Environ. Sci. Technol. 2010, 44, 3621–3628
Anaerobic Biodegradation of BTEX by Original Bacterial Communities from an Underground Gas Storage Aquifer SABRINA BERLENDIS,† JEAN-FRANC ¸ OIS LASCOURREGES,‡ BLANDINE SCHRAAUWERS,‡ PIERRE SIVADON,† AND M I C H E L M A G O T * ,† Equipe Environnement et Microbiologie, IPREM UMR 5254 Universite´ de Pau et des Pays de l’Adour, 64013 PAU, France, and APESA, He´lioparc, 2 avenue du Pre´sident Angot, 64053 PAU, France
Received January 13, 2010. Revised manuscript received March 27, 2010. Accepted March 31, 2010.
BTEX biodegradation by an indigenous deep subsurface microbial community was evaluated in a water sample collected in the area of an underground gas storage. Five different sulfatereducing microbial communities able to use at least either benzene, toluene, ethylbenzene, or xylene (BTEX) compounds were studied. A total of 21 different bacterial phylotypes were identified, each community containing three to nine bacterial phylotypes. Archaeal phylotypes were retrieved from only three communities. The analysis of 16S rRNA gene sequences showed that i) these consortia were mainly composed of novel species, some of which belonging to bacterial groups not previously suspected to be involved in BTEX anaerobic degradation, ii) three consortia were dominated by an uncultured Pelobacter sp. previously detected in biodegraded oil reservoirs, iii) a deeply branching species distantly affiliated to Thermotogales was abundant in two consortia, and that iv) Firmicutes related to the Desulfotomaculum and Carboxydocella genera represented the only three detectable phylotypes in a toluene-degrading consortium. This work shows that subdominant microbial populations present in a deep subsurface aquifer used for seasonal underground gas storage could be involved in the natural attenuation of the traces of BTEX coinjected with methane in the deep subsurface.
Introduction For the last two decades, many studies have been devoted to the characterization of deep subsurface microflora in terrestrial (1) and oceanic ecosystems (2). Beside the scientific fundamental interest for the description of both the huge microbial diversity of the deep biosphere (3) and the metabolic activities in deep geological formations (4), many publications report investigations on the relationships that could occur between subsurface industrial activities and deep endogenous microflora. They mainly concern the oil industry (5) and the underground storage of nuclear waste (6). * Corresponding author phone: +33 559 407 482; fax: +33 559 407 494; e-mail:
[email protected]. † IPREM UMR 5254 Universite´ de Pau et des Pays de l’Adour. ‡ APESA. 10.1021/es100123b
2010 American Chemical Society
Published on Web 04/09/2010
Natural gas is stored in deep geological formations in several countries such as Germany, Denmark, the United States, Russia, England, and France (7). The natural gas stored in deep aquifers is mainly composed of methane, associated with trace amounts of higher molecular weight hydrocarbons, some of which being found in produced water (8). Benzene and alkylbenzenes are the most soluble monoaromatic hydrocarbons in water and thus represent the best candidates as electron donors for anaerobic microbial activities in such deep environments. Nevertheless our knowledge of the microbiology of underground gas storage is limited to a few studies (9-13) although microbial processes might play a key role in natural attenuation of these compounds. Studying deep subsurface environments must conciliate technical difficulties for sampling at high depth with specific procedures to collect the most representative biological samples (14). Such requirements were recently met and made it possible to describe the microbial diversity of a deep aquifer used for seasonal underground gas storage at a depth of 830 m below ground level (9). This aquifer, approximately aged 150 million years, is confined in a rauracian calcareous geological formation isolated from the surface by an impermeable geological limestone formation. It is poorly fed by surface water, the turnover of the entire water formation being estimated about 140,000 years. The microbial community characterized by Basso et al. (10) did not suggest evidence of any influence of gas storage on the microbial ecology of the aquifer, at least in the surroundings of maximum extension reached by the gas bubble where the water sample was collected. Many previous observations, resulting from many years of operation of underground gas storage facilities in different countries, nevertheless indicated that microbial activity within the zone of gas storage is different from that observed in the undisturbed aquifer (13). Active underground biological sulfate-reduction was frequently observed, sometimes needing gas treatment to remain below the commercial gas specified limits of 5 mg · m-3. Sulfur isotopic fractionation linked to sulfatereduction (J. Connan and D. Dessort, personal communication) suggested that H2S is produced through biological sulfate reduction resulting from the introduction of exogenous carbon and energy sources in a nutrient-limited ecosystem. Several questions arose from these observations: i) what are the bacterial species involved in these processes, ii) what is their origin, either indigenous or introduced in the aquifer as the consequence of cyclic gas storage, and iii) what are the carbon and energy sources for bacterial metabolism? The results reported in this paper showed that different anaerobic microbial communities able to use BTEX as carbon and energy sources can be selected through microcosm experiments from an indigenous microbial community of gas storage aquifer. This suggests that, although the microflora of the undisturbed aquifer is driven by lithoautotrophic metabolisms as shown by Basso et al. (10), BTEX-degrading subpopulations are also present in the environment and might be involved in the natural attenuation of hydrocarbons in the gas storage area.
