulf. In situ biodegradation processes are gaining attention because of the role that microorganisms may play in alleviating environmental pollution (68). Decades of academic, industrial, a n d federal microbiological research have contributed to knowledge of biodegradation-a field that strives to span the genetics, biochemistry, physiology, and ecology of microorganisms, as well as soil science, limnology, and environmental chemistry and engineering. Biodegradation processes are frequently exploited by mankind. In fact, they are the basis of conventional engineering techniques for wastewater treatment (9). Because biodegradation reactions may break intramolecular bonds of organic compounds, their toxicity a n d transport properties are commonly altered. Often biodegradation will completely destroy, or mineralize, organic compounds (see “Definitions of Terms” box). The degradation of naturally occurring organic compounds by microorganisms in situ is of critical importance for the successful functioning of the biosphere. By enzymatically attacking high-molecularweight biomass (such as plant materials) in terrestrial and aquatic ecosystems, microorganisms complete the cycling of carbon begun by photosynthesis. Furthermore, nutrient elements N, P, S, and so forth are released during the decay of car-
Eugene L. Madsen Cornell University Ithaca, NY 14853-8101
bonaceous materials and are made available to subsequent generations of growing biota. The maintenance of the biosphere provided by microorganisms over evolutionary time is testimony to their effectiveness i n catalyzing these ecologically essential biogeochemical reactions ( 1 6 - 1 9 ) . Bioremediation strategies for pollution control merely bring the broad biodegradative capabilities of microorganisms that have been proven by evolution into focus on a cluster of organic compounds deemed undesirable in the 20th (and probably 21st) centuries. Recent usage of the terms in situ bioremediation and environment01 biotechnology in both scientific and popular literature (6-9, 20-24) implies that knowledge of biodegradation has produced one or more reliable technologies that are fully operable and able to eliminate pollutants from contaminated sites. But the fundamental progression from rigorous pure science to problem-oriented applied science to bench-, pilot-, and full-scale engineering has rarely been completed for in situ bioremediation. Indeed, the subject of this article, determination of in situ biodegradationwhich is pivotal for verifying the efficacy of all putative “ i n s i t u microbiological technologies”-is an imperfect science, not a routine assay. Both spontaneous and commercially managed in situ biodegradation processes promise to play a major role in alleviating society’s environmental problems. Yet scientific efforts to verify the in situ bio-
degradative activities of microorganisms continue to be plagued by major methodological limitations. This article will critically evaluate many issues pertinent to in situ biodegradation by confronting its ambiguities a n d delineating its strengths and weaknesses. Scenarios and limitations The difficulty of proving in situ biodegradation may be illustrated by a simple but nontrivial example: How do we know that microorganisms are responsible for the decay of a fallen tree in the forest? Assuming the tree had not been diseased prior to falling, the only initial sign of the imminent decay is its horizontal orientation. But the tree gradually loses its integrity as its largely cellulosic and ligninic tissues are colonized by characteristic white-rot and brown-rot fungi (25,26), among other microorganisms. Independent laboratory tests have shown that a characteristic sequence in woody tissue deterioration occurs only in the presence of microorganisms that uniquely produce cellulase and ligninase enzymes (25, 26). Repeated field observations of these same microorganisms in decaying logs undergoing a similar sequence of deterioration events help to erect a convincing argument that the microorganisms are responsible for the decay. Of course, at a field site other higher organisms (lichens, mosses, insects, animals, etc.) as well as climatic influences physically disturb the decaying tree, thereby accelerating the eventual conversion of a portion of its biomass to CO, and incorporation of the remainder into the forest floor (25). In situ microbiological degradation of a fallen tree is inferred from
Environ. Sci. Technol., VoI. 25, NO. 10. 1991
1663
Definitionsof term
rest, biotic reactions may include uptake and metabolism o
of cometabolism ( 1 1). situ biodegradatlon. In Latin, in situ means “in its original place egradation focuses on activating microbial processes for the destr
