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Previous Article | Next Article 
Applied and Environmental Microbiology, January 2006, p. 622-627, Vol. 72, No. 1
0099-2240/06/$08.00+0 doi:10.1128/AEM.72.1.622-627.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
-Hexachlorocyclohexane
Laboratory of Microbial Ecology and Technology, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium
Received 14 July 2005/ Accepted 28 October 2005
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ABSTRACT |
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-hexachlorocyclohexane [-HCH]). Slow-release inoculation of Sphingomonas sp. 1-7 was established in both liquid and soil slurry microcosms using open-ended silicone tubes in which the bacteria are encapsulated in a protective nutrient-rich matrix. The capacity of the encapsulated cells to degrade lindane under aerobic conditions was evaluated in comparison with inoculation of free-living cells. Encapsulation of cells in tubes caused the removal of lindane by adsorption to the silicone tubes but also ensured prolonged biodegradation activity. Lindane degradation persisted 2.2 and 1.4 times longer for liquid and soil slurry microcosms, respectively, than that for inoculation with free cells. While inoculation of free-living cells led to a loss in lindane-degrading activity in limited time intervals, encapsulation in tubes allowed for a more stable actively degrading community. The loss in degrading activity was linked to the loss of the linA gene, encoding -HCH dehydrochlorinase (LinA), which is involved in the initial steps of the lindane degradation pathway. This work shows that a slow-release inoculation approach using a catabolic strain encapsulated in open-ended tubes is a promising bioaugmentation tool for contaminated sites, as it can enhance pollutant removal and can prolong the degrading activity in comparison with traditional inoculation strategies. |
INTRODUCTION |
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-hexachlorocyclohexane [-HCH]) constitutes a major environmental problem due to its widespread use in the past, its pronounced persistence against chemical and biological degradation, and its trend to bioaccumulate in the food chain. Although at present, the production and use of lindane are prohibited in most countries (38), several countries are still using -HCH and new sites are continuously being contaminated (16, 17, 38). Due to the introduction of lindane into the environment, the presence of this pollutant has been observed frequently in soil and (ground)water all over the world (12) at concentrations ranging from nanograms to as much as milligrams per kilogram near dumping sites (2, 13, 21, 30, 31, 39). Despite its persistence, HCH biodegradation has been reported in various environments, including aerobic and anaerobic soils. HCH-degrading microorganisms such as fungi, cyanobacteria, and anaerobic and aerobic bacteria, including members of the genera Sphingomonas, Rhodanobacter, and Pandoraea, have been widely described (for a review, see reference 25). A detailed characterization of the pathway for aerobic HCH degradation in Sphingomonas paucimobilis strain UT26 showed the involvement of lin genes encoding the HCH biodegradation pathway (24). These lin genes have since been detected in several other HCH-degrading bacteria (18, 32).
A frequently used bioremediation technique for the removal of xenobiotic chemicals from soils is bioaugmentation, i.e., the inoculation of specialized degrading bacteria. However, in most cases, the number of introduced cells decreases shortly after inoculation due to both abiotic and biotic stresses (3). Abiotic factors controlling the survival of introduced microorganisms include moisture content, temperature, pH, texture, and oxygen and nutrient availability (35). Biological factors include the predation by protozoa, a limited starvation resistance (1), and the lack of suitable niches for extended cell survival (36).
An approach to overcome some of the problems associated with microbial survival after inoculation is the formulation of inocula in protective carriers, for example, the immobilization of cells by encapsulation (37). The capsule matrix can buffer against environmental stress and pollutant toxicity (5, 23) and protects the inoculum from predation and indigenous microorganisms (29) while allowing the diffusion of gases and liquids (37). Carrier materials to encapsulate cells for biodegradation include, among others, alginate, gelatin, and 
-carrageenan gel (36). A particular successful encapsulation method using open-ended silicone tubes was applied for the degradation of 3-chloroaniline in an activated sludge bioreactor (9). This immobilization in a nutritive matrix efficiently maintained a 3-chloroaniline-degrading inoculum within the activated sludge community. Ten percent of the degradation activity was estimated to be due to the cells in the tubes, while the continuous slow release of active cells from the tubes was responsible for about 90% of the degradation.