Experimental Procedures Samples. Anoxic water samples of the deep aquifer formation (-830 m) situated at about 100 km west of Paris (France) were aseptically collected at the wellhead of a peripheral monitoring well after a specific cleaning procedure as described previously (9). The anaerobic liquids and samples filtered on-site used in this study were designated sample S4 by ref 9. Samples were transported to the lab under anaerobic VOL. 44, NO. 9, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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conditions and processed the day after, as previously described (9, 10). Microcosm Experiments and Subcultures. BTEX biodegradation microcosms A, B, and C were prepared using the filter-concentrated biomass collected at the wellhead as inoculum (9). The Sterivex filters were aseptically dismantled in the anaerobic glovebox under an atmosphere of H2/CO2/ N2 (5/5/90). Thirty membranes fixed on their plastic support were dropped in 1 L of formation water and then vigorously agitated with a magnetic stirrer to detach bacteria. This approximately 160-fold concentrated biomass suspension was supplemented with 9 mM NH4Cl, 2 mM K2HPO4, and 2 mM KH2PO4, 1 mL of trace elements (15), and 1 mL of vitamin solution (16). Fifty-mL aliquots were transferred to 100-mL penicillin flasks sealed with thick butyl rubber stoppers, in which different electron acceptors had been added. Duplicates microcosms were carried out for each condition. A third flask was used as abiotic control after the addition of 5 mL of 1 M HCL. Microcosms B and C were supplemented with 20 mM oxihydroxide FeIII solution. Sodium fumarate was also added to microcosm C at the final concentration of 20 mM. A mixture of benzene, toluene, ethylbenzene, and m-, p-, and o-xylene (Sigma-Aldrich) was then added at a final concentration of 30 ppm (v/v) each. Subcultures VNA, VNB, and VNC1-3 were prepared in the same way using synthetic water (0.01 g L-1 NaF; 1 g L-1 MgCl2 (6H2O); 3.5 g L-1 Na2SO4; 0.09 g L-1 KCl; 0.84 g L-1 CaCl2 (2H2O); 4.7 g L-1 NaCl; 0.87 g L-1 NaHCO3), of which composition mimicked that of the formation water. pH was adjusted to 7.2. Culture media were sterilized by autoclaving prior to the addition of vitamins, FeCl2 solution (0.2 g L-1), and Na2S (9H2O) (0.08 g L-1). A 10% inoculum and the BTEX mixture were finally added. Incubations were carried out under static conditions at 37 °C in the dark. Analytical Procedures. BTEX concentrations were analyzed periodically by SPME/GC/FID with an autosampler CP-8200 (Varian) coupled with a gas chromatograph CP3800 (Varian) equipped with a flame ionization detector. Aqueous samples (0.3 mL) were transferred from microcosms to chromatographic vials and then acidified with 10 µL of a 3 N HCl. A microextraction fiber (100 µm PDMS, Supelco) was used to adsorb BTEX in headspace vials during 6 s. Desorption time inside the GC injector was 2 min. Compounds were separated through an AT-Wax (Alltech) column (30 m × 0.32 mm × 0.25 µm). Helium was used as a carriergas with a constant flow rate of 1 mL min-1. Since abiotic loss was observed during prolonged incubation times, BTEX biodegradation was estimated by measuring differential disappearance of these compounds in the test microcosm and in an acidified control microcosm prepared under the same conditions. Results are expressed as the residual percentage of BTEX as (Ct/Cc)*100, where Ct is the compound concentration in the test microcosm, and Cc the compound concentration in the abiotic control microcosm. DNA Extraction. Genomic DNAs were extracted in duplicate from microbial communities using the Ultraclean soil DNA isolation kit (Mo Bio Laboratories) by sampling 1.5-2 mL from each microbial cultures. Bacterial and archaeal ssu rRNA genes were selectively amplified from genomic DNA using the PCR Core Kit Plus (Roche). t-RFLP Analysis. Bacterial ssu rRNA genes were amplified with the 5′ end tet-labeled forward primer A8F (5′-tetrachlorofluorescein phosphoramidite-agagtttgatcctggctcag-3′) and the B926R (5′-ccgtcaattcctttgagttt-3′) reverse primer (17). Archaeal genes were amplified with 5′ end tet-labeled forward primers EK4F (5′-ctggttgatcctgccag-3′) (18) and A958R reverse primer (5′-yccggcgttgamtccaatt-3′) (19). For t-RFLP analysis, tet-labeled PCR products were excised and cleaned using the GFX DNA and Gel Band purification kit (Amersham). Six µL of each purified PCR product were 3622
