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Applied and Environmental Microbiology, April 2009, p. 2238-2245, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.02556-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Diverse and Novel nifH and nifH-Like Gene Sequences in the Deep-Sea Methane Seep Sediments of the Okhotsk Sea{triangledown} ,{dagger}

Hongyue Dang,1* Xiwu Luan,2* Jingyi Zhao,1 and Jing Li1

Centre for Bioengineering and Biotechnology, China University of Petroleum (East China), Qingdao 266555, China,1 Key Laboratory of Marine Geology and Environment, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China2

Received 10 November 2008/ Accepted 22 January 2009


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ABSTRACT
 
Diverse nifH and nifH-like gene sequences were obtained from the deep-sea surface sediments of the methane hydrate-bearing Okhotsk Sea. Some sequences formed novel families of the NifH or NifH-like proteins, of currently unresolved bacterial or archaeal origin. Comparison with other marine environments indicates environmental specificity of some of the sequences, either unique to the methane seep sediments of the Okhotsk Sea or to the general deep-sea methane seep sedimentary environments.


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INTRODUCTION
 
Shallowly buried methane hydrates sustain a significant biomass and productivity in the deep-sea environment, potentially requiring a large supply of fixed nitrogen (25). The largest reservoir of nitrogen in the ocean is dissolved dinitrogen gas, which is abundant in deep seawater (23). Microbial nitrogen fixation may provide a source of reactive nitrogen in the carbon-rich and nitrogen-limited methane seep environment (15). Besides bacteria, archaea were also found to possess the ability of nitrogen fixation (5), potentially important in oligotrophic open seas (21) and in hydrothermal vent and other deep-sea extreme environments (22-24). Archaeal nitrogen fixation may be important in the deep-sea methane seep environment, although very little is known currently (25). To test this hypothesis, a molecular study of the putative nitrogen-fixing microbial community was carried out for the surface sediments of the methane hydrate-bearing Okhotsk Sea.


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Sampling and DNA extraction.
 
Sediment core samples were collected from the LV39-25H and LV39-40H stations of the Okhotsk Sea during the CHAOS (hydro-Carbon Hydrate Accumulation in the Okhotsk Sea) international research expedition of 24 May to 18 June 2006 (20). Methane hydrates were discovered in shallow layers of both cores. Undisturbed surface sediments down to a 5-cm depth were sampled using sterile techniques and stored in liquid nitrogen during the cruise and at –80°C after returning to the laboratory. Sediment DNA was extracted by a previously established procedure (8).


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PCR, cloning, and sequencing.
 
Bacterial and archaeal nitrogenase reductase genes (nifH, including anfH and vnfH) were amplified with primers of Mehta et al. (23). Archaeal 16S rRNA genes were amplified with primers Arch21F and Arch958R (9). Methyl coenzyme M reductase A genes (mcrA) associated with methanogenic or anaerobic methane-oxidizing archaea were amplified with primers ME1 and ME2 (11). Primers AOM39_F and AOM40_R were also used to specifically amplify group b mcrA sequences (12). PCR products were cloned and sequenced by previously established protocols (6, 7), except that TaqI (MBI) was also used as a third restriction enzyme for nifH PCR product restriction fragment length polymorphism analysis.


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Diversity of nifH and nifH-like sequences.
 
Diverse nifH sequences were obtained (81 restriction fragment length polymorphism sequence types with 41.8 to 98.5% sequence identity among each other) (Table 1). The protein sequences deduced shared 39.2 to 100.0% identity among each other, and 47.1 to 100.0% identity to the closest match GenBank NifH and NifH-like sequences. Using DOTUR at 5% sequence distance cutoff (28), 35 NifH operational taxonomic units (OTUs) were identified (see Fig. S1 in the supplemental material). Twelve OTUs were shared between the two sediment samples, while 14 OTUs were unique to station LV39-25H and 9 OTUs to station LV39-40H.