of an organic molecule into its ino , PO,3-). Mineralization occurs wh ai catabolic and anabolic cellular
several convergent lines of indeper. dent evidence: a spatially distinct organic substrate (wood residing on the soil surface] is nearly completely eliminated because of alterations in its physical and chemical _ . structure: as the biomass of the wood disanpears, a portion is replaced by t6e growing biomass of microorganisms (and, later, by a succession of other organisms); and
1
1664 Environ. Sci. Technol., Vol. 25. No. 10,1991
some microorganisms, notably key fungi, aremorphologically recognizable, and their repeated occurrence on rotting trees and well-characterized biochemical capabilities implicate their participation in the decay process. Thus, the mass of the organic substrate (wood) diminishes, and microorganisms are the causative agents: In situ biodegradation has occurred. Let us now contrast the forest tree
scenario with a reasonably common environmental pollution event: contamination of soil and groundwater by gasoline. As with the fallen tree, proof of in situ biodegradation must show that the mass of the gasoline has decreased and that microorganisms are the causative agents. But these two pieces of information are exceedingly difficult to obtain. First, unlike the fallen tree, the gasoline does not remain spatially separated from soil and groundwater. Instead, gasoline infiltrates the porous soil matrix, partially dissolving into a moving aqueous phase and otherwise being dispersed, diluted, sorbed, or volatilized. Thus, mass balances may be unattainable because a variety of intractable abiotic processes contribute to the gasoline’s disappearance. Second, even if in situ gasoline biodegradation were occurring, there are no morphologically distinctive gasoline-degrading microorganisms whose growth can be readily discerned within the complex, heterogeneous soil and groundwater enviroGments. There are typically 10’ to 10 indigenous microorganisms per gram of soil and groundwater sediment, respectively (27-29).Many of these exist as spores or other resting stages so that their presence (as determined by direct microscopic counts or by viable counts on agar media requiring microbial growth) has little bearing on in situ microbial activity (26,29-32). With neither a means to account for the spilled gasoline nor a means to assess microbiological growth in response to the gasoline, in situ biodegradation cannot be demonstrated. The tasks of assembling mass balances at field sites and distinguishing biotic from abiotic attenuating processes are genuinely formidable. Many field-oriented microbiologists have confronted the situation and concluded that surmounting such adversities may not be possible (11, 33-36). The common way to contend with the uncertainties of in situ biodegradation is to initiate a flask assa in the laboratory which monitors CO, evolution from a radiolabeled pollutant compound (or one or more equivalent measures of chemical transformation) added to a sample of water, soil, or sediment gathered from a field site. Larger, more elaborate “model ecosystem” tests have also been developed to investigate both biodegradation and the partitioning of chemicals between various components of a given habitat (37-41).
L
Laboratory assays provide definitive evidence for microbial metabolism because sterilized treatments can be examined as abiotic controls, and mass balances are made possible by performing the assay in a sealed vessel (Figure 1, top). Indeed, this type of controlled laboratory experiment (using environmental samples, individual microbial cultures, or fractionated cellular components) bas provided the majority of information presently available on genetic (42). physiological ( I 7,43-451, biochemical (46, 471, kinetic (481, and ecological aspects (10,38, 41) of the biodegradation of organic compounds. Such
techniques are powerful because of the control attained in the laboratory and the use of experimental designs that can address specific hypotheses. But these benefits are counterbalanced by methodological artifacts that detract from the realism of resultant measurements. Environmental microbiologists must contend with their own version of the Heisenberg Uncertainty Principle (36): the closer a given process is examined, the more likely it is that artifacts will be imposed on measurements of that process. In situ biodegradation reflects a delicate and intricate balance of nutritional, physical, and biological rela-
