Bioremediation of groundwater pollution Ronald L. Crawford University of Idaho, Moscow, Idaho, USA Significant progress has been made in the past year towards an understanding of the microbial processes in subsurface environments that may allow natural microbial populations to be employed for bioremediation of groundwater pollution. Among the highlights were: the discovery of several previously unknown xenobiotic-degrading abilities in groundwater microorganisms; progress in using the unique abilities of methanotrophs to oxidize halogenated solvents; and characterizations of microbial populations from subsurface soils. Current Opinion in Biotechnology 1991, 2:436-439
Introduction Vast quantities of groundwater throughout the world have become contaminated with hazardous chemicals from industrial and agricultural activities. The magnitude of this problem is almost beyond comprehension. Abelson [1 °°] has summarized the grim situation as it exists in the USA, pointing out that as many as 375 000 leaking underground tanks have contaminated, or probably will contaminate, groundwater. There are at least 31000 identiffed waste sites in the USA, and groundwater contamination is common to most of them. Small halogenated hydrocarbons, benzene, toluene, and the xylenes, are the predominant contaminants at many sites, whereas heavy metals or radioactive substances contaminate some sites, and mixtures of most or all of these pollutants contaminate the worst sites. The situation in Europe and elsewhere is equally discouraging. Abelson also points out that cost-effective clean-up of groundwater pollution will often require 20-40 years of remedial action. Eff.ective remediation will almost always require the close cooperation of multidisciplinary teams of earth scientists, engineers and biologists. In the USA, costs of groundwater remediation over a period of 3 or 4 decades may reach $500 billion. A technique that promises to keep costs down, yet ensure effectiveness, is bioremediation, the use of biological processes to destroy or immobilize pollutants. This review will discuss what I consider to be the most significant papers on the bioremediation of groundwater pollution that have appeared in the literature since December 1, 1989. Bioremediation of groundwater can be accomplished in various ways. Water can be pumped from an aquifer and treated on the surface before being recharged into the subsurface or discharged above ground. Alternatively, bioremediation may be performed
in sire by properly stimulating populations of pollutantdegrading microorganisms that exist naturally in subsurface environments, or by introducing surface organisms with unique catabolic abilities to detoxify specific target chemicals. I n sire approaches promise to be relatively inexpensive and fast-acting, requiring treatment times of months to years, rather than years to decades. At the same time, such methods are more challenging to develop than pump-and-treat schemes. Here, I will focus on papers that discuss i~7 situ approaches to groundwater bioremediation.
Many of the current efforts to develop in situ bioremediation techniques are still at preliminary stages. Studies of aquifer microcosms most often involve soil slurry reactors or soil perfusion columns. Other work looks at aquifer microorganisms, both consortia and pure cultures, that have been isolated from subsurface soils or waters. There appear to be relatively few reports in the mainstream, peer-reviewed literature concerning work carried out .within actual aquifers. (I will not discuss papers that appear in trade joumals or the popular science press.) Papers summarized here encompass all of the above: microcosms, isolated cultures and field work.
Pure or mixed subsurface microbial cultures Dissimilatory iron-reducing microorganisms Two recent papers from the US Geological Survey's Water Resources Division [2-.,3] indicate the potential use of a little-known group of microorganisms in bioremediation of polluted groundwaters. This group includes microorganisms that oxidize organic substrates, using Fe3+ as an electron acceptor. As Lovley et al. [2 o'] discuss, the literature has established that many ancient sediments harbor anaerobic aquifers where organic mat-
Abbreviations BTX--benzene/toluene/xylene; DC~ichloroethylene; DO--dissolved oxygen; MMO---methane mono-oxygenase; TCE~t richloroethylene. 436
(~) Current Biology Ltd ISSN 0958-1669
Bioremediation of groundwater pollution Crawford 437 ter is being oxidized with the simultaneous reduction of Fe3 + to Fe2 +. It has been suggested that microorganisms are agents that couple these two processes [3], and microbes have been shown to link mineralization of organic compounds to CO2 with the reduction of Fe3+ [4-,5°]. tovley et al. [2..] have im'estigated the possibility that microorganisms are catalyzing the ongoing reduction of Fe3+ in deeply buried (20--250m) sediments of the Atlantic coastal plain. The authors enriched and isolated a pure culture of an anaerobe that oxidized acetate to CO2, while reducing Fe3+ to Fe2+. Such microorganisms were present only in sedimentary materials from regions in which biogeochemical evidence indicated that Fe3+ reduction was taking place. The culture appeared to oxidize acetate as follo~vs:
Two types of biMO are thought to occur in methanotrophs - - a particulate (membrane-bound) MMO and a soluble MMO. The soluble form of the enzyme appears to be synthesized under copper-limited conditions, and many researchers feel that only the soluble bilVlO is responsible for TCE oxidation. Henry and Grb]c-Gal~c [8"] have isolated a Metbylomonas sp. from an uncontaminated aquifer, and have presented evidence that transformation of TCE by this strain is catalyzed by a particulate MbiO, although TCE degradation was stimulated in the presence of a copper chelator. The implication of these observations for bioremediation specialists is that the role copper might play in regulating methanotrophic oxidation of TCE in groundwaters is somewhat confused.
