Environ. Sci. Technol. 2010, 44, 5159–5164
Effect of Dechlorination and Sulfate Reduction on the Microbial Community Structure in Denitrifying Membrane-Biofilm Reactors HUSEN ZHANG,† MICHAL ZIV-EL, BRUCE E. RITTMANN, AND ROSA KRAJMALNIK-BROWN* Center for Environmental Biotechnology, Biodesign Institute, Arizona State University, Tempe, Arizona, 85287
Received March 3, 2010. Revised manuscript received May 18, 2010. Accepted May 25, 2010.
Recent studies showed that the chlorinated solvents trichloroethene (TCE), 1,1,1-trichloroethane (TCA), and chloroform (CF) were reductively dehalogenated in a H2-based membrane biofilm reactor (MBfR) under denitrifying conditions. Here, we describe a detailed phylogenetic characterization of MBfR biofilm communities having distinctly different metabolic functions with respect to electron-acceptor reduction. Using massively parallel pyrosequencing of the V6 region of the 16S rRNA gene, we detected 312, 592, and 639 operational taxonomic units (OTU) in biofilms of three MBfRs that reduced nitrate; nitrate and TCE; or nitrate, sulfate, and all three chlorinated solvents. Comparative community analysis revealed that 13% of the OTUs were shared by all MBfRs, regardless of the feed, but 65% were unique to one MBfR. Pyrosequencing and realtime quantitative PCR showed that Dehalococcoides were markedly enriched in the TCE+nitrate biofilm. The input of a mixture of three chlorinated compounds, which coincided with the onset of sulfate reduction, led to a more diverse community that included sulfate-reducing bacteria (Desulfovibrio) and nitrate-reducing bacteria (Geothrix and Pseudomonas). Our results suggest that chlorinated solvents, as additional electron acceptors to nitrate and sulfate, increased microbial diversity by allowing bacteria with special metabolic capabilities to grow in the biofilm.
Introduction Contamination of groundwater by chlorinated solvents such as trichloroethene (TCE), 1,1,1-trichloroethane (TCA), and chloroform (CF) is widespread (1, 2). Of the 1,673 National Priorities List (NPL) sites identified by the U.S. Environmental Protection Agency, 382 were contaminated with TCE, 450 contained TCA, and 387 contained CF (3). The drinking water maximum contaminant levels (MCL) for TCE, TCA, and CF are 5, 200, and 80 µg/L, respectively (4). Under anaerobic conditions, chlorinated solvents can be biologically reduced to less chlorinated forms via a process called reductive dechlorination (5), in which the chlorine atoms are sequentially replaced with hydrogen atoms. Early * Corresponding author phone: 1-480-727-7574; fax: 1-480-7270889; e-mail:
[email protected]. † Present address: Department of Civil, Environmental, and Construction Engineering, University of Central Florida, Orlando, Florida 32816. 10.1021/es100695n
2010 American Chemical Society
Published on Web 06/04/2010
studies showed that mixed microbial cultures could dechlorinate some chlorinated compounds (6, 7). Later, it was shown that TCE-dechlorinators are phylogenetically diverse and include members of the genera Dehalobacter, Desulfitobacterium, Sulfurospirillum, Desulfuromonas, Geobacter, and Dehalococcoides (8, 9). To date, complete reductive dechlorination of chlorinated ethenes has been exclusively linked only to members of the genus Dehalococcoides (10–14). TCAdechlorinators include Dehalobacter (15–17), sulfate-reducing bacteria (18), and methanogens (19). Interactions among the biological reductions of TCE, TCA, and CF are of great interest, since these compounds may be found together in contaminated groundwater (20) and one compound might be inhibitory to the biodegradation of another. For example, TCA and CF inhibited TCE degradation in a Dehalococcoidescontaining consortium (21). The H2-based membrane biofilm reactor (MBfR) can be used to reduce and detoxify multiple oxidized contaminants (22, 23), including chlorinated solvents (24–26). The MBfR delivers H2 gas by diffusion through a hollow-fiber membrane to a biofilm growing on the outer surface. Oxidized contaminants diffuse through the biofilm from the opposite direction. Microorganisms in the biofilm use H2 as the electron donor and the oxidized contaminants as electron acceptors. We previously demonstrated that a denitrifying MBfR was capable of dechlorinating ∼93% of the incoming TCE to ethene after 110 days (24). We also demonstrated that a mixture of TCE, TCA, and CF was bioreduced in an MBfR (25). In that study, 83% of the TCE, 95% of the TCA, and 99% of CF was dechlorinated to ethene, chloroethane, and chloromethane, respectively, after 120 days. Although sulfate was present in the feed to all these reactors, sulfate reduction was only observed in the MBfR fed with the solvent mixture. The objective of this study was to understand the impacts of different electron acceptors on the structure of dechlorinating bacterial communities of MBfR biofilms. Based upon the performance of MBfR (24, 25), which is summarized in the previous paragraph, we hypothesized that Dehalococcoides would thrive in a TCE-dechlorinating community, but a TCA-, CF-, and TCE-dechlorinating community would be more diverse, since Dehalococcoides is not known to reduce TCA or CF. To test this hypothesis, we quantified total Bacteria, Archaea, and members of Dehalococcoides in the biofilms using quantitative real-time PCR. In parallel, we used massively parallel pyrosequencing to define the structures of the microbial communities developed in the biofilms. We placed a special focus on microorganisms that were enriched or depleted in response to dechlorination.
Materials and Methods MBfR Operation and Biofilm Sampling. The biofilms analyzed in this study were taken from the MBfRs whose performance was reported previously (24, 25). Briefly, the three bench-scale MBfRs were inoculated with the effluent taken from a pilot-scale denitrifying MBfR reactor at La Puente, California. The MBfRs were cylindrical with a total volume of 24 mL. H2 gas was supplied from inside the fiber in a bubble-less fashion at 0.17 atm (2.5 psi) throughout the experiments. The MBfRs had a main bundle of 32 hollow fibers and a single sampling fiber outside the main bundle. Dissolved oxygen was removed from the feed medium by periodically purging N2 gas. The feed medium, containing 0.36 mM nitrate and 0.8 mM sulfate, was supplied at a flow rate of 1.0 mL/min to each MBfR until complete denitrification occurred, which took about 20 days. One MBfR (MBfRVOL. 44, NO. 13, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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NS) continued to receive the same influent. A second MBfR (MBfR-TCE) had 7.6 µM TCE added to its influent, and a mixture of TCE (7.6 µM), TCA (7.5 µM), and CF (8.4 µM) was added to the feed medium of the third MBfR (MBfR-mix). We took biofilm samples from MBfR-NS, MBfR-TCE, and MBfR-mix at 60, 110, and 120 days, respectively, after the start-up of the MBfRs, or day 0. A schematic of the MBfR can be found elsewhere (24, 26). We took the biofilm samples when the reactors’ performance was at steady-state, as evidenced by the stable concentrations of electron acceptors and reduced products in the effluent. 