The Sequence Capture by Hybridization

The sequence capture by hybridization: a new approach for revealing the potential of mono-aromatic hydrocarbons bioattenuation in a deep oligotrophic aquifer Magali Ranchou-Peyruse, Cyrielle Gasc, Marion Guignard, Thomas Aüllo, David Dequidt, Pierre Peyret, Anthony Ranchou-Peyruse

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Magali Ranchou-Peyruse, Cyrielle Gasc, Marion Guignard, Thomas Aüllo, David Dequidt, et al.. The sequence capture by hybridization: a new approach for revealing the potential of mono-aromatic hydrocarbons bioattenuation in a deep oligotrophic aquifer. Microbial Biotechnology, Wiley, 2017, 10 (2), pp.469-479. 10.1111/1751-7915.12426. hal-01658765

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The sequence capture by hybridization: a new approach for revealing the potential of mono-aromatic hydrocarbons bioattenuation in a deep oligotrophic aquifer

Magali Ranchou-Peyruse,1 Cyrielle Gasc,2 this innovative strategy demonstrated the presence Marion Guignard,1 Thomas Aullo,€ 3 David Dequidt,4 of the bssA and bssA-like genes in this oligotrophic Pierre Peyret2 and Anthony Ranchou-Peyruse1,* ecosystem, probably harboured by Peptococcaceae. 1Universite de Pau et des Pays de l’Adour, Equipe The sequence capture by hybridization shows signifi- Environnement et Microbiologie, IPREM-CNRS 5254, cant potential to reveal the presence of genes of F-64013 Pau, France. functional interest which have low-level representa- 2Universited ’Auvergne, EA 4678 CIDAM, 63001 tion in the biosphere. Clermont-Ferrand, France. 3TIGF – Transport et Infrastructures Gaz France, 40 Avenue de l’Europe, CS20522, 64000 Pau, France. Introduction 4STORENGY – Geosciences Department, Bois-Colombes, France. The degradation of toluene and m, p and o-xylenes (TX) under anoxic conditions has been demonstrated in numerous marine and continental environments. It can Summary be associated with the reduction of nitrate, sulphate, iron and CO (Dolfing et al., 1990; Beller and Spormann, The formation water of a deep aquifer (853 m of 2 1997; Harms et al., 1999; Kane et al., 2002; Kube et al., depth) used for geological storage of natural gas was 2004; Morasch et al., 2004; Morasch and Meckenstock, sampled to assess the mono-aromatic hydrocarbons 2005; Washer and Edwards, 2007; Aullo€ et al., 2016). In attenuation potential of the indigenous microbiota. all cases, the initial addition of fumarate to the hydrocar- The study of bacterial diversity suggests that Firmi- bon molecule is catalysed by either benzylsuccinate syn- cutes and, in particular, sulphate-reducing bacteria thase (toluene) or benzylsuccinate synthase-like (Peptococcaceae) predominate in this microbial com- enzymes (xylenes). For about 15 years, the bssA gene, munity. The capacity of the microbial community to which encodes for the alpha subunit of this protein, has biodegrade toluene and m- and p-xylenes was been used as a biomarker for the biodegradation of TX demonstrated using a culture-based approach after under anoxic conditions. Beller et al. (2002) were the several hundred days of incubation. In order to reveal first to design primers targeting this gene, using avail- the potential for biodegradation of these compounds able sequences from isolated strains. These primers within a shorter time frame, an innovative approach then preferentially targeted nitrate-reducing Betapro- named the solution hybrid selection method, which teobacteria. Subsequently, other primer sets enabled sul- combines sequence capture by hybridization and phate-reducing, iron-reducing and syntrophic bacteria to next-generation sequencing, was applied to the same be targeted (Winderl et al., 2007; Beller et al., 2008; original water sample. The bssA and bssA-like genes Staats et al., 2011; Fowler et al., 2014). There was a diffi- were investigated as they are considered good culty in amplifying the bssA gene of some sulphate-redu- biomarkers for the potential of toluene and xylene cing bacteria belonging to the Clostridia which was partly biodegradation. Unlike a PCR approach which failed solved by the subsequent design of specific primers for to detect these genes directly from formation water, this class (von Netzer et al., 2013; for the latest review on bssA-like gene diversity, see von Netzer et al., 2016). Received 25 July, 2016; revised 15 September, 2016; accepted 18 In oligotrophic and stable environments, like deep con- September, 2016. *For correspondence. E-mail anthony.ranchou- [email protected]; Tel. +33 540 175 164; Fax +33 559 407 494. tinental aquifers ( 500 to 1200 m), the input of exoge- Microbial Biotechnology (2017) 10(2), 469–479 nous TX represents a potential source of carbon which doi:10.1111/1751-7915.12426. is likely to modify the growth and survival strategies of Funding Information Storengy and TIGF are acknowledged for funding for this research microorganisms. Over the last several years, the poten- project. tial for biodegradation of monoaromatic hydrocarbons in