CH3COO- + 8Fe3+ + 2H20 .-+ 2CO2 + 8Fe2+ + 7H +
Methanogenic consortia
Under culture conditions, the reduced iron frequently appeared as magnetite. These data are apparently the first to confirm a mechanism for oxidation of organic compounds in groundwater coupled to the production of large amounts of dissolved Fe2+ under anaerobic conditions.
Three related papers from Suflita et at [9"-11"] have examined numerous anaerobic aquifer slurry systems for their ability to degrade several important halogenated groundwater contaminants. A halogenated nitrogen heterocTclic herbicide (bromacfl) was shown to be debrominated under methanogenic, but not denitrifying or sulfate-reducing, conditions [9"°]. This observation expands the list of aryl-reductive dehalogenation reactions to include nitrogen heterocyclic molecules. Di-, tri- and tetrachloroaniline were shown to be reductively dehalogenated by methanogenic groundwater microorganisms in the form of aquifer slurries [10-]. Acclimation periods for the microfloras were long (8-10 months). Acclimated cultures had significantly decreased lags before dehalogenation activity appeared. Dehalogenations appeared to cease at the level of monochloroanilines, which persisted. Obviously this has serious implications for any in situ bioremediation schemes proposed for this class of molecules. Finally, 2,4,5-trichlorophenoxyacetic acid was shown to be dehalogenated by methanogenic aquifer slurries to form 2,4- and 2,5-dichlorophenoxyacetic acids as the first detected intermediates [11o.]. A series of sequential dehalogenations and side chain cleavages ultimately yielded phenol and ring cleavage. Addition of short-chain organic acids stimulated the process, whereas additions of sulfate were inhibitory. This series of papers provides a wealth of new information for bioremediation researchers who are considering manipulating anaerobic groundwater conditions to favor destruction of halogenated pollutants.
The existence of microorganisms capable of coupling the anaerobic reduction of Fe3+ to the oxidation of organic compounds that are groundwater pollutants, could open a new avenue for potential bioremediation of contaminated groundwaters. Stimulation or introduction of these microorganisms are possible mechanisms for catalyzing the destruction of pollutants in anaerobic aquifers. Lovley and Lonergan [6"] have confirmed the existence of such microorganisms, by isolating a dissimilatory Fe3 +reducer (strain GS-15) that degrades toluene, phenol and pcresol. Such organisms could potentially effect desirable transformations in anaerobic-aquifers contaminated by hydrocarbons.
Methanotrophic microorganisms One of the most significant contaminants of groundwater throughout the world is trichloroethylene (TCE). Thus far, all of the numerous aerobic microorganisms that have been shown to be capable of degrading TCE appear to utilize oxygenases that fortuitously oxidize TCE in place of their normal substrates. Methane-oxidizing bacteria and their non-specific TCE-attacking oxygenase, methane mono-oxygenase (MMO), have received the most attention as possible candidates for in situ bioremediation of TCE in groundwater. MMO requires a reductant in order to perform !ts oxidative functions. Methane, the physiological reductant for methanotrophs is, unfortunately, a competitive inhibitor of TCE oxidation and is therefore not an ideal candidate for field-stimulation of TCE degradation. Henry and Grblc-Gal~c [7°o] have shown that lipid storage granules can serve as a source of endogenous • electrons for TCE degradation by starved methanotrophs, a phenomenon with significant implications for the economical and effective use of methanotrophs for in situ bioremediation of TCE-contaminated groundwaters.
Aquifer studies Bioremediation studies Actual bioremediation studies within aquifers are still fairly rare. One such study involved observations of soluble hydrocarbon biodegradation in a shallow aquifer that had been punctured with 42 monitoring wells [12..]. A strong point of this study was that material mass balances were obtained for inputs, outputs, and destruction of molecules such as benzene over a period of 3 years. The first-order attenuation rate for benzene in
138 Environmentalbiotechnology this aquifer was shown to be 0.0095 per day. Spatial relationships between dissolved oxygen (DO) and total benzene/toluene/xylene (BTX) were confirmed statistically, and laboratory microcosms were employed to confirm the DO/BTX biodegradation relationship. This is an excellent example of the thorough characterization of a spill where natural bioremediation of groundwater contamination is occurring.
versity of groundwater microbial communities. The usefulness of microorganisms for i n situ bioremediation of groundwater pollutants looks more promising each time another subsurface secret is revealed.