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supplemented with 2 µL of sterile Milli-Q water and then digested for 3 h with restriction enzyme RsaI (Biolabs). The tet-labeled PCR fragments were digested in duplicate to ensure good repeatability and to distinguish unspecific fragments from putative partial digestions. One µL of restriction digests was then mixed with 20 mL of deionized formamide and 0.5 mL of a TAMRA-labeled Genescan 500 bp internal size standard (Applied Biosystems), denatured for 5 min at 95 °C, and immediately transferred to ice. Restriction samples were then analyzed in a Genscan ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The statistical analysis of t-RFLP data (20) was used to discriminate signal noise and “true” peaks within t-RFLP electrophoregrams according to a critical value for noise ratio equal to 3. On the basis of t-RFLP data, a Hierarchical Agglomerative Cluster analysis (HAC) based on the minimum variance criterion of Ward was done (21) by using the Ade4 package (22) with the R software (http://www.R-project.org). Construction and Analysis of ssu rRNA Libraries. 16S bacterial rRNA genes were amplified with the forward bacterial primer 8f and reverse universal primer U1492R (5′ttccggttgatccygccgga-3′) (17). DNA amplicons of appropriate sizes were purified using the GFX DNA and Gel Band purification kit (Amersham), cloned with the Topo TA cloning 10F’ kit (Invitrogen) in TOP 10 E. coli cells according to the manufacturer’s instructions. Inserts of appropriate size were sequenced by QIAGEN Genomic Services (Hilden, Germany). A preliminary screening of the 16S rRNA gene sequences was done with the ClustalW and MEGA 4.1 softwares (23). Putative chimera sequences were detected using the Chimera Check Software program of the Ribosomal Database project (http://rdp.cme.msu.edu). Sequences displaying more than 97% sequence identity were assembled as a single phylotype (24). The selected phylotypes were analyzed for nearest neighbor by the BLAST software on the National Center for Biotechnology Information database (http://blast.ncbi.nlm.nih.gov/). The ssu-rRNA sequences reported in this study were deposited in GenBank database with Accession No. GU339468 to GU339488. Diversity Analysis. EstimateS 8.2 software (www.viceroy.eeb.uconn.edu/EstimateS) was used to estimate the richness (Chao2) applied on the total number of t-RFs estimated after statistical treatment of t-RFLP data and total number of phylotypes retrieved on rRNA genes libraries (25). Coverage was deduced using Good’s equation (26): C ) (1ni/N)*100 where ni is the number of singletons within a library, and N is the total number of clones in the library.
Results and Discussion Selection of Anaerobic Microbial Communities with Distinct BTEX Degradation Capacities. In order to assess the potential for anaerobic biodegradation of BTEX by the gasstorage aquifer indigenous microflora, in vitro microcosm experiments were set up using a mixture of benzene (B), toluene (T), ethylbenzene (E), and the three isomers of xylene (o-X, m-X, p-X) as carbon and energy sources. Microcosms were prepared in the sampled aquifer water containing 90 mg · L-1 sulfate and supplemented with various terminal electron acceptors including nitrate, Fe(III), or CO2. Fumarate was also added in some experiments. A large variety of anaerobic metabolisms was consequently investigated depending on the type of microorganisms involved, including denitrification, iron and sulfate reduction, fermentation, and methanogenesis. Because of nutrient limitation in the deep subsurface (5) the microbial population was extremely low in the aquifer (i.e., 104 bacteria per mL). A filtered-concentrated biomass was thus used as inoculum as described earlier (10), giving an initial bacterial concentration of approximately 106 bacteria per mL in microcosms.
TABLE 1. BTEX-Degrading Microbial Consortia culture conditionsb consortia A B C VNA VNB VNC1 VNC2 VNC3
origina S4 S4 S4 A2 B4 C2 C2 C1
degradation profilesc (days)
pH
TEA
[BTEX] (ppm)
B
T
E
o-X
p-X
m-X
7.7 7.7 7.7 7.6 7.7 7.7 7.7 6.5
2-
30/cpd 30/cpd 30/cpd 10/cpd 80 10/cpd 10 8
∞ ∞ ∞ ∞ ∞ 50,120
100,200 10,100 10,100 100,130 20,60 30,60 -
200,300 ∞ ∞ 300,650 ∞ -
200,300 ∞ ∞ 300,650 ∞ -
100,150 ∞ 500,600 100,130 125,160 -
50,100 ∞ 500,600 30,50 120,130 20,60
SO4 Fe3+ Fe3+ fum SO42SO42SO42SO42SO42-
a Origin of the inocula. S4, Sample aquifer water collected at the sample point called S4 (9); An, Bn, and Cn, consortia isolated from the corresponding consortia listed in the first column. Subscript gives the number of enrichments from the initial culture. b TEA, terminal electron acceptor; [BTEX], BTEX compound concentrations; cpd, compound. c Numbers describe culture times at which degradation was detected and was completed, respectively. B, benzene; T, toluene; E, ethylbenzene; o-X, ortho-xylene; p-X, para-xylene; m-X, meta-xylene; ∞, no biodegradation observed; -, BTEX compound not tested.