In this study we investigated the applicability of the encapsulation technique in open-ended silicone tubes for the biodegradation of the important soil pollutant lindane. The -HCH-degrading strain Sphingomonas sp. 1-7, isolated from an HCH polluted site (22), was chosen as inoculum. The degradation after inoculation of encapsulated cells was evaluated relative to freely suspended cells in liquid and soil slurry microcosms. Degradation activity of Sphingomonas sp. 1-7 and the role of the degradative lin genes described herein were assessed in order to elucidate differences in lindane removal by both inoculation procedures.
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MATERIALS AND METHODS |
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-HCH was purchased from Sigma-Aldrich Chemie (Steinheim, Germany). Lindane has an octanol-water partition coefficient of 3.8, and its solubility in water (20°C) is 10 mg liter–1 (United Nations Environment Programme). Due to this relatively low solubility, lindane concentrations above 10 mg liter–1 cannot be monitored. It was therefore chosen to express lindane removal in percentages of added amounts of lindane.
Inoculum preparation.
For use as free-living cells, Sphingomonas sp. 1-7 was grown for 40 h at 28°C in 50 ml of a growth medium composed of mineral salts (6) and 10% Luria-Bertani broth (27), containing 50 mg of -HCH liter–1. Afterwards, 1 ml of this culture was washed twice and resuspended in 1 ml saline (0.85% NaCl) (cell density was 1.6 x 108 CFU ml–1, determined by a standard curve based on the optical density at 610 nm). The inoculum of Sphingomonas sp. 1-7 encapsulated in silicone tubes was prepared following a procedure previously described (9). One milliliter of a 40-h-grown culture (28°C) of Sphingomonas sp. 1-7 was washed twice with saline, resuspended in 1 ml saline (1.6 x 108 CFU ml–1), and mixed with 2 ml of the same growth medium as described previously but this time containing 30 g agar liter–1. Subsequently, while still fluid, the mixture was injected into a sterile silicone tube (product code 990.0040.007, 4-mm pore size, 0.7-mm wall size; Watson-Marlow, Cornwall, England). When the mixture was solidified, the tube was cut into eight pieces 2 cm long each. The silicone pieces remained open at both ends.
Adsorption and diffusion of -HCH to/through silicone tubes.
To assess adsorption of -HCH to silicone tubes, an Erlenmeyer flask containing 25 ml of distilled H2O and a 5-cm-long sterile silicone tube filled with agar suspension (the inoculum was replaced by sterile saline) was shaken (110 rpm, 28°C) with an initial amount of 0.5 mg -HCH. When -HCH was depleted, additional amounts of -HCH were added until no further removal was observed.
The diffusion of -HCH through silicone tubes was assessed by continuously (during 5 days) pumping water saturated with -HCH through an empty autoclaved silicone tube, which was then placed in an Erlenmeyer flask with 150 ml of distilled H2O. The ends of the tube were placed outside the flask to prevent the stock solution from coming in direct contact with the water. -HCH was measured in the water phase of the Erlenmeyer flask.
Biodegradation in liquid microcosms.
Liquid microcosms for biodegradation experiments were set up (in duplicate) as follows. Erlenmeyer flasks containing 50 ml liquid medium were inoculated with either an inoculum of free-living cells or an inoculum of encapsulated cells in tubes. Control flasks consisted of (i) 50 ml medium supplemented with 1 ml saline and (ii) 50 ml medium supplemented with noninoculated tubes, containing 1 ml saline in 2 ml agar medium. To each setup, 2.5 mg -HCH was added. All setups were subsequently incubated on a rotary shaker (110 rpm, 28°C). -HCH was added regularly (doses of 2.5 mg every 2 days). Duplicate samples were taken from each flask (four samples in total per treatment).
Biodegradation in soil slurry microcosms.