Semprini et al. [13"'] have presented the results from a multiyear field study" that documented the in situ biotransformations of TCE, ct~-dichloroethylene (DCE), tran~DCE and vinyl chloride in a saturated, semiconfined aquifer at the Moffett Naval Air Station, Mountain View, California [14.]. This site is not completely representative of many natural groundwater situations because it is aerobic, contains very homogeneous sands and gravels, and has relatively high water flow rates (8-10 litermin-1 at the extraction well). The data generated here, however, provide a very useful general guidance for designing bioremediation schemes using indigenous populations of methanotrophic bacteria. Indigenous methanotrophs were stimulated to degrade the target contaminants, without plugging the system with biomass, by pulsed additions of methane plus oxygen-supplemented water into an injection well. All target compounds were biotransformed, but only during times of active methane oxidation. In this system, pollutant oxidation could not be sustained under conditions of methane starvation (see [7"'1).
Papers of special interest, published within the annual period of review, have been highlighted as: • of interest •. of outstanding interest
References and recommended reading
1. ABELSONP: Cleaning Hazardous Waste Sites. Science 1989, •. 246:1097. An editorial discussing the extent of em-ironmental contamination, particularb, of ground~-ater, by toxic chemicals. Discusses the prospects for clean-up o f waste dumps and associated groundwater pollution, stressing the potential of bioremediation as a clean-up technology. 2. •,
LO~I.EYD, CH.~ELLE F, PttI1MPS E: Fe(llI)-Reducing Bacteria in Deeply Buried Sediments o f the Adantic Coastal Plain. Geology 1990, 18:954-957. Reports the existence vdthin deep subsurface aquifers of microbial populations able to couple the oxidation of organic matter to carbon dioxide, with the reduction of Fe3 +. Such populations may be useful for in situ groundwater bioremediation. 3.
LO%a.EYD: Organic Matter Mineralization with the Reduction o f Ferric Iron: a Review. GeomicrobiologyJourna11987,
5:375-399. 4. •
Loxa.EvD, B.MZDECKERM, LONERGAND, CO72AREI.H I, PHILLIPS E, SIEGELD: Oxidation of Aromatic Contaminants Coupled to Microbial Iron Reduction. Nature 1989, 339:297-299. Links mineralization of organic compounds to CO 2 with reduction of
Fe3+.
Deep subsurface microbial populations As a part of its Subsurface Science Program, the US Department of Energy has begun to characterize the diversity of bacteria in the deep sediments of the US Atlantic Coastal Plain and the basalts of the US Pacific Northwest. Fredrickson et al. [15"'] have presented a compendium of the results of the Atlantic coast studies. Bacteria were isolated from aseptically obtained core materials at periodic depths down to about 500 m. Aerobic chemoheterotrophs were present, even at great depth, to levels as high as 6.4 log col6ny-forming units per gram of sediment. A total of 198 pure cultures were compared using 108 different physiological tests. Cluster analysis grouped these bacteria into 21 different biotypes. The subsurface isolates degraded a large variety of organic acids, lipids, carbohydrates and amino acids. Only a few isolates could be readily assigned to known genera, and these tended to be representatives of the genus Pseudomonas. The great diversity observed among these bacteria implies that h~ situ bioremediation of groundwater pollution, even in the deep subsurface, may be possible.
Conclusion The work discussed here makes it clear that scientists have traditionally underestimated the capabilities and di-
5. •
IDVLEYD, PIIIIMPS E, 1.ONERG.~ND: Hydrogen and Formate Oxidation Coupled to Dissimilatory Reduction of Iron o r Manganese by Alteromonas putrefaciens~ Appl Environ Mi¢robiol 1989, 55:700-706. Reports the isolation of a pure bacterial culture capable of oxidizing formate by coupling the oxidation to reduction of Fe3+. 6. •.
LOXlEYD, LONERG&N D: Anaerobic Oxidation of Toluene, Phenol, and />Cresol by the Dissimilatory Iron-Reducing Microorganism, GS-15. Appl Environ Microbiol 1990, 56:1858-1864. Describes the first microorganism known to couple the oxidation of aromatic compounds to the reduction o f Fe3+. The bacterium is also the first described pure culture to anaerobically oxidize an aromatic hydrocarbon, toluene, indicating its potential for in situ bioremediation of anaerobic groundwaters contaminated by gasoline spills. 7. •.