ª 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 470 M. Ranchou-Peyruse et al. such environments has been demonstrated through cul- Table 1. Physico-chemical parameters and constituents of the for- ture-based approaches (Morasch et al., 2004; Berlendis mation water sampled from a deep aquifer (aquifer 1 in this study) € at 853 m of depth below groundwater and analysed at atmospheric et al., 2010; Aullo et al., 2016). The low biomass present pressure. in these ecosystems makes the demonstration of fi Physico-chemical parameters biodegradation capacities dif cult (i.e. detection of bssA ° fi Temperature ( C) 36 genes) in formation water (FW). It is currently dif cult, pH 8.25 indeed impossible, to assess the degradation potential of Conductivity at 25°C(lScm 1) 6000 FW sampled from a deep aquifer without having Redox potential (mV) 363 Pressure (bars) 93 recourse to laboratory-based degradation assays. In our Total suspended solids (mg l1) 16.0 previous studies, amplifications of the bssA gene using Constituentsa Carbonates (mg l1) < 20 the primers cited previously have unfortunately often Sulphates (mg l 1) 2186.5 proved to be unsuccessful, as a result of the few targets Ammonium (mg l 1) 1.75 available and problems of non-specific amplification Calcium (mg l 1) 25.1 1 (Aullo€ et al., 2016). Next-generation sequencing (NGS) Magnesium (mg l ) 15.9 Sodium (mg l1) 1400 techniques allow a deeper analysis of genetic diversity Potassium (mg l1) 34.0 and thus offer the possibility of dispensing with culture- Chloride (mg l 1) 260 1 Silicates (mg SiO2 l ) 15.8 based techniques. However, diversity analyses are very Phosphorus (mg l 1) < 0.05 often unable to identify microbial populations with low- Nitrates (mg l 1) < 2 level representation. The sequence capture by hybridiza- Fluoride (mg l1) 1.51 Barium (mg l1) 0.013 tion (Gasc et al., 2016), therefore, constitutes an alterna- Total iron (lgl 1) 3200 fi tive for the ef cient detection of rare or unknown Ferrous iron (lgl 1) < 100 sequences in metagenomic samples (Denonfoux et al., Manganese (lgl 1)44 1 2013; Bragalini et al., 2014; Biderre-Petit et al., 2016). Organic carbon (mg l ) 1.0 In the context of this study, we sought to demonstrate a. Arsenic, cadmium, chrome, copper, tin, mercury, lead, vanadium the presence of bssA genes in a deep aquifer used to and zinc were also measured, but were below the limits of detection. store natural gas, which is associated with trace amounts of other hydrocarbons, in order to reveal a while maintaining anoxic conditions. Filters were used to potential for natural bioattenuation of TX. We hypothe- collect the microbial biomass over a period of 6.5 h from sized that the Clostridia, in particular members of the 500 l of FW. At the end of sampling, the filters were Peptococcaceae family, play a key role in the degrada- placed in bags maintaining anaerobiosis (GasPakTM EZ; tion of TX in deep aquifers (Basso et al., 2009; Berlendis BD, Franklin Lakes, NJ, USA). Several litres of FW were et al., 2010; Aullo€ et al., 2016). It is known that some also sampled in sterile glass bottles and degassed with members of Peptococcaceae are capable of degrading nitrogen in the laboratory prior to subsequently preparing mono-aromatic hydrocarbons directly or via syntrophic and running microbial cultures. The set of samples (fil- relationships (Morasch et al., 2004; Taubert et al., ters and water) were immediately placed at 4°C and 2012). A conventional approach to amplify the bssA transported to the laboratory. The samples to be used gene using the sets of primers available in the literature for molecular analysis were then frozen at 20°C and is compared with the sequence capture by hybridization those for the cultures used the following day. approach. This study represents the first instance of its use in this type of environment and in an industrial Biodegradation assays context. Two biodegradation assays of a mixture of toluene, m-, Materials and methods p- and o- xylenes, here referred to as TX (with a final concentration of 10 ppm for each hydrocarbon; Sigma- Sampling Aldrich, Saint Quentin Fallavier, France) were carried out In 2011, water samples were obtained from a deep aqui- at the same time with FW alone (FW condition) or fer (853 m of depth) used for geological storage of natu- enriched with the concentrated biomass (FWCB condi- ral gas (aquifer 1, Paris Basin, France). Several tion). The concentrated biomass was obtained for the lat- physicochemical parameters of the FW are indicated in ter condition by resuspending 67 Sterivex filters (EMD; Table 1 (IPL Sante, Environnement Durables, Ile De Millipore) in 250 ml of anoxic FW under agitation. Ten France). After the tubing was cleaned as described pre- per cent (v/v) of this concentrated biomass was used to viously (Basso et al., 2005), the biomass from the FW inoculate the FWCB. For each condition, that is, without was collected at the wellhead by filtration through 70 addition of concentrated biomass (FW+TX) and with Sterivex filters (EMD; Millipore, Molsheim, France), addition of concentrated biomass (FWCB+TX), an abiotic