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  		TABLE 1. nifH, 16S rRNA, and mcrA gene clone libraries constructed for the deep-sea methane seep sediments of the Okhotsk Sea

Phylogenetic analysis indicated that almost all the NifH sequences might be obtained from currently uncultured or uncharacterized bacteria or archaea (Fig. 1). Seven NifH and NifH-like clusters were identified (Fig. 1), including three newly defined putative clusters (tentatively named clusters III-x, V, and VI) and all four previously established clusters (clusters I to IV) (23). Cluster V sequences were unique to the deep-sea methane seep sediments of the Okhotsk Sea, and cluster III-x sequences were unique to the general deep-sea methane seep sedimentary environments, including the Okhotsk Sea, Nankai Trough, and Eel River Basin so far studied (25).


Figure 1
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  		FIG. 1. Phylogenetic analysis of the NifH and NifH-like sequences obtained from the methane seep sediments of the Okhotsk Sea. Sequences were aligned using the Clustal_X program (29), and the phylogenetic tree was constructed with the amino acid residues corresponding to positions 17 to 130 of the Klebsiella pneumoniae NifH sequence (GenBank DNA accession no. J01740) using the software package Phylip via the distance and neighbor-joining methods (10). The tree branch distances represent the amino acid substitution rate, and the scale bar represents the expected number of changes per homologous position. Bootstrap values (no less than 50%) of 100 resamplings are shown near nodes. The Rhodobacter capsulatus chlorophyllide reductase iron protein subunit BchX sequence was used as an outgroup. The NifH and NifH-like sequences obtained in this study are shown in bold. *, the NifH sequences Bca2c_nif1g, Bca2c_nif3h, and Bca2c_nif9h recovered from the isolated ANME-2 consortia of the Eel River Basin deep-sea methane seep sediments (25) are highlighted in bold. ERB, Eel River Basin deep-sea methane seep sediments; NT, Nankai Trough deep-sea anoxic methane seep sediments.

A single OTU (25H-0N-1) was affiliated within cluster I (Fig. 1), sharing 95.2% identity with a bacterial NifH sequence obtained from the dead biomass of Spartina alterniflora (18). Another single OTU (40H-0N-26) was affiliated within cluster II (Fig. 1), which comprised the NifH sequences from certain methanogenic archaea and the alternative nitrogenase reductases encoded by anfH and vnfH (23).

Five OTUs were affiliated within cluster III, which could be divided into two subclusters (IIIa and IIIb) (Fig. 1). Four OTUs were affiliated within subcluster IIIa and one within IIIb. Subcluster IIIa comprised sequences mainly from anaerobic bacteria, and IIIb comprised those mainly from Methanosarcina methanogens. OTU 40H-0N-5 shared 100.0% identity with its closest match GenBank NifH sequences (accession no. BAF96834, ACD50919, and ACD50920) obtained from the deep-sea methane seep sediments of the Nankai Trough and Eel River Basin (25). OTUs 40H-0N-5 and 40H-0N-6 also shared 86.0 to 86.8% identities to the NifH sequence of Desulfovibrio gigas. Thus, both OTUs were putatively obtained from deep-sea sulfate-reducing bacteria. OTU 40H-0N-1 shared 89.1 to 89.9% identities with its closest match NifH sequences of Methanosarcina barkeri strain Fusaro, Methanosarcina acetivorans C2A, and Methanosarcina mazei Go1. This OTU was putatively obtained from a methanogen.

Five NifH OTUs were affiliated within the putative cluster III-x, and they shared very high sequence identities (98.3 to 99.2%) with some sequences obtained from the methane seep sediments of the Nankai Trough and Eel River Basin (25) (Fig. 1). However, all of the sequences in this cluster had low identity (<79.7%) to the GenBank sequences obtained from other environments or microorganisms, indicating that these NifH sequences might be specific to the deep-sea methane seep sedimentary environments. This new NifH cluster is monophyletic and supported by bootstrapping statistics. Due to the lack of NifH sequences from culturable strains, no conclusive taxonomic information can be obtained for this NifH cluster.