IIENRYS, GRBic-GAtlC D: Influence of Endogenous and Exogenous Electron Donors and Trichloroethylene Oxidation Toxicity on Trichloroethylene Oxidation by Methanotrophic Cultures from a Groundwater Aquifer. Appl Environ Microbiol 1991, 57:236-244. Evaluates the influence o f trichtorethylene-transforming methanotrophs on TCE oxidation, and the effect of reductant axxilability on TCE transformation rates during methane starvation. Presents information required for the effec~e design of in situ bioremediation processes employing methane-oxidizing bacteria. 8. •
IIENRYS, GRBIc-GAtiC D: Effect of Mineral Media on Trichloroethylene Oxidation by Aquifer Methanotrophs. Microb Ecol 1990, 20:151-169. Presents evidence to implicate particulate I~LMOin the oxidation o f TCE by an aquifer methanotroph. Considers the role of copper in regulating TCE oxidation by this microorganism. 9. ,,
ADRL~NN, 8UFLITAJ: Reductive Dehalogenation of a Nitrogen tleterocyclie H e r b i c i d e in Anoxic Aquifer Slurries. Appl Environ Microbiol 1990, 56:292-294.
Bioremediation of groundwater pollution Crawford Demonstrates the debromination of the nitrogen heteroc3"clic herbicide bromacil by aquifer microbial populations under methanogenic, but not denitrif)ing or sulfate-reducing, conditions. Extends the range of halogenated compounds known to be reduce'ely dehalogenated in groundwater emironments. 10. •,
Kul~ K, TOWNSENDT, SUFIIr^J: Effect of Sulfate and Organic Supplements on Reductive Dehalogenation of Chloroanilines in Anaerobic Aquifer Slurries. Appl Environ Microbiol 1990, 56:2630-2637. Shows that di-, tri- and tetrachloroanilines are reduce-ely dehalogenated in methanogenic aquifer slurries, with apparent accumulation of to~c monochloroanilines as end products, indicating a problem in methanogenic bioremediation using this class of compounds. 11. •.
GmSON S, SUFIITA J: Anaerobic Biodegradation of 2,4,5TrichlorophenoxTacetic Acid in Samples from a Methanogenie Aquifer: Stimulation by Short-Chain Organic Acids and Alcohols. Appl Environ Microbiol 1990, 56:1825-1832. Describes the pathway for degradation of 2,4,5-trichlorophenox3~cetic acid in anoxic aquifers. Apparently the first report to show that aty1 dehalogenafion reactions may be stimulated in aquifer materials by supplementation with specific organic amendments. 12. •.
CHIANGC, SAL&\qTROJ, CHAI E, COLTtlARTJ, KLEINC: Aerobic Biodegradation of Benzene, Toluene, and Xylene in a Sandy Aquifer - - Data Analysis and Computer Modeling. G r o u m l Water 1989, 27:823-834. One of the most thorough studies yet of a self-purif)ing groundwater system. BTX levels in a hydrocarbon-contaminated aquifer were monitored over a 3-year period. Negative correlations for contaminant concentrations and dissolved ox)gen levels within the contaminant plume are reported; these parameters are successfully modeled by computer simulation.
13. •.
SEMPRI,\qto ROBERTSP, ItOPKLNSG, MCCARTYP: A Field Evaluation of I n Situ Biodegradation of Chlorinated Ethenes; Part 2, Results of Biostimulation and Biotransformation Experinaents. Ground Water 1990, 28:715--727. Documents the in situ biotransformation of TCE, c/~DCE, trat~DCE and xin)tchloride in a saturated semiconfined aquifer. Biotransformadon ~as effected by stimulation of indigenous methanotrophs ~ith pulsed additions of methane- and ox~!gen-supplemented w~ter introduced ~ia an injection well. 14. •
ROBERTSP, HOPKL\'SG, ~t~CKAYD, SEMPRL'qL: A Field Evaluation of In Si t u Biodegradation of Chlorinated Ethenes: Part 1, Methodology and Field Site Characterization. G r o u n d lira. ter 1990, 28:591-604. Describes the details of experimental methods and site characterization information for the aquifer experiments described in [13"]. 15. •.
FREDRICKSONJ, BAIKWILLD, ZAC~L-LqAJ, D S-M, BROCKMAN F, SL~tMONSM: Physiological Diversity and Distributions of Heterotrophic Bacteria in Deep Cretaceous Sediments of the Atlantic Coastal Plain. Appl Environ Microbiol 1991, 57:402-411. Describes the distribution of chemoheterotrophic bacteria within the deep subsurface of the Atlantic coastal plain of the US, to a depth of about 500m. Almost 200 pure cultures were isolated from aseptically obtained drilling cores. These strains were placed into 21 biotyw.s by cluster anal)sis. The great physiological diversity of observed bacteria is encouraging, with respect to future attempts at in situ bioremediation of groundwater pollution.