ª 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology, Microbial Biotechnology, 10, 469–479 Sequence capture by hybridization applied for bssA 471 control was performed by the addition of 5% v/v of 1 M were eliminated. The final OTUs were subsequently clas- HCl. The four microcosms, with a final volume of 40 ml, sified taxonomically using BLASTn against a curated were prepared in 100 ml Wheaton serum bottles sealed database derived from RDPII and NCBI. with butyl rubber stoppers (Bellco Glass, Vineland, NJ). All manipulations were performed in a glovebox (Getinge Cloning and sequencing of benzylsuccinate synthase La Calhene, France) in an atmosphere of 95% N and 2 alpha-subunit (bssA) genes 5% H2. The hydrogen is necessary for the palladium cat- alyst can react to remove oxygen traces. All cultures PCR amplifications of the bssA gene were performed for were incubated at 37°C, in the dark without agitation. different sets of primers found in the literature. The primer Periodically, TX degradation was monitored in the four sets 7772F/8546R (Winderl et al., 2007), 7768F/8543R microcosms using SPME/GC/FID as described in Aullo€ (von Netzer et al., 2013), bss3F/bssAR (Staats et al., et al. (2016). 2011), and 997F/1230R (Brow et al., 2013) were used to target, respectively, the bssA gene of Deltaproteobacteria, Clostridia, Geobacter and nitrate-reducing Betaproteobac- Bacterial enumeration teria (Table S1). For each positive amplification, the PCR Eighteen millilitres of FW were fixed on site with 2 ml 10% fragment corresponding to the expected size was purified borax-buffered formaldehyde (37%; Sigma-Aldrich) and on an agarose gel and cloned using the TOPO TA Cloning stored at 4°C until quantification. Furthermore, after resus- kit (Fisher Scientific, Hampton, NH, USA) before being pension of the SterivexTM filters in FW for the biodegrada- sequenced by Qiagen Genomic Services (Hilden, tion assays, 18 ml of the concentrated biomass was Germany). sampled and fixed the same way. In all cases, 500 llof 40,60-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) Capture probe design and synthesis stock solution (200 lgml1) was added to 10 ml of fixed sample, and then vacuum filtration was performed using A set of five 31- to 38-mers degenerate probes covering 0.2 lm pore-size black polycarbonate filters (Millipore). the bssA gene was designed from nine bssA nucleic Ten fields were selected at random for each filter, and the sequences from strains belonging to Deltaproteobacteria, cells were counted on an Axio Observer.Z1 inverted Firmicutes (EF123665, EF123667, EU780921, microscope (Zeiss, Oberkochen, Germany) equipped with FO203503, EF123663, EF123662) and environmental a639 oil immersion objective (Plan APO, N.A. 1.4, M27). sequences obtained with 7772F/8546R primers set. Images were obtained with a Zeiss Axiocam 506 mono These three latter amplicons were obtained at the end of CCD camera via the Zeiss ZEN 2012 interface. TX degradation from enrichment cultures with autochthonous microbiota of different deep aquifers (aquifer 1: KX576576, aquifer 2: KX576577, aquifer 3: KX576575). Bacterial community analysis The deep aquifer 2 was studied by Berlendis et al. Microbial diversity was investigated on a SterivexTM filter (2010) (Paris Basin). The third aquifer (aquifer 3) is previously stored at 20°C. The filter was crushed in liq- located in the Aquitaine Basin (southwest of France). uid nitrogen and the DNA extracted with the Powersoil These sequences were processed using the KASpOD DNA Isolation kit (MoBio Laboratories, Inc., Carlsbad, CA, software (Parisot et al., 2012) (Table S1). Adaptor USA) following the manufacturers’ specifications. Geno- sequences were added at each extremity of the probe to mic DNA was sent to a commercial company (MR DNA, enable their PCR amplification, resulting in “ATCGCAC