Within the DNA fragments analyzed, 10 highly conserved key amino acid residues previously identified to be potentially important in NifH structure and function were examined, including Lys15, Ser16, Arg100, Thr104, Asp125, Asp129, and four Cys residues (no. 38, 85, 97, and 132) (18, 27). All of these key amino acid residues were conserved in our cluster III-x NifH sequences, except for 25H-0N-12 and 25H-0N-9, each with a single-amino-acid residue substitution (see Fig. S2 in the supplemental material). The newly defined cluster III-x sequences are likely to encode functional nitrogenase reductases.

Seven OTUs were affiliated within cluster IV. OTU 40H-0N-24 shared 95.9 to 100.0% identities with some NifH sequences obtained from the methane seep sediments of the Nankai Trough and Eel River Basin (GenBank accession no. ACD50923 and BAF96826) (25). OTU 25H-0N-3 shared 97.3% identity with a Nankai Trough methane seep NifH sequence (GenBank accession no. BAF96825) and 87.1% identity with the NifH sequence of Methanococcoides burtonii (Fig. 1). Both OTUs were probably obtained from deep-sea archaea. However, it is difficult to determine the exact taxonomic affiliations of most of our cluster IV NifH sequences, especially for the subcluster made exclusively by OTUs 25H-0N-15, 40H-0N-33, and 40H-0N-37. Cluster IV NifH-like sequences are polyphyletic, including many atypical methanogen NifH-like sequences without detectable nitrogen fixation activity (23, 24, 26). This "cluster" is actually a sequence assemblage. Currently most of these sequences are not characterized (25, 26).

Thirteen OTUs were affiliated within the putative cluster V, which comprised novel NifH-like sequences detected only from the methane seep sediments of the Okhotsk Sea. These sequences shared quite low identity (47.1 to 59.6%) with their closest match sequences, the hyperthermophilic methanogen FS406-22 NifH2 (GenBank accession no. ABK78685) (22), the Geobacter sp. strain FRC-32 NifH (GenBank accession no. EAT62753), and the Desulfitobacterium hafniense Y51 putative NifH (GenBank accession no. BAE85651). The detection of these sequences in both stations indicates that these sequences probably were not artifacts. Most of the 10 highly conserved key amino acid residues examined (18, 27) were present in these sequences, except for residues Cys85, Arg100, and Thr104. OTUs 25H-0N-39 and 25H-0N-40 had a replacement of Cys85 with Gly85. Substitutions at this position were also found in other environmental NifH sequences (18). All of the cluster V sequences had a double mutation: Arg100 was replaced by Lys100 and Thr104 by Glu104 (see Fig. S2 in the supplemental material). These two residues are located at the interaction interface between the Fe-protein and the MoFe-protein (27). A single replacement of Arg100 with Lys100 was found to render a loss of the NifH activity in Azotobacter vinelandii (30), while in the NifH-like light-independent protochlorophyllide reductase subunit BchL or ChlL, Arg100 was replaced by Tyr100 (4). Whether the double mutation would render any change to the enzyme activity or function is currently unknown. The cluster V NifH-like sequences also contained a 6-amino-acid insertion at position 78 (see Fig. S2 in the supplemental material). Previous studies also found insertions at this position (23). Molecular modeling with Swiss-Model (1) indicated that this insertion formed a loop on the exterior surface of the NifH homodimer structure, remote from the subunit-subunit interface (see Fig. S3 in the supplemental material) (4). For the NifH-like BchL, a recent study indicates that essentially no conservation of amino acids is observed on the surface of the BchL monomer outside of the BchL-BchL interface (3). BchL is not only a homolog but also a functional analog to NifH (26). The insertion at position 78 on the outside surface might not influence the NifH-like protein activity or function, if any ever exists for the cluster V sequences.