RL Cmvdord, Center for Hazardous Waste Remediation Research and Department of Bacteriology and Biochemistry, University of Idaho, Moscow, Idaho 83843, USA.
439
Introduction Vast quantities of groundwater throughout the world have become contaminated with hazardous chemicals from industrial and agricultural activities. The magnitude of this problem is almost beyond comprehension. Abelson [1 °°] has summarized the grim situation as it exists in the USA, pointing out that as many as 375 000 leaking underground tanks have contaminated, or probably will contaminate, groundwater. There are at least 31000 identiffed waste sites in the USA, and groundwater contamination is common to most of them. Small halogenated hydrocarbons, benzene, toluene, and the xylenes, are the predominant contaminants at many sites, whereas heavy metals or radioactive substances contaminate some sites, and mixtures of most or all of these pollutants contaminate the worst sites. The situation in Europe and elsewhere is equally discouraging. Abelson also points out that cost-effective clean-up of groundwater pollution will often require 20-40 years of remedial action. Eff.ective remediation will almost always require the close cooperation of multidisciplinary teams of earth scientists, engineers and biologists. In the USA, costs of groundwater remediation over a period of 3 or 4 decades may reach $500 billion. A technique that promises to keep costs down, yet ensure effectiveness, is bioremediation, the use of biological processes to destroy or immobilize pollutants. This review will discuss what I consider to be the most significant papers on the bioremediation of groundwater pollution that have appeared in the literature since December 1, 1989. Bioremediation of groundwater can be accomplished in various ways. Water can be pumped from an aquifer and treated on the surface before being recharged into the subsurface or discharged above ground. Alternatively, bioremediation may be performed
in sire by properly stimulating populations of pollutantdegrading microorganisms that exist naturally in subsurface environments, or by introducing surface organisms with unique catabolic abilities to detoxify specific target chemicals. I n sire approaches promise to be relatively inexpensive and fast-acting, requiring treatment times of months to years, rather than years to decades. At the same time, such methods are more challenging to develop than pump-and-treat schemes. Here, I will focus on papers that discuss i~7 situ approaches to groundwater bioremediation.
Many of the current efforts to develop in situ bioremediation techniques are still at preliminary stages. Studies of aquifer microcosms most often involve soil slurry reactors or soil perfusion columns. Other work looks at aquifer microorganisms, both consortia and pure cultures, that have been isolated from subsurface soils or waters. There appear to be relatively few reports in the mainstream, peer-reviewed literature concerning work carried out .within actual aquifers. (I will not discuss papers that appear in trade joumals or the popular science press.) Papers summarized here encompass all of the above: microcosms, isolated cultures and field work.
Pure or mixed subsurface microbial cultures Dissimilatory iron-reducing microorganisms Two recent papers from the US Geological Survey's Water Resources Division [2-.,3] indicate the potential use of a little-known group of microorganisms in bioremediation of polluted groundwaters. This group includes microorganisms that oxidize organic substrates, using Fe3+ as an electron acceptor. As Lovley et al. [2 o'] discuss, the literature has established that many ancient sediments harbor anaerobic aquifers where organic mat-
Abbreviations BTX--benzene/toluene/xylene; DC~ichloroethylene; DO--dissolved oxygen; MMO---methane mono-oxygenase; TCE~t richloroethylene. 436
(~) Current Biology Ltd ISSN 0958-1669
Bioremediation of groundwater pollution Crawford 437 ter is being oxidized with the simultaneous reduction of Fe3 + to Fe2 +. It has been suggested that microorganisms are agents that couple these two processes [3], and microbes have been shown to link mineralization of organic compounds to CO2 with the reduction of Fe3+ [4-,5°]. tovley et al. [2..] have im'estigated the possibility that microorganisms are catalyzing the ongoing reduction of Fe3+ in deeply buried (20--250m) sediments of the Atlantic coastal plain. The authors enriched and isolated a pure culture of an anaerobe that oxidized acetate to CO2, while reducing Fe3+ to Fe2+. Such microorganisms were present only in sedimentary materials from regions in which biogeochemical evidence indicated that Fe3+ reduction was taking place. The culture appeared to oxidize acetate as follo~vs:
Two types of biMO are thought to occur in methanotrophs - - a particulate (membrane-bound) MMO and a soluble MMO. The soluble form of the enzyme appears to be synthesized under copper-limited conditions, and many researchers feel that only the soluble bilVlO is responsible for TCE oxidation. Henry and Grb]c-Gal~c [8"] have isolated a Metbylomonas sp. from an uncontaminated aquifer, and have presented evidence that transformation of TCE by this strain is catalyzed by a particulate MbiO, although TCE degradation was stimulated in the presence of a copper chelator. The implication of these observations for bioremediation specialists is that the role copper might play in regulating methanotrophic oxidation of TCE in groundwaters is somewhat confused.