Similar to the setup for liquid microcosms, soil slurry microcosms consisted of Erlenmeyer flasks (in duplicate) containing 50 g (dry weight) of soil (a nonpolluted sandy loam soil from an experimental field located near Melle, Belgium [28]), supplemented with 50 ml medium to obtain a soil slurry. The composition of the soil slurry was based on the procedures described by Bachmann et al. (4). Fifty percent water (wt/wt) was used instead of 30% water (wt/wt) to provide conditions for gentle mixing. The microcosms were inoculated with either an inoculum of free-living cells or an inoculum of encapsulated cells in tubes. Control flasks consisted of (i) 50 g soil with 50 ml medium supplemented with 1 ml saline or (ii) 50 g soil with 50 ml medium supplemented with noninoculated tubes, containing 1 ml saline in 2 ml agar medium. Initially, 5 mg -HCH was added (a higher initial amount of -HCH was applied to soil slurry microcosms than to liquid microcosms due to the expected adsorption of -HCH to soil particles). All setups were subsequently incubated on a rotary shaker (110 rpm, 28°C). -HCH was added regularly (doses of 2.5 mg every 6 days; this long time interval compared to the 2-day interval in liquid microcosms was chosen due to the expected slower degradation in soil slurry microcosms). Duplicate samples were taken from each flask (four samples in total per treatment).
-HCH degradation by encapsulation in non-open-ended tubes.
To evaluate the encapsulation of cells in non-open-ended silicone tubes for -HCH degradation relative to open-ended tubes, the following experiment was set up: an open-ended inoculated silicone tube of 10 cm in length was placed in a sterile Erlenmeyer flask with 50 ml of sterile medium spiked with 5 mg -HCH (Fig. 1A). In a second sterile Erlenmeyer flask containing 50 ml of sterile medium spiked with 5 mg -HCH, an inoculated silicone tube was placed with the ends of the tube outside the flask so that the inoculum was not in direct contact with the water (Fig. 1B). The length of the tube in contact with the medium was 10 cm (only the part in contact with the medium contained inoculated agar). Since in both setups, the lengths of the tubes in contact with the medium were the same, adsorption of -HCH to the silicone tubes was considered to be identical. A control flask containing 50 ml sterile medium with a noninoculated 10-cm tube was included. The flasks (in duplicate) were incubated at 28°C and shaken at 110 rpm. Removal of -HCH from the medium was measured.
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1-7 at pH 5 was assessed by adjusting the growth medium with a 1 N HCl solution.
-HCH-degrading activity of Sphingomonas sp. 1-7.
At regular time intervals, coinciding with the sampling for -HCH analysis, 1-ml samples were taken from the liquid microcosms of the different setups inoculated with the strain in suspension and in tubes, respectively. Of these samples, 10-fold dilution series were prepared, of which 100 µl was plated on agar plates consisting of growth medium added with 50 mg liter–1 -HCH. At this -HCH concentration, part of the -HCH was insoluble and thus visible as a white precipitate. Cells that have grown on such a plate and which are able to degrade -HCH therefore create a halo of -HCH clearance around the colony. Cells grown on the plate but without the capacity to degrade -HCH lack such a halo. This feature was therefore used to distinguish between actively -HCH-degrading and nondegrading CFU.
DNA extraction and PCR amplification of linA.
Colonies with and without a halo were picked from the plates and cultured in growth medium. After growth, DNA was extracted using the Wizard Genomic DNA purification kit (Promega, Madison, WI) according to the manufacturer’s instructions. linA genes were subsequently amplified by PCR using the following primer set and temperature program: LinA F33 (5'-CGCGATTCAGGACCTCTACT-3') and LinA R418 (5'-CCAGCGGGGTGAAATAGTTC-3') (32); denaturation at 94°C for 2 min, 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, final extension at 72°C for 10 min, and hold at 4°C (8). Final concentrations in the PCR Mastermix were as follows: 0.5 mM (each) primer, 200 mM (each) deoxynucleoside triphosphate, 1.5 mM MgCl2, 10 ml thermophillic DNA polymerase 10x reaction buffer, 2.5 U of Taq DNA polymerase (Promega), 400 ng ml–1 of bovine serum albumin (Hoffman-La Roche, Basel, Switzerland), and DNase- and RNase-free filter-sterilized water (Sigma-Aldrich Chemie) to a final volume of 100 ml. Both DNA extraction and PCR amplification were verified by agarose gel electrophoresis.