Three OTUs were affiliated within the putative cluster VI (Fig. 1). However, the low bootstrap support (14%) indicates the uncertainty about the phylogenetic position of this cluster. These OTUs shared low identity (<56.1%) to known NifH or NifH-like sequences. The occurrence of these sequences in both sampling stations indicated that they were probably not artifacts. The preservation of the most conserved key amino acid residues (18, 27), including Lys15, Ser16, Arg100, Asp125, Asp129, and four Cys residues (no. 38, 85, 97, and 132) (see Fig. S2 in the supplemental material), suggests that these sequences may encode functional nitrogenase reductases. Some studies indicate that the loss of nitrogen fixation capability in some of the related microorganisms, such as Fusobacterium nucleatum, may be caused by the lack of a complete nif operon (26), probably not by the nifH-encoded enzyme per se. However, the phylogenetic position and the functionality and activity of the "cluster VI" NifH-like proteins need to be determined.


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Environmental classification of the nifH-carrying microbial communities.
 
UniFrac principal coordinate analysis and environment clustering analysis (19) of the nifH-carrying microbial assemblages in various marine environments indicated that environmental characteristics might have a strong influence on the composition and structure of the nifH-carrying microbial community (Fig. 2; and see Fig. S4 in the supplemental material). The nifH-carrying microbial assemblages of the deep-sea methane seep sediment environments from the Okhotsk Sea, Nankai Trough, and Eel River Basin (25) were distinct from those of the marine water and hydrothermal vent environments (21, 23, 24). The occurrence of the unique cluster V and the putative cluster VI NifH-like sequences in the Okhotsk Sea indicated the environmental specificity of these novel sequences and differentiated the nitrogen-fixing community of the Okhotsk Sea from those of the Nankai Trough and Eel River Basin methane seep environments (Fig. 2; and see Fig. S4 in the supplemental material).


Figure 2
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  		FIG. 2. Principal coordinate plot of UniFrac principal coordinate analysis of the nifH-containing microbial assemblages from various marine environments where nifH clone libraries were constructed with the PCR primers of Mehta et al. (23). 25H and 40H, Okhotsk Sea deep-sea methane seep sediments of stations LV39-25H and LV39-40H (this study); NT, Nankai Trough deep-sea anoxic methane seep sediments (GenBank accession no. BAF96788, BAF96793, BAF96798, BAF96803, BAF96808, and BAF96824 to BAF96838); ERB, Eel River Basin deep-sea methane seep sediments (25); HV20, hydrothermal vent fluid of Juan de Fuca Ridge sampled in 2000; HV99, hydrothermal vent fluid of Juan de Fuca Ridge sampled in 1999; DVP, diffuse vent fluid near Puffer on Endeavor segment of the Juan de Fuca Ridge; DSW, deep seawater on Endeavor segment (23); NPD, Northeast Pacific deep seawater (24); H01, H04 and H05, surface seawater samples collected in stations H01, H04, and H05 of the eastern Mediterranean Sea (21).


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Archaeal 16S rRNA gene diversity.
 
A total of 63 unique archaeal 16S rRNA gene sequences and 27 OTUs (based on 3% sequence distance cutoff) were obtained from the methane seep sediments of the Okhotsk Sea (Table 1; and see Fig. S1 in the supplemental material). These sequences fall into diverse archaeal clusters, including the uncultured marine group I; marine benthic groups B, C, and D; marine hydrothermal vent group; miscellaneous crenarchaeotic group; novel group of crenarchaea; terrestrial miscellaneous euryarchaeotic group; and ANME-2 (Fig. 3). The diversity of archaeal 16S rRNA gene sequences from the Okhotsk Sea is comparable to or higher than those of some of other deep-sea methane seeps or like environments (13, 14, 17).