CH3COO- + 8Fe3+ + 2H20 .-+ 2CO2 + 8Fe2+ + 7H +
Methanogenic consortia
Under culture conditions, the reduced iron frequently appeared as magnetite. These data are apparently the first to confirm a mechanism for oxidation of organic compounds in groundwater coupled to the production of large amounts of dissolved Fe2+ under anaerobic conditions.
Three related papers from Suflita et at [9"-11"] have examined numerous anaerobic aquifer slurry systems for their ability to degrade several important halogenated groundwater contaminants. A halogenated nitrogen heterocTclic herbicide (bromacfl) was shown to be debrominated under methanogenic, but not denitrifying or sulfate-reducing, conditions [9"°]. This observation expands the list of aryl-reductive dehalogenation reactions to include nitrogen heterocyclic molecules. Di-, tri- and tetrachloroaniline were shown to be reductively dehalogenated by methanogenic groundwater microorganisms in the form of aquifer slurries [10-]. Acclimation periods for the microfloras were long (8-10 months). Acclimated cultures had significantly decreased lags before dehalogenation activity appeared. Dehalogenations appeared to cease at the level of monochloroanilines, which persisted. Obviously this has serious implications for any in situ bioremediation schemes proposed for this class of molecules. Finally, 2,4,5-trichlorophenoxyacetic acid was shown to be dehalogenated by methanogenic aquifer slurries to form 2,4- and 2,5-dichlorophenoxyacetic acids as the first detected intermediates [11o.]. A series of sequential dehalogenations and side chain cleavages ultimately yielded phenol and ring cleavage. Addition of short-chain organic acids stimulated the process, whereas additions of sulfate were inhibitory. This series of papers provides a wealth of new information for bioremediation researchers who are considering manipulating anaerobic groundwater conditions to favor destruction of halogenated pollutants.
The existence of microorganisms capable of coupling the anaerobic reduction of Fe3+ to the oxidation of organic compounds that are groundwater pollutants, could open a new avenue for potential bioremediation of contaminated groundwaters. Stimulation or introduction of these microorganisms are possible mechanisms for catalyzing the destruction of pollutants in anaerobic aquifers. Lovley and Lonergan [6"] have confirmed the existence of such microorganisms, by isolating a dissimilatory Fe3 +reducer (strain GS-15) that degrades toluene, phenol and pcresol. Such organisms could potentially effect desirable transformations in anaerobic-aquifers contaminated by hydrocarbons.
Methanotrophic microorganisms One of the most significant contaminants of groundwater throughout the world is trichloroethylene (TCE). Thus far, all of the numerous aerobic microorganisms that have been shown to be capable of degrading TCE appear to utilize oxygenases that fortuitously oxidize TCE in place of their normal substrates. Methane-oxidizing bacteria and their non-specific TCE-attacking oxygenase, methane mono-oxygenase (MMO), have received the most attention as possible candidates for in situ bioremediation of TCE in groundwater. MMO requires a reductant in order to perform !ts oxidative functions. Methane, the physiological reductant for methanotrophs is, unfortunately, a competitive inhibitor of TCE oxidation and is therefore not an ideal candidate for field-stimulation of TCE degradation. Henry and Grblc-Gal~c [7°o] have shown that lipid storage granules can serve as a source of endogenous • electrons for TCE degradation by starved methanotrophs, a phenomenon with significant implications for the economical and effective use of methanotrophs for in situ bioremediation of TCE-contaminated groundwaters.
Aquifer studies Bioremediation studies Actual bioremediation studies within aquifers are still fairly rare. One such study involved observations of soluble hydrocarbon biodegradation in a shallow aquifer that had been punctured with 42 monitoring wells [12..]. A strong point of this study was that material mass balances were obtained for inputs, outputs, and destruction of molecules such as benzene over a period of 3 years. The first-order attenuation rate for benzene in
138 Environmentalbiotechnology this aquifer was shown to be 0.0095 per day. Spatial relationships between dissolved oxygen (DO) and total benzene/toluene/xylene (BTX) were confirmed statistically, and laboratory microcosms were employed to confirm the DO/BTX biodegradation relationship. This is an excellent example of the thorough characterization of a spill where natural bioremediation of groundwater contamination is occurring.
versity of groundwater microbial communities. The usefulness of microorganisms for i n situ bioremediation of groundwater pollutants looks more promising each time another subsurface secret is revealed.