16S rRNA-Gene Pyrosequencing. We placed a section of the sampling fiber into the bead-solution in the MoBio Ultra Clean Soil DNA kit, and extracted DNA from biofilms on the sampling fiber (2-3 cm sections) following manufacture’s instructions. The DNA concentrations were: 5.1 ng/µL for MBfR-NS, 7.4 ng/µL for MBfR-TCE, and 7.9 ng/µL for MBfRmix. Using extracted DNA as the template, we generated the PCR-amplicons of the V6 region of the 16S rRNA-gene with the primer 967F and 1046R (27) using the PCR conditions described earlier (28). We pooled three PCR reactions starting from 0.2, 1, and 2 µL of templates to reduce PCR bias (29). The amplicons were pyrosequenced with a massively parallel 454 GS-FLX according to standard protocols involving emulsion PCR and sequencing by synthesis (28, 30). After removing low-quality pyrosequencing reads and trimming off low-quality bases (27, 28, 31), we classified the high-quality pyrosequences by finding their full-length matches in a reference database (32, 33), which is annotated according to the bacterial taxonomy maintained by the Ribosomal Database Project (RDP) Release 10 (34). We deposited the pyrosequencing reads obtained in this paper to the NCBI Short Read Archive (http://www.ncbi.nlm.nih.gov/Traces/ trace.cgi?) under accession no. SRA010187. Analysis of Community Structures Using OTU-Based Approaches. We aligned all pyrosequencing tags using the “Infernal” aligner (34, 35), which uses the secondary structure information of the 16S rRNA to help achieve a correct alignment. We calculated distance matrices using the above aligned pyrosequences. By setting a 0.05 distance limit (equivalent to 95% similarity), we binned the very high number of pyrosequences into “genus-based” operational taxonomic units (OTUs). For each individual MBfR, we calculated rarefaction, the Shannon diversity index, and the Chao1 estimator, which estimates the true total number of OTUs with infinite sampling (36). To describe similarity and difference between the three MBfR communities, we obtained OTUs that were shared between communities and OTUs that were unique to a community. We use the computer program “mothur” to calculate the Chao1 and Shannon values, as well as to calculate shared and unique OTUs (37). Quantification of Bacteria, Dehalococcoides, and Archaea with Real-Time Quantitative PCR. The primer and TaqMan probe sequences and the annealing temperatures used in QPCR amplification were described earlier in (24, 38, 39) for quantifying total Bacteria and Dehalococcoides, and in ref 40 for quantifying total Archaea. We used plasmid DNA containing the 16S rRNA gene of Dehalococcoides strain BAV1 as standards for quantifying Bacteria and Dehalococcoides, and we used plasmid DNA containing the 16S rRNA gene of Methanosarcina thermophila (GenBank accession: M59140) as standards for quantifying Archaea. Negative controls included water instead of template DNA in the PCR reaction mix. We performed triplicate PCR reactions for all samples and negative controls.
Results MBfR Performance. The performance of the three MBfRs was previously reported in detail (24, 25). Here, the fluxes of electron acceptors (in e- eq m-2 d-1) are summarized in 5160
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TABLE 1. Summary of Electron-Acceptor Fluxes in the Three MBfRsa chemical fluxes (eq m-2 d-1)
nitrate sulfate TCE TCA CF
MBfR-NS
MBfR-TCE
MBfR-mix
0.33 0b NP NP NP
0.14 0b 0.0162c NP NP
0.33 1.18 0.0033 0.0042 0.0048
a The flux values are summarized from previous studies (24, 25). b The flux is zero because sulfate reduction did not occur in these conditions. c Cumulative flux as TCE to ethene. NP: not present. eq: electron equivalents.
Table 1. Chlorinated solvents represented small fractions of the total fluxes, the majority of which came from either nitrate or nitrate + sulfate. MBfR-NS and MBfR-TCE established full denitrification within 20 days, but no sulfate reduction occurred (24). MBfR-mix, which had mixed solvents, achieved denitrification and sulfate reduction within 5 days (25). At the time of biofilm sampling, MBfR-TCE and MBfR-mix had partially dechlorinated TCE to vinyl chloride and ethene. MBfR-mix also had partially dechlorinated TCA and CF to dichloroethane and chloromethane, respectively. Effect of Chlorinated Solvents on MBfR Biofilm Community Structures. Pyrosequencing produced a total