Lindane analysis.
Analysis of -HCH from liquid matrices was performed by first extracting -HCH from a 2-ml sample using C18 SPE columns (product code 12102025, Bond Elut; Varian, Harbor City, CA) according to the manufacturer’s instructions. Subsequently samples were analyzed by gas chromatography (GC, CP-3800; Varian) with an electron capture detector (ECD). GC conditions were an injection temperature of 225°C, detector temperature of 300°C, and initial column temperature of 100°C (hold for 2 min); increase to 160°C at a rate of 15°C min–1 and increase to 270°C at a rate of 5°C min–1; and column pressure of 16 lb/in2. The column used was a Factor Four low-bleed capillary column (VF-5ms, 30 m by 0.25 mm inner diameter, film thickness = 0.25 µm; Varian) (method 8081A; U.S. Environmental Protection Agency [EPA]). For analysis of -HCH from soil slurry matrices, soil slurry samples were extracted with acetone-hexane (1:1) using the Soxhlet extraction method (method 3540; EPA). After clean-up with Florisil-based packed SPE tubes (product code 57053, Supelclean, Supelco; Sigma-Aldrich, Bornem, Belgium) (method 3620; EPA), samples were analyzed by GC-ECD as described above.
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RESULTS |
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-HCH to/through silicone tubes.-HCH that could be adsorbed to the silicone tubes used for encapsulation was estimated in a separate adsorption experiment. A known amount of sterile silicone tubes filled with agar suspension without inoculum was incubated with -HCH until maximal adsorption occurred. This revealed that sorption by agar-filled silicone tubes was 0.273 ± 0.001 mg per cm of tube. Since 16 cm of tubes were used in each setup of the biodegradation experiments, removal of -HCH by sorption to tubes accounted for 4.37 ± 0.01 mg.
To assess diffusion of -HCH through the silicone tubes, a saturated -HCH solution in water was continuously pumped through a silicone tube, placed in a recipient with pure water (with the tube’s ends outside the recipient). After 5 days of running saturated water through the tube, a concentration of 2.60 mg liter–1 -HCH in the recipient was measured; this indicated that diffusion of -HCH through the silicone tubes took place.
Biodegradation in liquid microcosms.
Lindane removal was evaluated for both free-living and encapsulated cells in liquid microcosms. The amount of -HCH added during the first two time intervals (5 mg) was completely removed by both free-living and encapsulated cells (Fig. 2, time interval from 0 to 4 days and 4 to 6 days). In the case of encapsulated cells in tubes, however, adsorption of -HCH to the tubes most likely caused the majority of this -HCH removal. Indeed, as depicted above, adsorption to 16 cm of tubes can account for 4.37 ± 0.01 mg, which amounts to 87.4% of the added dose. This can also be seen from Fig. 2, where -HCH removal by noninoculated tubes is shown. The amount of -HCH subsequently added was also successfully removed by both free-living cells and cells in tubes (Fig. 2, time interval from 6 to 12 days). At this point, however, removal by encapsulated cells could not merely have been caused by adsorption, since the amount of lindane added exceeded the amount that could adsorb to the tubes. Indeed, Fig. 2 shows no further removal by noninoculated tubes. After further addition of lindane (Fig. 2, time interval from 12 to 14 days on), free-living cells failed to remove the added -HCH. Encapsulated cells, on the other hand, continued lindane removal over a period of 26 days, during which five more consecutive additions of -HCH occurred. In the reference flasks, in which the inoculum was replaced by saline, no -HCH removal was observed.
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-HCH added during the first two time intervals (7.5 mg) was removed rapidly by both free-living and encapsulated cells (Fig. 3, time interval from 0 to 13 days and 13 to 22 days). Again, adsorption of -HCH to the tubes needs to be taken into account (Fig. 3). After the third addition of lindane, however, adsorption to the silicone tubes no longer contributed to the lindane removal (Fig. 3, time interval from 22 to 30 days). The removal percentage of additional lindane doses (Fig. 3, time interval from 30 to 37 days and 37 to 41 days) by freely suspended cells dropped drastically (from 80% to 13% and further to 8%), while lindane removal by encapsulated cells remained substantial (55%), although it decreased to 25% in the final time interval.