Figure 3
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  		FIG. 3. Phylogenetic analysis of the archaeal 16S rRNA gene sequences obtained from the deep-sea methane seep sediments of the Okhotsk Sea. Sequences were aligned using the Clustal_X program, and the phylogenetic tree was constructed with 964 aligned nucleotide positions using the software package Phylip via the distance and neighbor-joining methods. The tree branch distances represent nucleotide substitution rate, and the scale bar represents the expected number of changes per homologous position. Bootstrap values no less than 70% of 100 resamplings are shown with solid circles on the corresponding nodes, and those less than 70% but greater than or equal to 50% are shown with open circles on the corresponding nodes. The bacterial 16S rRNA gene sequence from Thermotoga maritima MSB8 was used as an outgroup. The archaeal 16S rRNA gene sequences obtained in this study are shown in boldface. DSAG, deep-sea archaeal group; MBG, marine benthic group; MCG, miscellaneous crenarchaeotic group; MG I, marine group I; MHVG, marine hydrothermal vent group; NGC, novel group of crenarchaea; TMEG, terrestrial miscellaneous euryarchaeotic group.


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Archaeal mcrA gene diversity.
 
A total of 33 unique mcrA gene sequences and 28 McrA OTUs were obtained based on sequence uniqueness from the methane seep sediments of the Okhotsk Sea (Table 1; and see Fig. S1 in the supplemental material). Diverse tentative ANME mcrA gene sequences, including groups a, c, d, and e (12), were detected via phylogenetic analysis (Fig. 4). No group b or f mcrA sequence could be obtained with the primers used (11, 12, 17). The mcrA analysis results (Fig. 4) detected much higher ANME diversity of both sampling stations LV39-25 and LV39-40H than the archaeal 16S rRNA gene analysis results (Fig. 3). For the sampling station LV39-25H, a methanogen mcrA sequence (25H-0A-8) affiliated within Methanosarcinales was also obtained (Fig. 4). However, for both mcrA clone libraries, sequences putatively affiliated within ANME were much more predominant than those affiliated within methanogens.


Figure 4
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  		FIG. 4. Phylogenetic analysis of the archaeal McrA sequences obtained from the methane seep sediments of the Okhotsk Sea. Sequences were aligned using the Clustal_X program, and the phylogenetic tree was constructed with 244 aligned amino acid residues using the software package Phylip via the distance and neighbor-joining methods. The tree branch distances represent the amino acid substitution rate, and the scale bar represents the expected number of changes per homologous position. Bootstrap values no less than 70% of 100 resamplings are shown with solid circles on the corresponding nodes and those less than 70% but greater than or equal to 50% are shown with open circles on the corresponding nodes. The McrA sequence from Methanopyrus kandleri AV19 was used as an outgroup. McrA sequences obtained in this study are shown in boldface.

In both stations LV39-25H and LV39-40H, methane hydrates were directly obtained from shallow sediments, usually less than 2 m below the seafloor. The existence of intense methane plumes above the seafloor indicated a sufficient in situ CH4 supply for the sedimentary microbial ecosystem and a potential oxygen-limited environment of the surface sediments (20). ANME might be a major functional component of the in situ microbial community. Previous studies indicate that cultivated archaeal nitrogen fixers are all affiliated within methanogenic Euryarchaeota, including Methanosarcinales, Methanococcales, Methanomicrobiales, and Methanobacteriales (16). However, recent studies also indicate that some of the deep-sea novel nifH and nifH-like sequences might be obtained from ANME (25). In line with this, most of our nifH sequences had quite high identity with sequences obtained from anaerobic methanotrophic consortia of other deep-sea methane seep sedimentary environments, such as the Nankai Trough and Eel River Basin (25). The recent GenBank publications of several nearly complete nif operons putatively affiliated with anaerobic methane-oxidizing archaea from the Nankai Trough methane seep sediments (GenBank DNA accession no. AB362194, AB362195, and AB362197; corresponding NifH protein accession no. BAF96793, BAF96798, and BAF96808) indicate that some methanotrophic archaea may possess the genetic potential for nitrogen fixation (Ken Takai, personal communication). This was further demonstrated by several NifH sequences, namely Bca2c_nif1g, Bca2c_nif3h, and Bca2c_nif9h, recently obtained from the isolated ANME-2 consortia of the Eel River Basin methane seep sediments (25). All of these NifH sequences show a high degree of identity to the cluster III-x sequences obtained from our study (Fig. 1). In Pernthaler and colleagues' study (25), in situ nitrogen fixation by methane seep sediment methanotrophic consortia was demonstrated via a 15N2 incubation study, which also showed that ANME might be the major contributors to the diazotrophic activity of the consortia. The ANME microorganisms still escape from being cultivated for physiological and genetic research. Cultivation-independent molecular approaches have significantly advanced our understanding of their distribution and ecophysiology in natural and engineering environments. In bacterial methanotrophs, the genetic potential and nitrogen fixation activity have already been identified (2). It is reasonable to speculate that the genetic potential and diazotrophic activity might also exist very commonly in anaerobic archaeal methanotrophs, especially in deep-sea methane-rich sedimentary environments. Recent studies also indicate the possibility that some of the deep-sea novel nifH sequences might be obtained from other archaeal or proteobacterial lineages (24, 25). Further investigations should be carried out to test these hypotheses.