Twenty-two duplicate BTEX biodegradation experiments and their abiotic controls were initially set up, using different electron acceptors and substrate mixtures, under otherwise identical conditions. Biodegradation was detected in only 3 conditions (Table 1, conditions A, B, and C). Under sulfatereducing conditions (condition A) with no additional terminal electron acceptor added, all BTEX but benzene sequentially disappeared after approximately 300 days of incubation, starting by m-X after a 50-day lag period, then p-X, T, o-X, and finally E. In condition B with Fe(III) added, the biodegradation of toluene was achieved in less than 100 days. No other aromatic compound disappeared upon subsequent incubation. Another biodegradation pattern was observed in condition C with fumarate, known to play as cosubstrate in benzyl succinate synthase-driven metabolization of alkylbenzenes (27, 28), and Fe(III). Toluene disappeared first, followed by the very late disappearance of p-X and m-X after a 500-day lag period. The production of iron sulfide black precipitates in the three experimental conditions indicated active sulfate-reduction. Several outlines suggested that the disappearance of BTEX in these experiments could be assigned to biodegradation. First, BTEX loss in degradation experiments and not in abiotic controls was observed in only three out of the 22 independent experiments, ruling out the possibility of abiotic loss of any compound. Second, the fact that the degradation process was sequential and selective, depending on the type of molecule and the experimental condition, also excluded the involvement of an abiotic mechanism, considering that all chemical and physical characteristics of these molecules are similar. Last, the reproducible biodegradation profiles upon subcultures, as described below, reinforced the hypothesis that they were the result of a biological phenomenon. In order to select stable anaerobic BTEX-degrading microbial communities, subsequent subcultures of microcosms A, B, and C were prepared using artificial synthetic water mimicking the mineral composition of the aquifer water. This led to the selection of five microbial communities designated VNA to VNC3 (Table 1). VNA was obtained after two successive transfers under sulfate-reducing conditions similar to those used to obtain microcosm A (Table 1). The BTEX biodegradation pattern is identical to that of consortium A (Figure 1) and thus designates VNA as a deep subsurface wide spectrum BTEX-degrading microbial consortium. Since the presence of bacterial spores was observed in microcosm B, an aliquot of this microbial community was heated at 80 °C for 10 min to kill vegetative cells prior to inoculation of a subculture with BTEX and sulfate as electron donor and acceptor, respectively. Three subsequent transfers under the same conditions were done, leading to the selection of the
microbial community VNB (Table 1), a consortium able to rapidly degrade toluene (Figure 1). Three additional distinct communities were selected from microcosm C. Community VNC1 was transferred twice with sulfate as electron acceptor and 100 mM each of the BTEX mixture. Its biodegradation pattern was identical to that of C. VNC2 was subcultured twice under the same conditions but with m-X as the single electron donor. VNC3 was selected in a more complex manner: when microcosm C was reinoculated under sulfatereducing conditions at a lower pH (6.5), the sequential biodegradation of toluene and benzene was observed. This community was then subcultured with benzene as single electron donor and designated VNC3, a consortium slowly degrading benzene in 120 days (Table 1 and Figure 1). This biodegradation activity was then shown to be reproducible upon subcultures (data not shown). The anaerobic microbial community sampled from the deep aquifer sample presented a wide degradation potential for T, E, o-X, p-X, and m-X. Moreover, benzene degradation was observed through selection at a lower pH. Diversity Analysis of the BTEX-Degrading Consortia. t-RFLP analysis was carried out in order to investigate the biodiversity of the microbial communities involved in the anaerobic biodegradation of BTEX as well as to estimate the similarities between the five microbial consortia. t-RFLP profiles of the bacterial 16S rRNA genes amplified from the five microbial communities and digested with the restriction enzyme RsaI, displayed a limited number of t-RFs, indicating a low microbial diversity (Table 2). Species richness determined according to Chao2 model on the basis of t-RFs abundance showed that the observed species richness was in agreement to the average of the theoretical values (25). The size heterogeneity of the t-RFs observed suggested that different bacterial species are involved in BTEX biodegradation in the different consortia (Supporting Information). Interestingly, consortia VNC1, VNC2, and VNC3 shared two major bacterial t-RFs (456 and 485 bp) (Supporting Information). Communities VNC1-3 and