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-HCH degradation by encapsulation in non-open-ended tubes.-HCH degradation by cells encapsulated in non-open-ended tubes relative to open-ended tubes. While the total amount of 5 mg -HCH that was spiked to flasks with an inoculated open-ended tube was completely removed after 2 days of incubation, non-open-ended inoculated tubes were not able to completely degrade this amount (Fig. 4).
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-HCH-degrading activity of Sphingomonas sp. 1-7.
At the start of the biodegradation experiment in liquid microcosms, cells inoculated as free-living cells were more numerous in suspension than cells released from the inoculated tubes (Fig. 5A). At this point, most cells that were inoculated in tubes were still encapsulated inside the tubes and few cells were released from the tubes. The first days of the experiment, the percentage of actively degrading cells was significantly less (P < 0.01) for cells inoculated as free-living cells compared to cells released from tubes (Fig. 5B). After day 12, the point at which no more lindane degradation was observed in the setups inoculated with free-living cells (Fig. 2), cell density in microcosms inoculated with free-living cells dropped (Fig. 5A) although all surviving cells were active (Fig. 5B). Microcosms inoculated with cells in tubes preserved a relatively high cell density (Fig. 5A); moreover, the cells all remained active degraders (Fig. 5B).
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DISCUSSION |
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1-7, was increased by encapsulation of the strain in open-ended, nutrient-rich silicone tubes. In both liquid microcosms and soil slurry microcosms, encapsulation of cells in tubes ensured sustained biodegradation of -HCH. In liquid microcosms inoculated with encapsulated cells, the ability to degrade lindane remained for more than twice the duration relative to inoculation with free-living cells. Though adsorption to silicone tubes caused the removal of the initially spiked -HCH, removal of additional lindane was attributed to biodegradation. Similar results were observed in soil slurry microcosms, although the effect of prolonged biodegradation of lindane was less pronounced (extension of 1.4 times the period of degradation activity), which is most probably related to a low level of bioavailable lindane in the soil slurry relative to the aqueous solution (7).
Previous research has shown that encapsulation often increases the rate of survival of the introduced microbial inoculum (19, 26, 33). Results showed that initially, Sphingomonas sp. 1-7 was more numerous in suspension when inoculated as free-living cells than with cells inoculated in tubes. Interestingly, shortly after inoculation, the percentage of actively degrading cells—as monitored by the occurrence of lindane clearance around the colonies—was only about 10% for cells inoculated as free-living cells. At the time when lindane degradation was not observed any more in the setups inoculated with free-living cells, cell density in microcosms of the latter dropped significantly, although all remaining surviving cells were active. Microcosms inoculated with cells in tubes showed a more stable cell density, with cells that remained active degraders. It has been described earlier that the lindane degradation phenotype can be lost rather easily (11). Furthermore, studies have revealed that lindane degradation stability is strain dependent and correlates to the presence or absence of degradative lin genes (18). Since our results indicate that encapsulation preserves the degradation activity of cells compared to freely inoculated cells, it was hypothesized that encapsulation of the unstable lindane degrader Sphingomonas sp. 1-7 better preserved its degradative lin genes. This hypothesis was supported by PCR amplification analysis that showed that the loss in degrading activity was related to the loss of the degradative gene linA.
Increased metabolic activity with encapsulated cells has been observed in several other studies (14, 15), although the precise mechanisms responsible for this effect are not known. The main advantage of the use of encapsulation in open-ended silicone tubes for the biodegradation of 3-chloroaniline was previously described to be attributed to the continuous release of nonstarved cells, resulting in a higher metabolic rate (9). In this study, the nutritive environment inside the tube and the continuous presence of lindane, due to adsorption to the tubes, may contribute to an increased metabolic activity. In the case of inoculation of the soil slurries, protection against predation could be an additional advantage of encapsulation (1).