tionships that can change rapidly in response to environmental factors such as oxygen, water, pH, and nutrients 115). When a sample of water, soil, or sediment is removed from the field, it cannot be assumed that the physiological status of the accompanying microorganisms is unaltered. Indeed, it has been shown that determination of microbial activity in disturbed, displaced environmental samples incubated in the laboratory is likely to be quantitatively, even qualitatively, different from the same determination in situ (49-52). The problems of extrapolating from laboratory data to the field have never been solved
Comparison of typical biodegradation experiments perf flasks and in replicated, controlled-releasefield plots
at control treatment 100% recovery Actual control treatment. Small abiotic I
plicated field plots
Environ. Sci. Technol., Vol. 25, No. 10, 1991 1665
-
despite decades of debate ( 5 3 . 5 4 ) . The specific scenarios discussed above have shown that under some circumstances in situ biodegradation can (e.g., the fallen tree) or cannot (e.g., the gasoline contamination) be determined. Furthermore, both the powerful insights and the possible artifacts characteristic of laboratory flask assays need to be acknowledged. Suggestive studies The majority of biodegradation investigations that utilize environmental samples (water, soil, and sediments from c o n t a m i n a t e d a n d uncontaminated sites) in laboratory flask assays demonstrate biodegradation potential, not in situ biodegradation (Figure 1, top). The reason for performing these physiological studies is clear-the control over mass balances and ability to distinguish biotic from abiotic reactions allow definite conclusions to be drawn. Furthermore, once microbial metabolism of contaminant organic compounds has been established, a variety of influential environmental and ecological parameters (i.e., pH, redox potential, nutrients, inoculation, contaminant concentration, predation, competition, etc.) can be investigated. Also, single organisms or defined consortia responsible for the biodegradation can be isolated from the environmental sample-enabling more rigorous reductionistic, physiological, biochemical, and genetic probing of the biodegradation process. Nonetheless, all such studies suffer from the Heisenberg Uncertainty Principle ( 3 6 ) discussed above. Case studies suggesting in situ biodegradation ofien involve chemical and microbiological measurements on samples from a contaminated field site which are performed in addition to flask studies of biodegradation potential (Table 1).The types of information from a contaminated field site that are meant to complement determinations of biodegradation potential include the following: Horizontal or vertical profiles that indicate diminishing concentrations of the contaminants (61):such measurements usually fail to distinguish biotic from abiotic loss mechanisms. Enhanced numbers of microorganisms able to grow on the contaminant in samples from the contaminated area: this information strengthens the case for putative in situ biodegradation, espe1666 Environ.
TABLE 1
Examples 01 invesiigarions suggesring in situ oioaegraaation Type of site
Contaminam
Methods
R~relIca
Soil habitat
Agricultural soil
55
Dicamba (2-
methoxy-3.6-
dichlorobenzoic acid),a pesticide
Production of %O, and intermediary metabpiites from radiolabeled dicamba added to soil in iaboratoryincubated flasks
51
Oil
Quaternary ammonium comoounds
oil from tubes fioatina on sea water flowing thiough open tanks Production of 14C0 and incorporation of ‘48into bacterial biomass in sealed iaboratorv-incubated flasks receivh ’ C labeled compounis; addition of compounds to vessels incubated in situ rior to initiating the above todegradation assay; enumeration of bacteria
51
g.
ses of field site waters organic and inorganic constituents: ’%Os Droduction from radiolabeled com unds in laboratory-incutkd flasks, enumeration of bactena Anal
kr
cially if comparisons inside and out of the contaminated area indicate that metabolic adaptation has occurred (25,30,591. Detection of changes in reactants (e+, oxygen and nutrients) and products (e.g., CO, or intermediary metabolites) that may be indicative of known metabolic processes (49, 62, 63). Although many of the investigations listed in Table 1 reported rigorous laboratory and site characterization data, they failed to prove in situ biodegradation (see “Definitions of Terms” box). The evidence for expression of microbial metabolic potential in the landscape was inadequate. This situation is the rule i n biodegradation studies. The methodological limitations that prevent extrapolation from laboratory data to the field and impede on-site mass balances constitute a major impasse.