Semprini et al. [13"'] have presented the results from a multiyear field study" that documented the in situ biotransformations of TCE, ct~-dichloroethylene (DCE), tran~DCE and vinyl chloride in a saturated, semiconfined aquifer at the Moffett Naval Air Station, Mountain View, California [14.]. This site is not completely representative of many natural groundwater situations because it is aerobic, contains very homogeneous sands and gravels, and has relatively high water flow rates (8-10 litermin-1 at the extraction well). The data generated here, however, provide a very useful general guidance for designing bioremediation schemes using indigenous populations of methanotrophic bacteria. Indigenous methanotrophs were stimulated to degrade the target contaminants, without plugging the system with biomass, by pulsed additions of methane plus oxygen-supplemented water into an injection well. All target compounds were biotransformed, but only during times of active methane oxidation. In this system, pollutant oxidation could not be sustained under conditions of methane starvation (see [7"'1).
Papers of special interest, published within the annual period of review, have been highlighted as: • of interest •. of outstanding interest
References and recommended reading
1. ABELSONP: Cleaning Hazardous Waste Sites. Science 1989, •. 246:1097. An editorial discussing the extent of em-ironmental contamination, particularb, of ground~-ater, by toxic chemicals. Discusses the prospects for clean-up o f waste dumps and associated groundwater pollution, stressing the potential of bioremediation as a clean-up technology. 2. •,
LO~I.EYD, CH.~ELLE F, PttI1MPS E: Fe(llI)-Reducing Bacteria in Deeply Buried Sediments o f the Adantic Coastal Plain. Geology 1990, 18:954-957. Reports the existence vdthin deep subsurface aquifers of microbial populations able to couple the oxidation of organic matter to carbon dioxide, with the reduction of Fe3 +. Such populations may be useful for in situ groundwater bioremediation. 3.
LO%a.EYD: Organic Matter Mineralization with the Reduction o f Ferric Iron: a Review. GeomicrobiologyJourna11987,
5:375-399. 4. •
Loxa.EvD, B.MZDECKERM, LONERGAND, CO72AREI.H I, PHILLIPS E, SIEGELD: Oxidation of Aromatic Contaminants Coupled to Microbial Iron Reduction. Nature 1989, 339:297-299. Links mineralization of organic compounds to CO 2 with reduction of
Fe3+.
Deep subsurface microbial populations As a part of its Subsurface Science Program, the US Department of Energy has begun to characterize the diversity of bacteria in the deep sediments of the US Atlantic Coastal Plain and the basalts of the US Pacific Northwest. Fredrickson et al. [15"'] have presented a compendium of the results of the Atlantic coast studies. Bacteria were isolated from aseptically obtained core materials at periodic depths down to about 500 m. Aerobic chemoheterotrophs were present, even at great depth, to levels as high as 6.4 log col6ny-forming units per gram of sediment. A total of 198 pure cultures were compared using 108 different physiological tests. Cluster analysis grouped these bacteria into 21 different biotypes. The subsurface isolates degraded a large variety of organic acids, lipids, carbohydrates and amino acids. Only a few isolates could be readily assigned to known genera, and these tended to be representatives of the genus Pseudomonas. The great diversity observed among these bacteria implies that h~ situ bioremediation of groundwater pollution, even in the deep subsurface, may be possible.
Conclusion The work discussed here makes it clear that scientists have traditionally underestimated the capabilities and di-
5. •
IDVLEYD, PIIIIMPS E, 1.ONERG.~ND: Hydrogen and Formate Oxidation Coupled to Dissimilatory Reduction of Iron o r Manganese by Alteromonas putrefaciens~ Appl Environ Mi¢robiol 1989, 55:700-706. Reports the isolation of a pure bacterial culture capable of oxidizing formate by coupling the oxidation to reduction of Fe3+. 6. •.
LOXlEYD, LONERG&N D: Anaerobic Oxidation of Toluene, Phenol, and />Cresol by the Dissimilatory Iron-Reducing Microorganism, GS-15. Appl Environ Microbiol 1990, 56:1858-1864. Describes the first microorganism known to couple the oxidation of aromatic compounds to the reduction o f Fe3+. The bacterium is also the first described pure culture to anaerobically oxidize an aromatic hydrocarbon, toluene, indicating its potential for in situ bioremediation of anaerobic groundwaters contaminated by gasoline spills. 7. •.