Shallowater, TX, USA). The hypervariable V4 region of CAGCGTGT-N(31-38)-CACTGCGGCTCCTCA” sequences, the 16S rRNA gene was amplified with PCR primers 515/ with N(31-38) representing the bssA-specific capture 806 and sequenced by the MiSeq 2 9 300 bp run (Illu- probe. Biotinylated RNA capture probes were then syn- mina, San Diego, CA, USA). The construction of DNA thesized as described by Ribiere et al. (2016). libraries with the amplicons and the sequencing were performed following the manufacturers’ specifications. The Preparation of biological samples and libraries sequence data were subsequently processed through the analysis pipeline designed by MR DNA. At first, the Next-generation sequencing libraries were constructed on sequences were demultiplexed and the barcodes and pri- genomic DNA extracted directly from aquifer 1 FW after mers were removed before eliminating all sequences concentration by means of ethanol precipitation. Libraries smaller than 150 bp. Sequences displaying ambiguous were prepared using the Nextera XT Kit (Illumina) using bases were also eliminated. Operational taxonomic units the manufacturer’s instructions (Genoscreen, Lille, (OTUs) were defined by clustering sequences displaying France) and were PCR amplified using the GC-RICH PCR 97% similarity. Thus, singleton sequences and chimeras system kit (RocheDiagnostics GmbH, Mannheim,

ª 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology, Microbial Biotechnology, 10, 469–479 472 M. Ranchou-Peyruse et al.