Sci. Technol., VoI. 25. No. 10, 1991
When indicators of known microbial metabolic processes from field samples are unequivocal, then the attempt to prove in situ biodegradation will be successful. But substantial ambiguities, especially at spatially heterogeneous field sites, often impair definitive statements attributing changes i n chemical constituents at the site uniquely to microorganisms [this will be discussed in greater detail below). Successful studies The uncontrollability of field sites and their spatial heterogeneity constitute major obstacles to determining in situ biodegradation. Successful strategies overcome these obstacles by finding evidence of microbiological involvement in contaminant disappearance from field sites. Ideally one would be able to account for each and every possible biotic and abiotic avenue of attenu-
ation for organic contaminants. In this way quantitative budgets could be assembled and rates of all field processes, including biodegradation, could be discerned. In an exceptional series of experiments utilizing an artificial but highly realistic test system (40, 64, 65),Crawford and co-workers were able to surmount virtually all of the obstacles that plague in situ biodegradation studies. Mississippi river water was diverted through replicated outdoor experimental channels dosed with pentachlorophenol (PCP). By monitoring the amount of PCP entering, leaving, and remaining within the channels, mass balances were obtained. Furthermore, the time lag (severalweeks) prior to metabolic adaptation of the microbial community to PCP provided an opportunity for PCP volatilization, sorption, and photolysis to be measured. Thus, in this unique test system biotic and abiotic pollutant attenuation processes were successfully quantified. However, at actual field sites, the multiplicity of competing mechanisms of disappearance can seldom, if ever, be deciphered. Thus, most successful efforts at documenting in situ biodegradation are qualitative. Controlled-release experiments, aimed at simulating an oil spill or some other pollution event, are the most common of all successful in situ biodegradation studies (Table 2). The only disadvantage of such experiments is that they may add potential pollutants to otherwise untainted field test areas. But the advantages are substantial: statistically valid, replicated randomized block experimental designs (66, 71, 72)can be achieved using a variety of experimental treatments that address mass balances, site heterogeneity, and both biotic and abiotic loss mechanisms. Microbiological cause of contaminant loss becomes apparent by virtue of an on-site manipulation that elicits an enhanced microbiological response that is distinguishable from an uncertain background of other unenhanced processes (Figure 1,bottom). For instance, Westlake et al. (67) and Raymond et al. (68)included experimental treatments with and without fertilizer, and Lamar and Dietrich (66) tested plots with and without fungal inocula (Table 2). The fertilized and inoculated treatments would be expected to stimulate biodegradation but not abiotic contaminant losses. The relative difference between enhanced and
unenhanced treatments found by these investigators was attributed to in situ microbial activity. Strictly speaking, however, the absolute q u a n t i t y of c o n t a m i n a n t l o s t through biodegradation could not be identified because biotic and abiotic contributions to contaminant loss in the unenhanced treatments were uncertain (Figure 1, bottom). The study by Spain et al. (69,Table 2) represents a rare instance where virtually no abiotic losses were found in the unenhanced treatment. In this case, loss of poro-nitrophe-
Replicated field plots
no1 from the previously exposed half of a pond was complete before any appreciable loss occurred in the previously unexposed half of the pond. Here, then, biodegradation operated exclusive of abiotic processes; consequently, quantitative rates of in situ biodegradation were measured (Figure 1,bottom). The report by Pritchard and Costa (5, Table 21 is a recent example of approaches to documenting in situ biodegradation of spilled crude oil in marine environments. A visually dramatic loss of dark surface mate-
Contamlnant
Evidama
PentachloroDhenol
Enhanced loss of extract-
6f
6i
6t
Pond divided Inr
rarrrllllr
0:
half of the pond, PNP was added to both halves PNP then disappeared only from the previously exposed, metabolically adapted hail of the pond. Ground water habitat
Biodearadation ootentiai and oiher microbial lca~ parameters indical2metabolic adaptation in samdes from contaminated. but not adiacent uncon-' laminated; areas. Enhanced tozoan numbers, o n r n contaminated area. in&ated in situ microbial growth resuiting from the contaminants. At a nighly instrumented mnf8nedaquifer site, mass balances were pos. Sible. Cometabolic oxidation 01 chlorinated ethenes
7c
Environ. Sci. TBChnol.. Vol. 25. No. 10, 1531 1667