The particular success of encapsulation in open-ended tubes was evaluated by a parallel inoculation with non-open-ended tubes. While complete -HCH removal was reached after 2 days with inoculated open-ended tubes, non-open-ended tubes failed to completely degrade the same amount. Since adsorption effects were equal in both setups, it could be concluded that slow release of highly active cells from open-ended tubes was the principal cause of degradation, although encapsulated cells were also active. The latter activity is plausible since the tube contains a nutritive agar sustaining cell growth and since lindane can diffuse through the silicone tubes. However, any degradation product that cannot diffuse through the silicone tube will accumulate inside the tube, possibly causing toxic or inhibitory effects on the catabolic strain (10). We showed that a pH drop occurred inside the non-open-ended tube, possibly causing the failure in degradation, which is supported by the fact that Sphingomonas sp. 1-7 could not be grown at pH 5. This problem did not arise when using the open-ended tubes, since any degradation product can freely diffuse through the open ends. It could also be argued that diffusion of lindane through the silicone wall of closed tubes is the rate-limiting step in lindane degradation, therefore causing reduced degradation compared to open-ended tubes. However, the pH drop observed inside the tubes indicates that a substantial amount of lindane can diffuse through the silicone walls. Stagnation in degradation in closed-ended tubes therefore seems to be due to a pH drop inside the tubes, which apparently does not occur in open-ended tubes.
This inoculation technique opens perspectives for future research. It is well known that the biodegradation of contaminants that are present in low concentrations is often very difficult (20). The adsorption characteristics of silicone tubes could be exploited to concentrate these contaminants to overcome this problem. The encapsulation in tubes was also shown to increase the stability of degradative genes, which could eliminate the need to inoculate only strains with very stable degradative genes. This technique has been applied before in a bioreactor setup (9). Further applications of such tubes could be directed toward pump and treat technology in which contaminated groundwater can be dealt with in an external reactor inoculated with encapsulated contaminant-degrading strains. However, since it often is not possible to locate and remove the residual -HCH, remediation also needs to focus on preventing further migration of the dissolved contamination. This plume control must be maintained for a long period of time. A cost-effective approach for the remediation of contaminated aquifers is the installation of permeable reactive zones or barriers within aquifers. As contaminated groundwater moves through a permeable reactive zone, the contaminants are scavenged or degraded, and uncontaminated groundwater emerges from the down-gradient side of the reactive zone (34). An HCH-degrading inoculum encapsulated in tubes could be used as a reactive barrier. A first step toward the development of this technology for application in soil bioremediation will, however, need to focus on longer time intervals more relevant to soil bioremediation. In addition, the use of biodegradable instead of silicone tubes needs to be investigated in order to develop an in situ bioremediation technique by mixing inoculated tubes with the soil.
In conclusion, this study showed the sustained removal of lindane by inoculation of a lindane-degrading strain, Sphingomonas sp. 1-7, encapsulated in open-ended silicone tubes. In both liquid and soil slurry microcosms, the biodegradation capacity was prolonged by encapsulation of the cells compared to inoculation of free-living cells. Encapsulation was shown to preserve the degrading activity of cells by increasing the stability of degradative genes. Since the tubes contain a nutritive agar which sustains cell growth while allowing contact with the pollutant, encapsulated cells have the opportunity to feed on the lindane in a nutrient-rich environment and can subsequently release highly active cells. These findings suggest that encapsulation in open-ended tubes is a promising tool for soil bioremediation, also for sites contaminated with compounds other than lindane.
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ACKNOWLEDGMENTS |
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Petra Vandamme is thanked for her technical assistance with molecular analysis. We further thank Vincent Denef, Bram Pauwels, and Korneel Rabaey for their helpful comments in redrafting the manuscript.
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FOOTNOTES |
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REFERENCES |
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-carrageenan-encapsulated and unencapsulated Pseudomonas aeruginosa UG2Lr cells in forest soil monitored by polymerase chain reaction and spread plating. FEMS Microbiol. Ecol. 16:71-82.-hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26 are localized in the periplasmic space without molecular processing. J. Bacteriol. 181:5409-5413.-hexachlorocyclohexane-degrading bacterium. J. Bacteriol. 178:6049-6055.
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