VNA were also weakly related, sharing the 456-bp major t-RF. On the other hand, the VNB consortium exhibited two major 489-bp and 459bp TRFs covering more than 80% of the specific richness, that were not shared with the other consortia. Archaeal ssu rRNA gene amplification, either by direct or nested PCR, was unsuccessful in consortia VNA, VNB, and VNC3. t-RFLP archaeal profiles of VNC1 and VNC2 were simpler than bacterial ones, with one dominant 76-bp t-RF representing more than 70% of the total fluorescence in consortia VNC1 and VNC2, out of a total of six T-RFs. To provide a deeper insight into the microbial diversity of the BTEX-degrading consortia, microbial diversity of each VOL. 44, NO. 9, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. BTEX biodegradation kinetics of VNA, VNB, and VNC3 consortia. a) Consortium VNA. BTEX values were normalized to the undegraded benzene values. b) Consortium VNB. Arrows indicate when VNB microcosm was spiked again with toluene. c) Consortium VNC3. BTEX biodegradation is expressed as the residual percentage of BTEX, (Ct/Cc) × 100, where Ct is the compound concentration in the test microcosm, and Cc the compound concentration in the abiotic control microcosm. Filled triangles: benzene; filled diamonds: toluene; filled squares: ethylbenzene; open squares: p-xylene, open diamonds: m-xylene, open triangles: o-xylene. BTEX-degrading selected microbial community was characterized by constructing and analyzing small-subunit rRNA clone libraries of the Bacteria and Archaea domains. Although the same DNA region than for t-RFLP analysis was amplified, a different set of primers was used in order to avoid similar putative methodological PCR bias between both approaches. Twenty-two unique phylotypes were identified from the sequencing of 465 clones (Table 3), confirming the overall low microbial diversity. Only three to seven and zero to two different bacterial and archaeal phylotypes were detected in the different consortia, respectively. The accumulation curves (data not shown) and the calculation of the coverage index C (Table 2) theoretically established that the bacterial clone libraries mostly covered the complete diversity of all BTEXdegrading consortia, with a good correlation between the number of phylotypes observed and the Chao2 index (Tables 2 and 3). Furthermore, the estimated total number of species correlated well with similar values predicted by t-RFLP 3624
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analysis (Tables 2 and 3), the VNA and VNC1 consortia displaying the highest diversity by both methods. A majority of the 22 characterized phylotypes belonged to the phyla δ-Proteobacteria and Firmicutes, most of them being affiliated with phylotypes previously detected in other subsurface environments (Table 3). This observation is consistent with data previously reported on the microbial diversity of this gas storage aquifer (10). Similarly, 78% of the phylotypes found in the BTEX-degrading consortia could not be affiliated to any cultured microorganism as they showed less than 97% of 16S rRNA sequence identity with their closest relatives (24). The identified phylotypes clearly separated the five BTEX-degrading microbial communities into three distinct groups significantly different in terms of microbial populations (Table 3). The main characteristic of the VNC1-3 consortia was the dominance of a Pelobacter phylotype (VNC3B001) representing approximately 70% of the clone library. Considering its low sequence similarity with validly described species, this phylotype most probably represents a new bacterial species within this genus. Interestingly, the closest related environmental gene sequence in databases, displaying 100% identity, was retrieved from a low-temperature biodegraded petroleum reservoir in Canada in which anaerobic hydrocarbon biodegradation was supposed to occur (29). Moreover, the second dominant phylotype in the benzene degrading consortium VNC3 (VNC3B005) represented 24% of the bacterial diversity and was distantly affiliated to Thermotogales. Theoretical t-RF sizes calculated from in silico digestion of 16S rRNA gene sequences of the Pelobacter- and a deeply branching Thermotogales-related phylotypes were identical to the major t-RFs observed in t-RFLP profiles (490 and 456 bp) (Supporting Information), thus suggesting that t-RFLP analysis and clone libraries, which gave consistent information, adequately described the actual microbial diversity of these consortia. Except for the presence of another Pelobacter-related phylotype in VNC2, the other bacterial signatures in these three consortia were mainly related to sulfate-reducing bacterial genera and species. The presence of these bacteria is consistent with the observed sulfatereduction in microcosms. Archaeal clone libraries were only constructed for consortia VNC1 and VNC3, revealing the presence of two equally abundant phylotypes (VNC3A001, VNC3A005; Table 3). One was related to the euryarchaeotal Methanolobus