Environ. Sci. Technol. 1994, 28, 1134-1 138
Behavior of Pollutant-Degrading Microorganisms in Aquifers: Predictions for Genetically Engineered Organisms Mary Lou Krumme,t Richard L. Smith,* Jorg Egestorff,t Suzanne M. Thiem,§ James M. Tiedje,l Kenneth N. Timmls,? and Daryl F. Dwyer’9t Molecular Microbial Ecology Group, Department of Microbiology, National Research Center for Biotechnology, Braunschweig, Germany, Water Resources Division, US. Geological Survey, Boulder, Colorado 80303, and Department of Microbiology, Michigan State University, East Lansing, Michigan 48824
Bioremediation via environmental introductions of degradative microorganisms requires that the microbes survive in substantial numbers and effect an increase in the rate and extent of pollutant removal. Combined field and microcosm studies were used to assess these abilities for laboratory-grown bacteria. Following introduction into a contaminated aquifer, viable cells of Pseudomonas sp. B13 were present in the contaminant plume for 447 days; die-off was rapid in pristine areas. In aquifer microcosms, survival of B13 and FR120, a genetically engineered derivative of B13 having enhanced catabolic capabilities for substituted aromatics, was comparable to B13 field results; both bacteria degraded target pollutants in microcosms made with aquifer samples from the aerobic zone of the pollutant plume. Results suggest that field studies with nonrecombinant microorganisms may be coupled to laboratory studies with derivative strains to estimate their bioremediative efficacy. Furthermore, laboratory strains of bacteria can survive for extended periods of time in nature and thus may have important bioremediative applications.
Introduction Aquifer contamination seriously compromises groundwater resources at a time of increasing demand ( I ) . Attempts to restore groundwater quality often rely on practices which are inefficient or merely transfer pollutants from one environment to another (2-5). Biostimulation is a relatively cost-effective and environmentally sound alternative technology in which pollutant-degrading microorganisms effect contaminant removal (4-7). Where such organisms are not present in sufficient density a t contaminated sites, the native microflora may be supplemented with organisms having appropriate degradative functions. Natural selection has been slow to yield microbes that degrade xenobiotic pollutants. In some cases, organisms expressing required catabolic activities can be designed in vitro (8-11), suggesting that some genetically engineered microorganisms (GEMs) may be useful for in situ bioremediation. Many laboratory-“trained” bacteria may not be sufficiently robust for in situ applications (12, 13). To remediate aquifers, introduced bacteria must survive in substantial numbers, be transported to contaminants, and provoke an increase in the rate and extent of pollutant
* To whom correspondence should be addressed. Present address: Dept. of Civil and Mineral Engineering, University of Minnesota, Minneapolis, MN 55455; e-mail: dwyer003@ maroon. tc.umn.edu. + National Research Center for Biotechnology. U.S. Geological Survey. f Michigan State University.
*
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degradation ( 4 , 5 ) . No information exists on these aspects for bacteria introduced into polluted aquifers. To begin remedying this situation, we used field trials to observe the fate of Pseudomonas sp. B13 (B13) in a polluted aquifer. B13 metabolizes 3-chlorobenzoate (3CB) via an ortho-cleavage pathway (14)and is the parental bacterium for several GEMs with novel catabolic pathways for pollutants. One derivative, P. sp. B13 FRl(pFCR20P) (FR120), contains a hybrid pathway that is stably expressed and regulated for the degradation of methyl- and chloro-subsititued aromatics which, via two incompatible pathways, are toxic for most bacteria (15). Unfortunately, measuring biodegradative activity in aquifers is difficult to due to limited accessibility of samples as well as sorption and abiotic transformation of contaminants (16, 17). For this reason, microcosms that simulate the aquifer environment were used to assess potential in situ pollutant-degrading activity by B13 and FR120. Microcosms have the advantages of being both accessible and relatively easy to manipulate. In addition, due to regulatory constraints on environmental applications of GEMS (13),microcosms can be particularly useful in studies aimed at predicting the in situ fate and activity of these microorganisms (18-20). The strategy developed and used for predicting in situ microbial fate and activity involved: (I) introducing B13 into an aquifer and monitoring its survival and transport; (11) testing survival and degradative activity of B13 and FR120 in aquifer microcosms; (111) comparing survival of B13 in the aquifer with its survival in microcosms; and (IV) based on the data obtained, making predictions concerning the survival and pollutant-degrading potential of FR120 in the aquifer.