of 82 706 high-quality V6 tags of the 16S rRNA-gene (Table 2). As a way of assessing to what extent the sequencing effort covered the species richness of a community, we generated rarefaction curves, which are presented in Figure 1. The rarefaction curve was based on an OTU definition of 95% sequence similarity. Despite the uneven number of sequences in the three MBfRs, more OTUs were observed in the MBfRs with TCE or mixed solvents than in the one with only nitrate and sulfate. Although we observed 312, 592, and 639 OTUs in MBfR-NS, MBfR-TCE, MBfR-mix, respectively, none of the rarefaction curves reached saturation, which means that, regardless of whether or not solvents were added, more sequences are needed to fully sample the communities’ diversity. Figure 2 shows the taxonomic breakdown at the bacterial class level for the three MBfR bacterial communities. Overall, the MBfR biofilm communities contained sequences from more than 20 bacterial classes. The majority of the sequences belonged to Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, and “Dehalococcoidetes”, with the remainder spread among Acidobacteria, Actinobacteria, Verrucomicrobiae, Bacilli, Clostridia, and other minor groups. Out of the three MBfRs, the TCE-fed community was most highly enriched with members of “Dehalococcoidetes”. This MBfR also had the most Gammaproteobacteria. The relative abundances of Deltaproteobacteria and Acidobacteria were markedly higher in MBfRs fed with chlorinated solvents than in the control MBfR, and this was particularly true for the MBfR receiving the mixture of solvents. Though a breakdown at the class level provides us a broad overview of the high bacterial diversity detected by pyrosequencing, a comparison of MBfR community structures at the genus level allowed us to infer functions, based on the assumption that phylogenetically closely related members share similar metabolic capabilities. Pyrosequencing detected 126 genera in the RDP taxonomy in the MBfR-TCE, while a previous clone-library approach detected only eight genera (24). MBfRs -NS and -mix had 80 and 109 genera, respectively. Thus, pyrosequencing provided much greater detail on the less abundant species, making it possible to observe that the MBfR biofilms were highly diverse.
TABLE 2. Chlorinated Solvents in the MBfR Influent, Dechlorination Products in the Effluent, Biofilm Sampling Time, and the Number of Sequences Obtained in Each Sample MBfRs (electron acceptors added)
biofilm sampling time from Day 0 (day)
dechlorination end products at the time of sampling
number of high-quality sequences (total )82,706)
MBfR-NS (nitrate + sulfate) MBfR-TCE (nitrate + sulfate + TCE) MBfR-mix (nitrate + sulfate + TCE + TCA + CF)
60 110 120
N/A ethene and vinyl chloride (TCE removal: 93%) TCE: ethene and vinyl chloride (removal: 83%); TCA: dichloroethane (removal: 95%); CF: chloromethane (removal: 99%)
10 971 31 850 39 885
Figure 3 shows the relative proportions of the most abundant bacterial genera in the three MBfRs. The first observation is that members of Dehalococcoides were highly enriched in the TCE-MBfR, but not in the MBfRs without TCE or with the mixed solvents. However, Dehalococcoides were present in all MBfRs. The second observation is that the most abundant genera in MBfR-NS and MBfR-TCE were
FIGURE 1. Rarefaction curves based on pyrosequencing of MBfR biofilm communities. Sequences that are 5% or less divergent to each other were combined as one OTU. SR ) sulfate reduction.
FIGURE 2. Taxonomic breakdown (at the class level) of pyrosequences from three MBfRs. The relative abundance of a class within a reactor community was defined as the number of sequences affiliated with that class divided by the total number of sequences in that reactor. MBfRs -NS, -TCE, and -mix had sequences affiliated with 15, 19, and 20 classes, respectively.