Germany) with primers complementary to the library adap- sampled after cleaning of the sampling well, to remove ters to obtain sufficient amounts of DNA to run the the microorganisms growing as a biofilm on the tubing sequence capture by hybridization. and the wellhead (see Basso et al., 2009 for Scheme of principle of a natural gas underground storage in an aquifer). During this study, the methane bubble was Hybridization capture and elution located close to the well and the presence of methane, Solution hybrid selection (SHS) was conducted on FW originally dissolved at reservoir depth, was observed in sample according to the protocol described by Ribiere the sampled water. At the time of sampling, the water et al. (2016). Briefly, 500 ng of heat denatured libraries was at a temperature of 36°C, with a pH of 8.25, and no was hybridized to the set of biotinylated RNA probes for oxygen was detected and a redox potential of 363 mV 24 h at 65°C. Probe/target heterodimers were trapped (Table 1). This aquifer displayed a total organic carbon by streptavidin-coated paramagnetic beads (Dynabeads concentration (TOC; 1 mg l 1) typical of this type of M-280 Steptavidin, Invitrogen, Carlsbag, CA, USA). After environment which has been little impacted by human several washing steps, the captured targets were eluted activity (Pedersen, 1997; Sahl et al., 2008). Mecken- from the beads using NaOH and purified using AMPure stock et al. (2015) report in their review dedicated to beads (Beckman Coulter Genomics, Takeley, Essex, microbial clean-up in contaminated aquifers that only United Kingdom). Enriched products were PCR amplified 0.5–5% of this TOC is likely to be directly used as car- using primers complementary to the library adapters and bon source by the microbial community which makes purified again using AMPure beads (Beckman Coulter this deep aquifer an oligotrophic environment. The analy- Genomics). To increase the enrichment, a second round sis of the chemical composition of this water reveals a of hybridization and amplification was performed using sulphate content of 2186.5 mg l 1, a total iron content of the obtained captured products. 3200 lgl1 but no nitrate. These data suggest that the indigenous microbial community is probably dominated by sulphate-reducing bacteria. Sulphate reduction is Illumina MiSeq sequencing and data analysis indeed an important metabolic process in deep subsur- Captured DNA products were sequenced using a single face environments and particularly in aquifers (Detmers MiSeq 2 9 300 bp run (Illumina) according to the manu- et al., 2004; Amend and Teske, 2005; Bombach et al., facturer’s specifications (Genoscreen). All raw reads 2010; Itavaara€ et al., 2011) where it has been demon- were scanned for library adaptors and quality filtered strated that sulphate-reducing bacteria also play a key using PRINSEQ-lite PERL script (Schmieder and role in the degradation of mono-aromatic hydrocarbons Edwards, 2011) prior to assembly and analysis. The (Basso et al., 2009; Berlendis et al., 2010; Aullo€ et al., clean reads were assembled de novo using IDBA-UD 2016). The concentrated biomass derived from the filtra- (v1.1.1) (Peng et al., 2012). Contigs generated were tion of FW was subjected to a diversity analysis in order combined for a second round of assembly using CAP3 to determine the predominant bacterial groups. For this to generate longer contigs (Huang and Madan, 1999). purpose, the V4 region of the sequence of the 16S rRNA The amino acid (AA) sequences were deduced from the gene was targeted by NGS (no archaea were detected). final assembled nucleotide contigs and then aligned with The taxa detected in this study are regularly found in reference open-reading frames sourced from public data- studies of deep subsurface ecosystems. Figure 1 shows bases using MEGA version 6 (Tamura et al., 2013). The that Firmicutes were clearly predominant (61.1%) among phylogenetic tree was constructed with the same soft- the 62 OTUs found (similarity ≥ 97%), with 29.3% of ware using the neighbour-joining method. The bootstrap Peptococcaceae (Carboxydothermus, candidatus Desul- analysis was performed for 1000 replicates. forudis, Pelotomaculum and Desulfotomaculum) and 31.2% of sequences belonging to Thermoanaerobacter- aceae (Thermoanaerobacter, Moorella, Sporotomaculum Nucleotide sequence accession numbers and Ammonifex). The detection thermophilic microorgan- Sixty-five sequences (16S rRNA and bssA genes) have isms signatures (e.g. Moorella or Thermoanaerobacter) been submitted to GenBank under accession numbers in a mesothermic environment may be surprising. How- KX576572 to KX576636. ever, similar results have already been reported in the study of another mesothermic aquifer (Berlendis et al., Results and discussion 2010; aquifer 2 in this study). Taking into account of the diversity observed, most sulphate reduction must be car- Characterization of the site ried out by Firmicutes since the Deltaproteobacteria, In the autumn of 2011, the FW of a deep aquifer (aquifer which include a large number of sulphate-reducing bac- 1) used for geological storage of natural gas was teria, represent less than 1% of total sequences. The