r i a l was observed o n l y in portions o f r o c k y beaches t o w h i c h oleop h i l i c fertilizer was applied. However, i t was n o t entirely clear if the fertilizer exerted i t s effect by acting as a nutrient source (stimulating microbial activity) or merely as a solvent (mobilizing t h e oil). More importantly, Pritchard a n d Costa (5) rocused o n the ratio o f t w o isomeric components of the crude oil that are
chemically similar but very different in their susceptibility t o microb i a l attack. The microbiologically resistant branched-chain isomer, phytane, served as an internal massbalance tracer w h i c h persisted relat i v e t o octadecane. Thus, a decrease in t h e octadecanelphytane r a t i o over time was proof of in s i t u biodegradation. Semprini et al. (70) (Table 31 were
able t o demonstrate mass balances in t h e i r highly instrumented conf i n e d aquifer f i e l d site by injecting a conservative tracer and monitoring its concentration at various distances f r o m the p o i n t o f release. These investigators also stimulated indigenous methane-oxidizing microorganisms by injecting methane and oxygen into the intensively characterized test area. In situ cometabolic
TABLE 3
A generalized stepwise strategy for determining in situodegradation processes 1IvBlapprmches
,_.,onduct
preliminaty u,,a,aL,v,,Lafiaid D I = E ~ , o , site investigation assess the following: Spatial, physical, and h drologic constraints for SuccessYU~~y understandingthe site
Type and degree of contaminationthat has occurred
Predominant physiological regimes that are already established by the indigenous microorganisms
A homogeneous geologic seuing may be amenable to a sampling scheme that provides interpretabledata demonstrating clear concentrationaradients and hydrologic flow pathi. However, a hydrologicallyand geologically complex setting may prevent interpretablemeasurements from being obtained. The identity of chemicals present and their concentrations should se a limited set of gPiode radation ssibilities, baseton estab&ed ohvsioloaicaland biochemical kn6wlediie If oxygen is already depleted, then attention to anaerobic processes may be warranted. It the site is marine, then sJifate reaunion ma be imponant and sulfides shourd be abundant. If the site is at hi h altitude or latitude, then cod temperatures mav severelv imDair microbial activity.
Site characterizationmethods are well roven. Geostatistics may be used:
Biodegradation principles and techniaues of analviical ChemistN are well proven. .
Physiologicalprinciples for surmising on-site microbial processes are well established.
. .
2. Develop in situ Based on existing biochemical knowledge. idenlilyke chemical biodegradation assessment strategy and b w i c a idtena &at match site characteristicswith bkdegradation processes.
PCBs be reductively dechlonnated(43). Many hydrocarbons are readily metabolizedaerobiilly (73, 74,
7% Toxic chemicals in high
contaminant(s)are diluted.
3. Conduct detailed
Refine the site characterization site characterization sampling Ian to provide optimally interpretah chemical and microbiologicalmeasurements.
eek proof 01 i Biodegradation
Disbnguish biotic from abiotic processes by seeking chemical and biological criteria that are unique1 indicative 01 microbial metaboh processes: Production of metabolic mtennediate compounds. especially as they contrast with contam!nants originally released at the sm
Site characterizationandlor statistical designs must rovide the framework for samptng. Analytical and sampling prqcedures should allow trends indicative of on-site processes to be discerned.
Highly feasible but not well proven
degradation of chlorinated ethenes (trichloroethylene, dichloroethylene, and vinyl chloride) b y methane-oxidizing microorganisms was proven by (1) a decreased concentration of these compounds coinciding with the onset of methane utilization, ( 2 ) t h e a p p e a r a n c e of a characteristic metabolic intermediate compound, trans-dichloroethylene oxide, and (3) cessation of (1)
a n d ( 2 ) w h e n methane addition was stopped (70). My colleagues a n d I were able to determine in situ biodegradation in a shallow unconfined aquifer without a n y on-site experimental man i p u l a t i o n s (30).W e measured a broad array of microbiological a n d chemical characteristics at a coalt a r w a s t e s i t e in s a m p l e s t h a t spanned c o n t a m i n a n t concentra-
t i o n gradients. At t h i s r e l a t i v e l y u n i f o r m sandy study site, clear patterns emerged w h i c h demonstrated in s i t u g r o w t h o f biodegradative microorganisms. Trophically stimulated protozoan biomass occurred e x c l u s i v e l y within a p l u m e of groundwater contamination and, in concert with metabolic adaptation and other determinations, served to indicate in situ biodegradation (30).
denitrifcation may or may not be tied to contaminant loss . Furthermore. trace amounts of NO , may be ucea during nitrilication camon
(%bus.