denitrifying bacteria, including Stenotrophomonas (41) and Dechloromonas (42–44). This reflects that the microbial ecology in the biofilms correlates appropriately with denitrification being the major electron-accepting process in these MBfRs (Table 1). Figure 3 also shows that the mixed solvents and sulfate reduction altered the community significantly, strongly enriching for three genera: Desulfovibrio, Geothrix, and Pseudomonas. The sulfate-reducing bacteria Desulfovibrio rose from undetectable with no solvent to 7.2% in relative abundance when mixed solvents were present, while the iron- and nitrate-reducing Geothrix increased from undetectable to 5.2%. Even more pronounced was the increase in Pseudomonas, which became the most abundant genus (26%) in the presence of mixed solvents and sulfate reduction. The 16S rRNA-gene copy numbers of Dehalococcoides measured with QPCR are presented in Figure S1 of the Supporting Information. They followed the same trends as the relative abundance of this group measured with pyrosequencing. The relative abundance of Dehalococcoides, measured as a fraction of total bacteria, was highest (12.1%) in MBfR-TCE and lowest (1.0%) in MBfR-mix. The number of Dehalococcoides in MBfR-TCE was (4.9 ( 2.3) × 106 (average ( standard error of the mean) copies per cm2 of biofilms, and was highest among the three reactors. The reactor MBfRNS had significant numbers (3.3 × 105 copies of the 16S rRNAgene per cm2 of biofilm) of Dehalococcoides, despite that there were no chlorinated solvents added in the influent. The number of Dehalococcoides in MBfR-mix was (1.2 ( 0.0026) × 106 (average ( standard error of the mean) copies per cm2 of biofilms. Archaea in all MBfR biofilms were below the detection limit, which was about 120 copies of the 16S rRNA-gene per cm2 of biofilm. These data suggest that methanogenesis did not play an important role as a H2 sink in our systems. We estimated the richness (number of OTUs) and diversity of a community with Chao1 and Shannon index, respectively. MBfR-mix had the highest richness (884 OTUs) and diversity (4.47) among the three MBfRs. In contrast, although the number of OTUs in MBfR-TCE was 816 and markedly higher than 473 in MBfR-NS, the Shannon indices of these two
FIGURE 3. Predominant bacterial genera whose relative abundance (%) was markedly increased or decreased by TCE, mixed chlorinated solvents, or sulfate reduction (SR). VOL. 44, NO. 13, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Venn diagram showing how the three MBfR communities overlapped and the taxonomic identities of the shared OTUs. Next to each MBfR’s name is the total number of OTUs in that reactor community. The sum of total observed OTUs in all three MBfRs was 1,043. The number of OTUs shared by all three MBfRs was 135. An OTU is defined by 5% sequence dissimilarity as a proxy for a genus. The phylum affiliations of OTUs shared by all MBfRs are shown in the pie chart. reactors were about the same (3.87 and 3.84, respectively) and much lower than that of MBfR-mix. Since the Shannon index depends on richness and evenness (the relative abundance of each OTU), and MBfR-TCE had higher richness than MBfR-NS, the similar Shannon index of MBfR-TCE and NS indicates that the OTUs in the TCE-fed community were not as evenly distributed as those in the no-solvents community, as seen by a highly enriched Dehalococoides (Figure 2). Figure 4 illustrates bacterial OTUs that are unique to an MBfR community, or shared by two or three MBfR biofilm communities. We used OTUs at the 5% sequence distance, which is the cutoff value commonly used as a proxy for a genus (45). A core community consisting of 135 OTUs, or 13% of the total OTUs, was shared by all three MBfRs. The majority (74%) of the 135 core OTUs were alpha, beta, and gamma subdivisions of Proteobacteria, which harbors denitrifying genera such as Stenotrophomonas and Dechloromonas. No Deltaproteobacteria were in this shared core. This agrees with the observation that sulfate reduction occurred only in the MBfR-mix. OTUs that were unique to each community numbered 107 (MBfR-NS), 264 (MBfR-TCE), and 307 (MBfR-mix), and together they accounted for 65% of the total number of observed OTUs.