genus and the other to a yet undescribed species. Analysis of the bacterial clone library from consortium VNA showed the predominance of two phylotypes (VNABa05 and VNC3B005). The most abundant one (VNABa05; 56% of the clones) is identical to the deeply branching bacterial phylotype found in the VNC1 and VNC3 consortia. The second dominant phylotype in the VNA population was distantly related to the genus Desulfomonile. However, considering their low sequence similarity, it may represent a still undescribed genus. Two other minor phylotypes displayed high nucleic acid sequence similarity with sulfate-reducers previously found in deep subsurface environments, including the Desulfomicrobium baculatum species (30). As expected, heat-treatment of the community VNB resulted in the selection of spore-forming Gram-positive bacteria after two successive transfers. Consistently with t-RFLP analysis, 16S rRNA gene cloning described VNB as the less complex bacterial consortia of the five BTEXdegrading microbial communities characterized in this work. It was composed of only three distinct phylotypes, one related to the genus Desulfotomaculum (VNC1B071), the two others being distantly related to other Firmicutes (VNBB003 and VNBB004). Interestingly, the closest related sequences in databases were characterized from other subsurface environments such as a water sample from the kalahari shield
TABLE 2. Comparison of Phylotype Diversity Estimated from t-RFLP Analysis and Small Subunit rRNA Gene Librariesf
consortia
VNA VNB VNC1 VNC2 VNC3 aquifer microflora (sample S4)
clone nba
Nb of phylotypes
Cb
Chao2 (phylotypes)d
total t-RFs detected in each consortiumc
Chao2e (TRFs Rsa1)
82 55 86 61 87 94
6 3 9 5 6 20
97.6 100 97.7 100 96.6 90.0
6 (13) 3 (7) 9 (19) 5 (11) 6 (13) 20 (101)
9 5 11 7 6 12
6 (13) 3 (7) 7 (15) 4 (9) 6 (13) 12 (24)
a Total number of clones that were sequenced. b Coverage of the bacterial clone libraries calculated according to the model of Good (26) where C ) (1-ni/N)*100 with ni as the number of singletons within a library, and N as the total number of clones in the library. c Total number of t-RFs detected after t-RFLP analysis and statistical treatment as proposed by Abdo et al. (20). d Species richness predicted by the Chao model (25) on the basis of the total number of OTUs, singletons, and doubletons observed in the bacterial clone libraries. e Species richness predicted by the Chao model on the basis of the total number of TRFs observed on each t-RFLP diversity profile. f Calculations of species richness were made using the EstimateS software. Values into brackets correspond to the upper bound of the 95% confidence interval for the Chao 2 estimator.
in South Africa (31). The sequence of the third phylotype (18% of the library) is closely related to that of another undescribed organism from a deep subsurface environment. Relationships between the Original Community, Degrading Consortia, and BTEX Biodegradation. The low species diversity in the BTEX-degrading consortia, but also their heterogeneous species composition and the occurrence of bacterial species shared by the original community described by Basso et al. (10), strongly suggested that the microbial BTEX-degrading populations were subsets too scarce to have been detected in the indigenous aquifer S4 community. This is best illustrated by VNB in which the most abundant phylotype (VNC1B071), representing 71% of the clone library, was previously detected as a minor component of the original aquifer community (VNc094, 2.5% of the S4 community). In this consortium, the selection pressure applied by the heat treatment led to a very simple composition of the bacterial community, with only three phylotypes phylogenetically related to the spore-forming Firmicutes, and probably representing still undescribed bacterial species. The high phylogenetic distances with validly described species even suggested that they might represent new genera (Table 3) and clearly established that they are different from the single Gram-positive anaerobe described for its ability to oxidize toluene, o- and m-xylene under sulfate-reducing conditions so far (32, 33). As such, these bacteria represent an original toluene-degrading community. In the subcultures originating from microcosm C, δ-proteobacteria dominated the species diversity, in particular in the m-xylene degrading consortium VNC2. Similar results were obtained by Nagakawa et al. (34) on a p-xylenedegrading consortium. However, a more detailed analysis showed that there was no common species between these two studies. The broad dominance of an uncultured Pelobacter phylotype in all three VNC consortia may suggest that this undescribed Pelobacter species likely plays a role in the BTEX degradation by the VNC consortia. Moreover, the fact that the same 16S rRNA gene has been cloned from different subsurface environments undergoing hydrocarbon biodegradation sustains this hypothesis (29). In an experiment which is not reported here, we showed that although selected with benzene as single electron donor, consortium VNC3 has also the ability to degrade toluene, and that toluene biodegradation in stopped when molybdate, a sulfate-reduction inhibitor, was added (Supporting Information). Thus, considering that Pelobacter species are not known as able to use sulfate as electron acceptor, it might be reasonably assumed that the Pelobacter species detected here could be involved in syntrophic degradation of BTEX with sulfate-reducing bacteria (Table 3). On the other hand, the methanogenic Archaea detected in the VNC3 consortia is not likely involved in this