Selective disap arance of micmbiologicalGdile isomers,
5. Gather microbiolo ical evidence tfat confirms in situ biodegradation.
Not w a y s reliable because Mane and phytane are redable (79,601 Pro sed for Exxon VaMer oil spilfih, 63. but limitations must be acknowledged (see text)
&lodeg
e melabdism of
The enanttomeric ratio of a chiral
Promising: likely to be highly effective 11contaminant-specific biochemist is thoroughty mcumenterd Not yet reliable lor bicdegradation; useful lor geocnemical reactions
Although man microbiolo(lical assays are in&idualiy of limited relevance to in situ processes, convergent lines 01 indirect evidence lend strength to arguments addressing in situ biodegradation.
Periorm laboratory tests demonstrating that the biodegradation occurs in biotic. but not abiotic, samples from the site (10, 15, 38).
Limited relevance to in situ biodegradation, but supportive auxiliary physiological and microbiological data can be very Useful
Isolate and enumerate microorganisms from the site which catalyze biodegradation (90). Use DNA or RNA gene probes to assess distribution of potential catabolic expression (91, 93
Environ. Sci. Technol., Vol. 25. No. IO, 1991 1559
e-+-
-.
Strategies Most of the studies discussed thus far that have successfully proven in situ biodegradation have been contrived. However, the majority of organic pollution events are accidental [e.g., leaking underground storage tanks, overturned tractor trailers, the Amoco Codiz oil spill (73, 741, the Exxon Voldez oil spill (4, 511. In contemplating accidental spills, it is essential to transfer the same principles for proving in situ biodegradation that were successful for the fallen-tree scenario and other experiments (above). But again one encounters the obstacles from the gasoline contamination scenario and the above studies which may have suggested, but failed to prove, in situ biodegradation. When accidental spills occur, where are the control treatments that allow biotic and abiotic losses to be distinguished? In the face of the adversity posed by accidental spills (uncharacterized sites of unknown history and unknown spatial heterogeneity, without contrived experimental designs), it seems prudent to accept the futility of quantitative mass halances while searching for on-site changes in reactants, metabolic products, or organisms such as protozoan predators that are uniquely characteristic of microbiological biodegradation processes. Table 3 presents a generalized strategy for determining in situ biodegradation that is applicable to both controlled-release and accidental spill sites. The rationale for each step, some examples, and their reliability are also. presented. The three portions of Step 1 are directed towards gaining a rough idea about the feasibility of obtaining useful field data, learning the identity and concentrations of the contaminants, and discerning broad categories of microbiological processes that may be operating on site, respectively. Step 2 focuses conceptually on pollutant-specific indicators of in situ biodegradation that would be obtained from chemical and microbiological analysis of site samples. Step 3 refines the site characterization, sampling, and analysis procedures (begun in Step 1) according to the detailed needs resultine from Step 2. Distinguishing biotic from abiotic urocesses lSteu 4 of Table 31 is the key issue for p;oving in situ biodegradation. As mentioned above, this is also the key issue that separates Y
L
I i
suggestive studies from conclusive ones. A wide variety of criteria for uniquely identifying microbial processes as active in situ have been proposed by various investigators. If the physiology and biochemistry of contaminant metabolism are sufficiently understood, detection of unique intermediary metabolites in samples from the site can serve as conclusive qualitative evidence for in situ biodegradation. However, a great deal of care must be exercised in drawing conclusions from putative intermediary metabolites because the rules of microbial metabolism and abiotic reactions are sometimes ambiguous. For instance, the intermediary degradation compounds produced from pentachlorophenol during sequential photolytic (93) and biodegradative (94, 95) processes may be the same. Furthermore, even if characteristic metabolic intermediates are produced in situ, they may be too short-lived to be detected before they enter possible biotic or abiotic reaction pathways. One approach, mentioned above, that has gained considerable attention is to focus on components of the environmental contaminants that serve as internal mass-balance indicators because they are not biodegradable but are transported and diluted similarly to the biodegradable components (Table 3, Step 4). If a decrease in the ratio of biodegradable to the nonbiodegradable