Discussion Our results agree with previous observations that Dehalococcoides were detected in environments that had not been exposed to TCE (46), suggesting their natural occurrence in the inoculating groundwater and in a variety of different environments. Dehalococcoides strain FL2 was isolated from river sediment that had not been exposed to chlorinated solvents (10). Other known dechlorinating bacteria, such as Dehalobacter (47), Desulfuromonas (48, 49), and Desulfitobacterium (50), were not detected in our MBfR communities. The nondetection of Dehalobacter could be due to the inhibitory effect from TCE, as shown in a recent report (16). The absence of added organic carbon in our experiments may have selected against dechlorinators that require organic electron donors. For example, Desulfuromonas chloroethenica and Desulfuromonas michiganensis use acetate, but not H2 as their electron donor (48, 49). Dehalococcoides uses H2 as the electron donor and acetate as the carbon source for dechlorination (51). In an autotrophic system such as ours, no acetate or other organic carbon were added. Dehalococcoides had to rely on acetate produced by other members of the microbial community. It has been speculated that homoacetogens such as Sporomusa (52) and Acetobacterium sp. strain HAAP-1 (53), played important roles in dechlorinating mixed cultures, possibly synthesizing 5162
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acetate autotrophically and providing it as a carbon source to Dehalococcoides. Spirochaetes also have been repeatedly found in Dehalococcoides-containing cultures (10, 53–55). We found low levels of Spirochaetes, 86 and 8 sequences in MBfR-TCE and MBfR-mix, respectively. The phylum Spirochaetes harbors homoacetogenic bacteria, such as some Treponema (56). The number of Spirochaetes OTUs was low despite that an earlier study (24) found them in a clone library having a much smaller sampling size. An inherent PCR primer bias may have limited our ability to detect Treponema, because our postsequencing analysis indicated that only 4 out of 1822 Treponema sequences in the RDP database (release 10) have perfect match to the forward PCR primer used in this study. The input of TCA and CF, the onset of sulfate reduction, or both seemed to have had an inhibitory effect on Dehalococcoides. The fraction of Dehalococcoides sequences decreased from 21% in MBfR-TCE to 1.3% in MBfR-mix. TCE reduction was completely inhibited by 3.8 µM of TCA and 5.2 µM of CF (21). Later, it was shown that dechlorination of cis-DCE and VC by the KB-1 type of Dehalococcoides was inhibited by TCA (15). CF (1.6 µM) was shown to inhibit cis-DCE dechlorination (51). The proliferation of the sulfate-reducing bacteria Desulfovibrio in MBfR-mix agrees with our observation of sulfate reduction in this reactor (25). In contrast, we did not detect Desulfovibrio in the biofilms from MBfR-NS and MBfR-TCE, which also agrees with the observation that no sulfate reduction occurred in those systems (24). The mixed solvents contained TCA, which can be tolerated by Desulfovibrio (15). Based on the nondetection of the Dehalobacter and the abundance of Desulfovibrio, it is conceivable that some Desulfovibrio could be responsible for TCA-degradation in MBfR-mix. Wrenn and Rittmann (18) found that sulfate reduction competed with TCA-dechlorination for electrons. Inhibiting sulfate reduction in MBfRs may be a viable way of increasing H2 availability to TCA dechlorination. In summary, although the electron equivalents used for reductive dechlorination were 100 times smaller than for the dominating electron-accepting processes of nitrate and sulfate reductions, the addition of low concentrations of chlorinated compounds significantly changed the composition of the biofilm community. The relative abundance of Dehalococcoides increased markedly in response to the addition of a low concentration of TCE as a single chlorinated solvent, but it decreased when a mixture of chlorinated solvents including TCA and CF were fed and sulfate reduction took place. High-throughput sequencing of the biofilm community enabled detection of other major community shifts related to the input of chlorinated solvents (in MBfRs-
TCE and -mix) and the onset of sulfate reduction (in MBfRmix). For example, the input of a mixture of three chlorinated compounds, which coincided with the onset of sulfate reduction, led to a more diverse community that included sulfate-reducing bacteria (Desulfovibrio) and nitrate-reducing bacteria (Geothrix and Pseudomonas). Pyrosequencing of PCR-amplified 16S rRNA-genes has improved the resolution of microbial community analysis for MBfR biofilms compared with the clone-library based approach. Sequencing the metagenome would provide more functional, as well as phylogenetic, information about the microbial community.
Acknowledgments We thank Dr. Frank Lo¨ffler for providing the plasmid controls containing the 16S rRNA gene of Dehalococcides BAV1. The funding of the study was provided by Aptwater (Pleasant Hill, CA).
Supporting Information Available Figure S1. This material is available free of charge via the Internet at http://pubs.acs.org.
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