biodegradation process, since toluene biodegradation is not inhibited by the addition of 2-bromoethanesulfonate. When subcultured with benzene as sole carbon and energy source at pH 6.5, the structure of the VNC3 bacterial community showed an important difference when compared to the VNC1 and VNC2 consortia. More particularly, the selection pressure exerted by benzene seemed to favor the development of a bacterial phylotype distantly related to the deeply branching Thermotogales (VNC3B005), which represented 24% of the VNC3 bacterial gene library. This observation might suggest the involvement of this phylotype in the biodegradation of benzene. This is nevertheless not supported by the biodegradation profile of VNA, which showed no benzene degradation although this phylotype represents the dominant (56%) bacterial population. Therefore, the role of this bacterial species should be analogous to that of the Pelobacter sp., i.e. the biodegradation of different BTEX in syntrophy with other bacterial populations in VN consortia. Finally, although this uncultivated bacterium is phylogeneticaly weakly related to deeply branching thermophilic bacterial groups, the culture conditions used during the course of this study suggest that it is a mesophilic organism, like the so-called “Mesotoga” whose existence has been postulated in several polluted environments (35). The only consortium described in this work as able to degrade ethylbenzene (VNA) is characterized by the high proportion of a sulfate-reducing-related δ-proteobacteria phylogenetically different from those associated with the Pelobacter species in consortia VNC1-3. Considering that we assumed that SRB in consortia VNC1-3 are likely involved in syntrophic biodegradation, the VNA SRB-related phylotypes should on the other hand be more directly involved in the TEX biodegradation in consortium VNA, as the ethylbenzene-degrading strain EbS7 (36). All these considerations represent new targets to design future research strategies to understand the role of each bacterial type in the biodegradation processes. The phylogenic considerations discussed here should allow, at least in some specific cases, to design culture media for the isolation and characterization of these uncultured microorganisms. This classical microbiological approach is nevertheless obviously not sufficient to link bacterial types to substrate consumption profiles. Several recent techniques aimed at deciphering the metabolic role of the different species present in a consortium have been developed during the past decade, including for instance stable isotope probing (37) or FISH analysis (32, 34). Their application to the study of bacterial consortia such as those described in this work nevertheless still faces several major challenges: the duration of biodegradation processes that can occur across years, the very small VOL. 44, NO. 9, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Phyla
closest related environmental sequences
b
89
Desulfotomaculum geothermicum (AJ621886) Bellilinea caldifistulae (AB243672) Anaerobaculum thermoterrnum (EU276415) Thermosipho melaniensis (FN556063) Cytophaga sp. AN-B14 (AM157648) Thermovirga lienii strain Cas60314 (DQ071273)
93 96 98 96 99 99
Methanolobus sp. R15 (EF202842) Thermococcus mexicalis (AY099181)
98 98
c
86
80
96
91
82
82
85
97
93
99 94
99
99
99
92
93
Desulfobulbus sp. BG25 (DSU85473) Desulfomonile limimaris sp. (NR_025079)
94 94
90
Carboxydocella ferrireducens strain 019 (EF092457)
Pelobacter acetylenicus (X70955)
98
98
Desulfotomaculum sp. Ox 39 (AJ577273)
Desulfuromonas alkaliphilus (DQ309326)
98
98 90
89
96
Desulfovibrio alkalitolerans (GQ863489) Desulfobulbus rhabdoformis (U12253)
90 94
99 100
Desulfobacca acetoxidans (AF002671)
91
95
%
Desulfomicrobium baculatum DSM 028T (AM419440) Delftia sp. PHD-6 strain (DQ301783) Desulfotomaculum geothermicum (AJ621886)
Pelobacter acetylenicus (X70955)
closest relative species
100
%
56
1
9
1
9
24
1
VNA
11
18
71
VNB
50
50
3
1
1
15
2 8
68
VNC1
3
11
5
81
VNC2
OTUs abundance (%)b
50
49
1
3
24
1
1
70
VNC3
Sequence accession number is given into brackets. OTUs detected in both t-RFLP and ssu rDNA cloning analyses are in bold. δ-Proteobacteria; β-Prot., β-Proteobacteria; Firm., Firmicutes; Chlor., Chloroflexi; Therm., Thermotogae; Bacter., Bacteroidetes; Synerg., Synergitetes; EuryA, Euryarchaeota. OTU, operational taxonomic unit.