4c70 Environ. Sci. Technol., VoI. 25. No. 10. 1991
u u i p o u n d s is discerned, in situ biodegradation is indicated. Although this idea is an excellent one, it is not always successful. For instance, decreasing ratios of C,, and C,, alkanes to their branched-chain isomers, pristane and phytane, have been used as evidence for in situ oil biodegradation (5,73, 74). But other independent studies have found that the supposedly persistent massbalance indicators, pristane and phytane, are themselves biodegradable (79,801. Thus when a microbial community impacted by spilled oil metabolizes pristane, the ratio of C,, alkanes to pristane will not necessarily decrease, and in situ biodegradation will be underestimated. It has recently been suggested that hopanes (microbially resistant pentacyclic alkanes] be utilized instead of pristane and phytane as internal mass-balance indicators in assessing in situ biodegradation that has ensued since the Exxon Voldez oil spill (R. C. Prince, Exxon Research and Engineering Company, personal communication, 1991; 81). Such an approach is promising, especiall y when coupled to detailed chemical composition analyses of the crude oil prior to its release. However, it is not obvious which biodegradable crude oil components should be compared to hopanes. Most crude oil components exhibit solubility, transport, photolytic, and volatility characteristics that differ from those of hopanes, there-
by diminishing the utility of hopanes as conservative tracers. Just as microorganisms can selectively metabolize certain isomers, there is recent evidence for stereoselective metabolism of a single a-hexachlorocyclohexane enantiomer (82).Thus, a change in the enantiomeric ratio of a chiral organic compound can serve to distinguish biotic from abiotic reactions (Table 3 , Step 4). Stable isotopic fractionation patterns have been used in geologic studies to identify microbiological sources of CH, and CO, in groundwaters (83, 84). This technique is powerful but relies on signature isotopic ratios of organic matter in geologic formations and therefore may not be transferable to recent releases of unaged, isotopically u n f r a c t i o n a t e d , organic contaminant chemicals. If a contaminant source does bear a characteristic ratio of stable isotopes, then fractionation patterns could be used to document in situ biodegradation by tracing substituents of contaminants through the food chain. Trends indicating diminishing concentrations of coreactants of biodegradation processes such as oxygen (for aerobic reactions), nitrate (for denitrifying reactions), or sulfate (for sulfate reduction) (Table 3, Step 4) are not, themselves, conclusive evidence but may add to arguments for in situ biodegradation. Trudell et al. (85) used the loss of nitrate relative to bromide (a conservation tracer) as evidence for in situ denitrification, but this electron-accepting process was not associated with loss of an organic contaminant nor were alternative mechanisms of nitrate loss considered in this study. By comparing laboratory flask biodegradation assays using samples from contaminated and uncontaminated areas of a site, metabolic adaptation can be documented. Adaptation, alone, is not conclusive evidence for in situ biodegradation. However, if linked to other evidence such as enhanced numbers of urotozoan uredators. adautation can provide essential auxiliary evidence for in situ biodegradation [see above, [30]1. If a field site is uniform enough to be spatially divided into comparable sections, responses to various on-site manipulations (e.g., fertilizers, conservative tracers, etc.) can be detected using chemical and microbiological assays, and interpreted according to microbiological rationale. When not restricted by regulatory
. .