In conclusion, our work obtained diverse and novel nifH and nifH-like gene sequences in the deep-sea methane seep sediments of the Okhotsk Sea. Almost all of the sequences were obtained from uncultured or uncharacterized bacteria or archaea. Comparison with other marine environments indicates certain environmental specificity of some of the sequences. Methane seep sediments may harbor diverse and unique microbial genetic potentials, important in nitrogen fixation and new production of the deep-sea ecosystem.


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Nucleotide sequence accession numbers.
 
The nifH gene sequences reported in this study have been deposited in GenBank under accession no. EU713922 to EU713990 and EU713992 to EU714003, the archaeal 16S rRNA gene sequences under accession no. EU713859 to EU713921, and the mcrA gene sequences under accession no. FJ403593 to FJ403625.


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ACKNOWLEDGMENTS
 
We thank the reviewers for valuable comments and suggestions. We also thank Linbao Zhang, Ying Zhang, Jian Sun, and Jin Sun for assistance with the project.

This work was supported by China National Natural Science Foundation grants 40576069 and 40776032, National Basic Research Program of China (973 Program) grant 2007CB411702, Hi-Tech Research and Development Program of China grant 2007AA091903, and China Ocean Mineral Resources R&D Association grants DYXM-115-02-2-6 and DYXM-115-02-2-20.


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FOOTNOTES
 
* Corresponding author. Mailing address for Hongyue Dang: Centre for Bioengineering and Biotechnology, China University of Petroleum (East China), Qingdao 266555, China. Phone: 86-532-86981561. Fax: 86-532-86981318. E-mail: danghy{at}upc.edu.cn. Mailing address for Xiwu Luan: Key Laboratory of Marine Geology and Environment, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China. Phone and fax: 86-532-82898536. E-mail: xluan{at}ms.qdio.ac.cn Back

{triangledown} Published ahead of print on 30 January 2009. Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back


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Applied and Environmental Microbiology, April 2009, p. 2238-2245, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.02556-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Miyazaki, J., Higa, R., Toki, T., Ashi, J., Tsunogai, U., Nunoura, T., Imachi, H., Takai, K. (2009). Molecular Characterization of Potential Nitrogen Fixation by Anaerobic Methane-Oxidizing Archaea in the Methane Seep Sediments at the Number 8 Kumano Knoll in the Kumano Basin, Offshore of Japan. Appl. Environ. Microbiol. 75: 7153-7162 [Abstract] [Full Text]  
  • Dekas, A. E., Poretsky, R. S., Orphan, V. J. (2009). Deep-Sea Archaea Fix and Share Nitrogen in Methane-Consuming Microbial Consortia. Science 326: 422-426 [Abstract] [Full Text]  

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