Experimental Methods Fate of B13 in the Aquifer. The aquifer chosen for this project is contaminated with secondarily treated sewage and serves as the U.S. Geological Survey’s Cape Cod Ground Water Contamination Study Site. The site (F347)for injection of B13 was 250m (1.5-2 yr groundwater travel time) from the contaminant source. Aquifer hydrology, the contaminant plume, and its effect on microbial populations have been described in detail (2126). During this study, specific conductivity and dissolved oxygen values at the injection site were determined as previously described (26). B13 was grown in minimal medium (27) containing 3CB (5 mM) in 100-L fermenters and harvested at mid-log phase. The bacterial slurry (4 L) was divided equally into six bottles. Bacteria in three bottles were stained with the fluorochrome 4’,6-diamidino-2-phenylindole(DAPI) (5 mM). One bottle each of DAPI-stained B13 and untreated, viable B13 were injected into the aquifer at 0013-936X/94/0928-1134$04.50/0
0 1994 American Chemical Society
Specific Conductivity
- 0
Groundwater
f
Flow
U 1.2 m
a 12-
14
Figure 1. Procedure for field testing B13. (A) Plan view of injection wells (Cl-C5). (B) Schematic showing the stepwise procedure for the injection of bacteria into the three aquifer depths. (C)Diagram of the end result: creation of 15 bacterial injectate-clouds(0.9-m diameter), five in each of the three target depths (Dl, D2, and D3) of the aquifer.
three depths (Figure l), corresponding to groundwater (i) above the contaminant plume and thus pristine (Dl), (ii) in the contaminant plume, exhibiting a reduced level of dissolved oxygen (D2), and (iii) deeper within the plume, exhibiting no measurable oxygen (D3) (Figure 2). The role of DAPI-stained B13 was primarily as an internal control used to assess the recovery of injected bacteria. To make the injections, five well points (Cl-C5), consisting of steel pipes (5 cm outer diameter) fitted with stainless steel wound wire screens (30 cm height), were driven into the aquifer at 1.2-m intervals perpendicular to the flow of groundwater (Figure 1A). The screened ends were initially placed a t the lowest depth of injection (D3), 7.9 m below surface (rnbs). B13 and the DAPIstained B13 were mixed into groundwater obtained from wells corresponding to this depth. This suspension was then pumped simultaneously into the aquifer via the five well points (Figure lB, step 1). The procedure was repeated at 6.8 and 5.4 mbs (D2 and D3, respectively) (steps 2 and 3). The result was the creation of 15 bacterial “injectate-clouds’’(Figure 1C) that were calculated, based on the amount of water injected and pore volume, to be 0.9 m in diameter. The injection depths were well below the water table, the depth ofwhich varies over time (Figure 2). Sediment samples from Dl-D3 were taken 2,6,30,70, and 158 days after injection of B13 into the aquifer using a split spoon sampler and hollow stem auger drilling rig. Cores were taken on day 2 from C1, on day 6 from C2, on day 30 from C3, on day 70 from C4, and on day 158 from C5. Both viable B13 and DAPI-stained B13 were
0
2
4 6 8 1 0 1 2 Dissolved Oxygen OWL)
Figure2. Vertical profllesfor specific conductivityand dissolvedoxygen, May 1990. Increasing values for conductivity and decreasing values for dissolved oxygen indicate the presence of the sewage plume. Injection and sampling depths are depictedas D1, D2, and D3. Profiles were simllar at later sampling dates. The water table was 4.0 mbs in May 1990 and 4.9 mbs in June 1991.
Table 1. Detection of B13 in Aquifer Samples. aquifer depth (m) 5.5-6.1 (Dl) 6.1-6.7 6.7-7.3 (D2) 7.3-7.9 7.9-8.5 (D3)
8.5-9.2
1586
sampling dates (days) 158C 158d 358e
-
-
-
-
2.98
NDh
ND
-
-
-
-
-
-
-
2.1
ND
ND
2.5 ND
ND
ND
+
+ + +
+
447f
+ + +
Taken from the following: b Injection point C5. 0.9 m downgradient from C5. d 1.8 m downgradient from C5. e Injection point C1. f 0.9 m downgradient from injection point C4.8 Log cfu/gof dws; enrichment cultures in which B13 was detected (indicated by plus sign) and not detected (indicated by minus sign). ND, dates for which enrichment cultures were not made. ~
enumerated as described below. Additional samples were taken at later dates, 158-447 days postinjection, as described in the legend to Table 1. These samples were analyzed to enumerate viable B13 at the injection sites (the DAPI stain does not fluoresce after these extended time periods) and to detect the transport of B13 downgradient from the injection sites. To detect DAPI-stained B13, bacteria were extracted from sediment (10 g) by shaking (45 min) with 90 mL of 1% Nap207 (pH 7.0) containing 1% polyvinylpyrrolidone. Aliquots of supernatant (10 mL) were filtered onto polycarbonate filters prestained with Sudan Black B. Fluorescent cells were enumerated using epifluorescence microscopy (488nm). Viable bacteria were extracted from sediment (20 g) by shaking (30 min) with 20 mL of 0.1% Na4P207, concentrated 10-fold by centrifugation, and serially diluted. Aliquots of dilutions were spread onto Environ. Scl. Technol., Vol. 28, No. 6, 1994
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agar containing 3CB (5 mM) as the substrate to selectively Positive enrichment cultures were obtained from the enumerate B13. The detection limit was 50 cfu/g of dry 4-week sample indicating that B13 was present at a density weight sediment (dws). Procedures were modified from of