IIENRYS, GRBic-GAtlC D: Influence of Endogenous and Exogenous Electron Donors and Trichloroethylene Oxidation Toxicity on Trichloroethylene Oxidation by Methanotrophic Cultures from a Groundwater Aquifer. Appl Environ Microbiol 1991, 57:236-244. Evaluates the influence o f trichtorethylene-transforming methanotrophs on TCE oxidation, and the effect of reductant axxilability on TCE transformation rates during methane starvation. Presents information required for the effec~e design of in situ bioremediation processes employing methane-oxidizing bacteria. 8. •
IIENRYS, GRBIc-GAtiC D: Effect of Mineral Media on Trichloroethylene Oxidation by Aquifer Methanotrophs. Microb Ecol 1990, 20:151-169. Presents evidence to implicate particulate I~LMOin the oxidation o f TCE by an aquifer methanotroph. Considers the role of copper in regulating TCE oxidation by this microorganism. 9. ,,
ADRL~NN, 8UFLITAJ: Reductive Dehalogenation of a Nitrogen tleterocyclie H e r b i c i d e in Anoxic Aquifer Slurries. Appl Environ Microbiol 1990, 56:292-294.
Bioremediation of groundwater pollution Crawford Demonstrates the debromination of the nitrogen heteroc3"clic herbicide bromacil by aquifer microbial populations under methanogenic, but not denitrif)ing or sulfate-reducing, conditions. Extends the range of halogenated compounds known to be reduce'ely dehalogenated in groundwater emironments. 10. •,
Kul~ K, TOWNSENDT, SUFIIr^J: Effect of Sulfate and Organic Supplements on Reductive Dehalogenation of Chloroanilines in Anaerobic Aquifer Slurries. Appl Environ Microbiol 1990, 56:2630-2637. Shows that di-, tri- and tetrachloroanilines are reduce-ely dehalogenated in methanogenic aquifer slurries, with apparent accumulation of to~c monochloroanilines as end products, indicating a problem in methanogenic bioremediation using this class of compounds. 11. •.
GmSON S, SUFIITA J: Anaerobic Biodegradation of 2,4,5TrichlorophenoxTacetic Acid in Samples from a Methanogenie Aquifer: Stimulation by Short-Chain Organic Acids and Alcohols. Appl Environ Microbiol 1990, 56:1825-1832. Describes the pathway for degradation of 2,4,5-trichlorophenox3~cetic acid in anoxic aquifers. Apparently the first report to show that aty1 dehalogenafion reactions may be stimulated in aquifer materials by supplementation with specific organic amendments. 12. •.
CHIANGC, SAL&\qTROJ, CHAI E, COLTtlARTJ, KLEINC: Aerobic Biodegradation of Benzene, Toluene, and Xylene in a Sandy Aquifer - - Data Analysis and Computer Modeling. G r o u m l Water 1989, 27:823-834. One of the most thorough studies yet of a self-purif)ing groundwater system. BTX levels in a hydrocarbon-contaminated aquifer were monitored over a 3-year period. Negative correlations for contaminant concentrations and dissolved ox)gen levels within the contaminant plume are reported; these parameters are successfully modeled by computer simulation.
13. •.
SEMPRI,\qto ROBERTSP, ItOPKLNSG, MCCARTYP: A Field Evaluation of I n Situ Biodegradation of Chlorinated Ethenes; Part 2, Results of Biostimulation and Biotransformation Experinaents. Ground Water 1990, 28:715--727. Documents the in situ biotransformation of TCE, c/~DCE, trat~DCE and xin)tchloride in a saturated semiconfined aquifer. Biotransformadon ~as effected by stimulation of indigenous methanotrophs ~ith pulsed additions of methane- and ox~!gen-supplemented w~ter introduced ~ia an injection well. 14. •
ROBERTSP, HOPKL\'SG, ~t~CKAYD, SEMPRL'qL: A Field Evaluation of In Si t u Biodegradation of Chlorinated Ethenes: Part 1, Methodology and Field Site Characterization. G r o u n d lira. ter 1990, 28:591-604. Describes the details of experimental methods and site characterization information for the aquifer experiments described in [13"]. 15. •.
FREDRICKSONJ, BAIKWILLD, ZAC~L-LqAJ, D S-M, BROCKMAN F, SL~tMONSM: Physiological Diversity and Distributions of Heterotrophic Bacteria in Deep Cretaceous Sediments of the Atlantic Coastal Plain. Appl Environ Microbiol 1991, 57:402-411. Describes the distribution of chemoheterotrophic bacteria within the deep subsurface of the Atlantic coastal plain of the US, to a depth of about 500m. Almost 200 pure cultures were isolated from aseptically obtained drilling cores. These strains were placed into 21 biotyw.s by cluster anal)sis. The great physiological diversity of observed bacteria is encouraging, with respect to future attempts at in situ bioremediation of groundwater pollution.
RL Cmvdord, Center for Hazardous Waste Remediation Research and Department of Bacteriology and Biochemistry, University of Idaho, Moscow, Idaho 83843, USA.
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