a
Archaeal ssu rRNA Gene Libraries deep coal seam groundwater of VNC3A001 EuryA northern Japan (AB294253) anaerobic digestion of VNC3A005 Unknown EuryA sludge (CU916807)
Bacterial ssu rRNA Gene Libraries production waters of a VNC3B001 δ-Prot. low-temperature biodegraded oil reservoir (AY570613) microbial populations in contaminated VNC3B026 δ-Prot. sediments (DQ404843) VNC1B049 δ-Prot. LCFA-degrading microbial communities (DQ984657) VNC1B050 δ-Prot. Pearl River estuarine sediments (EF999343) alkaliphilic Fe(III), Mn (IV) -reducing bacterium VNC1B013 δ-Prot. from soda lake (DQ309326) hydrocarbon-degrading consortia from oil VNC2Bg11 δ-Prot. sands tailings settling basin (EU522642) VNC2Bb08 δ-Prot. Pearl River estuarine sediments (EF999343) perchloroethene-respiring microorganisms VNABa05 δ-Prot. in anoxic river sediment (EF667559) dolomite aquifer 896 m below VNABa08 δ-Prot. the surface (AY604056) VNAB098 β-Prot. deep-sea methane vents (EU622281) VNC1B071 Firm. ridge flank crustal fluids (DQ079650) subsurface water of the Kalahari VNABa03 Firm. Shield, South Africa (DQ234639) subsurface drilled fluid at VNBB003 Firm. 3-4 km depth (AY768822) subsurface water of the Kalahari VN3BB004 Firm. Shield, South Africa (DQ234639) a muddy hot spring in VNC3B043 Chlor. southwestern Taiwan (FJ638525) thermophilic anaerobic hybrid reactor VNABd11 Chlor. degrading terephthalate (AY297966) volatile fatty acid -degradiing VNC3B005 Therm. microbial community (AB195914) fault bordered aquifers in the Miocene VNC3B008 Bacter. formation of northernmost Japan (AB237702) production water from an Alaskan VNC3B092 Synerg. mesothermic petroleum reservoir (EU721809)
OTUs
ssu rDNA nucleic acid sequence identitya
TABLE 3. Phylogenetic Affiliation and Abundance of Phylotypes Retrieved from Bacterial and Archaeal Small Subunit rRNA Gene Librariesc
population of microorganisms, and the small culture volumes necessary to manipulate to ensure good anaerobic conditions. Natural Attenuation of BTEX in Gas Storage Aquifers. Two types of investigations have been conducted on a unique water sample obtained from a Rauracian aquifer collected on a peripheral control well of an underground storage of natural gas in October 2002 (9, 10). The first study described the bacterial biodiversity of the noncontaminated aquifer (10). It showed that the dominant bacterial population was composed of hydrogenotrophic, autotrophic bacteria. The work presented here focused on the in vitro potential for biodegradation of monoaromatic hydrocarbons by this microbial community. It showed that besides lithoautotrophic bacteria that are the primary producers of biomass in the undisturbed deep aquifer, there are bacterial subpopulations displaying genetic and physiological potentials to biodegrade hydrocarbons in anaerobic conditions. The selection pressure imposed by BTEX as electron donors led tosignificantchangesinthestructureofbacterialcommunities. These in vitro findings may explain, at least in part, common observations made during routine operation of underground gas storages. Indeed, studies on several sites have shown that traces of BTEX associated with stored gas in the area of operation underwent an isotopic fractionation of carbon and hydrogen during storage (D. Dessort, personal communication). These observations strongly suggest the occurrence of anaerobic degradation of BTEX within deep geological formations. This leads to the conclusion that at shutdown of industrial operations, a natural attenuation process in underground gas storages would allow the gradual elimination of these compounds from the environment, possibly making the aquifer returning to its original state as a lithoautotrophic ecosystem. Our results provide novel and useful indications on the involvement of indigenous microorganisms from deep aquifer gas storage on the ability to anaerobically degrade BTEX compounds, which were not previously taken into consideration in this context.
Acknowledgments We are grateful to Dr. Anthony Ranchou-Peyruse for his help during the preparation of the manuscript.
Supporting Information Available One additional table (correspondences between terminal restriction fragments (TRFs) obtained in silico from 16S rRNA gene sequences and TRFs detected by t-RFLP analysis) and three additional figures (effects of inhibitor addition on toluene biodegradation in a consortium originated from successive enrichments of VNC, electrophoregrams from t-RFLP analysis of bacterial 16S rRNA gene of BTEX-degrading consortia VNA, VNB, VNC1, VNC2, and VNC3, and electrophoregrams from t-RFLP fingerprinting profiles from archaeal 16S rRNA gene of BTEX-degrading consortia VNC1 and VNC2). This material is available free of charge via the Internet at http://pubs.acs.org.
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