agencies, intentional release of traceable isotopically labeled contaminants can provide proof of in situ biode adation. Lee et al. (86) measured %Oz produced from radioactive hexadecane released within a cylinder anchored in coastal waters. This constituted strong evidence for in situ biodegradation. However, in this and other (87, 88) studies, the efficiency of recovering the labeled compounds from open systems may be quite low. Step 5 of the strategy (see Table 3) is to seek confirmatory microbiological evidence. Such supplemental information is not essential, but it can be a valuable addition to the typical rationale for proving in situ biodegradation. Both laboratoryincubated flask assays and the isolation and enumeration of biodegrading microorganisms (as discussed above) provide confirmatory support. The use of DNA and RNA gene probes for pollutant catabolic pathways (90, 91) may provide useful mechanistic information about biodegradation potential. When available, these assays rely on extensive and detailed genetic and biochemical knowledge of specific metabolic pathways. Before gene-probing techniques can be applied with confidence to uncharacterized naturally occurring microbial populations, several methodological impediments [e.g., inefficient extraction of cells, DNA, and RNA from environmental samples (95-97) and divergence between nucleotide sequences obtained from laboratory and naturally occurring microorganisms] must be overcome. The biochemical nuances of cellular processes that precede biodegradation may seem peripheral to documenting the chemical transformation itself. But when performed in conjunction with field and laboratory measurements described above, gene probing promises to reveal both practical and evolutionarily important insights about how and w6y biodegradition activity is controlled and exuressed.
Conclusions The evolutionary role of microorganisms as effective agents causing the destruction and recycling of organic materials in the biosphere is well established. Nonetheless, in situ biodegradation of organic contaminant compounds is very difficult to prove because the uncontrollability of field sites a n d their spatial heterogeneity may prevent
IN situ
Giodegrudution of organic contaminants is very difficult to prove, the assembly of mass balances and the identification of microbiological contributions to contaminant loss. A typical approach to determining in situ biodegradation relies on several convergent lines of independent evidence from a field site. These include: laboratory-incubated flask assays which may show that samples from a contaminated portion of the site contain metabolically adapted microorganisms: concentration profiles at the site suggesting both contaminant losses which exceed those expected for abiotic processes and changes in reactants or products indicative of microbial metabolism; and an unequivocal distinction between biotic and abiotic attenuating processes. This latter requirement may be attained i n controlled-release experiments which determine an enhanced loss of contaminants in fertilized or inoculated field nlots. Alternatively, biotic processes may be distinguished from abiotic ones by detecting unique microbial metabolites on site, a stimulation of protozoa by the growth of contaminant-fed bacterial prey, or decreasing ratios of biodegradable to nonbiodegradable contaminants. Determining in situ biodegradation is an essential step in the development and validation of many
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types of technologies aimed at allev i a t i n g environmental pollution. But despite decades of research exploring microbial m e t a b o l i s m of organic compounds, d o c u m e n t a t i o n of in situ biodegradation is relatively rare and almost always qualitative. The s h o r t - t e r m challenge to inv e s t i g a t o r s a t t e m p t i n g to prove in situ biodegradation is t o devise n e w , more reliable approaches for distinguishing biotic f r o m abiotic reactions in the field. There are no set formulas for a c h i e v i n g this goal. Successful approaches will probably rely on unique circumstances at each field site as well as an expanding pool of knowledge provided by m i c r o b i o l o g i s t s , g e n e t i c i s t s , biochemists, p h y s i o l o g i s t s , analytical chemists, and ecologists about microbial metabolism o f s p e c i f i c organic compounds. Three long-term challenges for biodegradation scientists that need to be addressed in the future are q u a n t i t a t i v e determination of in situ biodegradation rates, extension of this d e t e r m i n a t i o n to complex mixtures of organic compounds, and a c h i e v e m e n t of a level of contaminant attenuation that complies with health standards set by regulatory agencies. The importance of these challenges cannot be o v e r e m phasized. Acknowledgments Research support for pre aration of this article was provided by t l e U.S. Department of Energy, Office for Health a n d Environmental Research, Subsurface Science Program, a n d the Electric Power Research Institute. Expert assistance i n manuscri t preparation from Patti Lisk is gratefufiy acknowledged.
Eugene L. Modsen is a senior research associate in the Section of Microbiology within the Division of Biological Sciences a t Cornell University. His Ph.D., from Cornell, is in microbiology, ecology, a n d soil science. After completing appointments a t Rutgers a n d Penn State universities a n d MSI Detoxificotion, Inc., Mads e n r e t u r n e d to C o r n e l l to p u r s u e research interests i n groundwater microbiology, microbial metabolism of organic pollutants, a n d strategies formeaw r i n g microbiological processes in situ.
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