Fig. 1. Composition of the bacterial community in the formation water collected from a deep aquifer (853 m). The pie chart represents the per- centage of each taxon within the whole microbial species analysed. dominance of the sulphate-reducing community by Firmi- dominant compared with pelagic microorganisms (Whit- cutes has been shown in a 120 m deep aquifer display- man et al., 1998; Griebler and Lueders, 2009). Even if ing low sulphate concentrations (≤ 17.1 mg l1; Detmers the diversity between the pelagic and the attached et al., 2004). Salinity seems to play a role in the distribu- microorganisms can differ (Roling€ and van Verseveld, tion of Deltaproteobacteria and sulphate-reducing Firmi- 2002), the biomass supplemented condition tended to cutes (Leloup et al., 2005). Here, the dominance of simulate the influence of the bacterial concentration in Firmicutes can probably be further explained by their our biodegradation assays. In the two microcosms of this capacity to sporulate, their capacity to survive in olig- study, toluene and m- and p- xylenes (Tm, pX) were otrophic environments and their metabolic versatility degraded simultaneously (Fig. 1). Toluene is assumed (Sass et al., 1997; Spring and Rosenzweig, 2006; Orsi to be the most easily biodegraded aromatic hydrocarbon et al., 2016). Moreover, some studies have suggested or in anoxic conditions, which induces that it is degraded demonstrated the key role of some Peptococcaceae, first and therefore delay the degradation of other hydro- and in particular representatives of the genera Desulfo- carbons (Haag et al., 1991; Edwards et al., 1992; Phelps tomaculum, Desulfosporosinus and Pelotomaculum,in and Young, 1999; Meckenstock et al., 2004; Morasch the degradation of BTEX (Robertson et al., 2000; Liu et al., 2004). However, this interpretation must be tem- et al., 2004; Morasch et al., 2004; Cupples, 2011; Abu pered since it is evident that the biodegradation potential Laban et al., 2015). depends on the pool of key genes present. Indeed simultaneous biodegradation of toluene and xylene has already been observed (Herrmann et al., 2009; Shah, Biodegradation assays 2014). It is interesting to observe that while the m- and The day after sampling, assays of biodegradation of p-xylenes were degraded, o-xylene resisted degradation toluene and the 3 xylene isomers were initiated either throughout the incubation period. In the FW degradation directly on the water obtained from the site (FW+TX) or assay, the degradation of Tm, pX began after a lag in water supplemented with concentrated indigenous phase of about 800 days. Degradation was almost four bacterial biomass (FWCB+TX). A cell-count carried out times faster (237 days) when the biomass was concen- in FW using epifluorescence (DAPI) showed a cell con- trated fourfold, which implies that the number of microor- centration of 4.5 9 105 2.3 9 105 cells ml1, while ganisms influences Tm, pX degradation. This result the concentration of microorganisms in FWCB was four suggests that the in situ biodegradation process, with a times greater; 1.8 9 106 cells ml 1. While it is difficult to supposed higher concentration of microorganisms in bio- quantify microorganisms attached to the mineral matrix film structures, could be faster than revealed by in deep aquifers, it is commonly accepted that the biodegradation studies in the laboratory (Botton and Par- indigenous biomass growing in a biofilm is very largely son, 2006; Berlendis et al., 2010; Higashioka et al.,

ª 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology, Microbial Biotechnology, 10, 469–479 474 M. Ranchou-Peyruse et al.

2012; Aullo€ et al., 2016). Indeed, only the pelagic frac- explore the methanogenic communities present in a tion of the aquifers can be harvested, which underesti- lacustrine environment by targeting the methyl coenzyme mates the potential for in situ biodegradation. M reductase subunit A (mcrA) gene with a set of non- overlapping probes, which targeted both known sequences and potential undescribed variants of the BssA detection assay by PCR mcrA gene. The mcrA sequences represented more than Numerous studies have shown that the bssA and bssA- 40% of the obtained sequences after two cycles of cap- like genes can serve as biomarkers for the in situ anaero- ture, revealing enrichment compared with shotgun bic degradation of toluene and xylenes (Krieger et al., sequencing, in which only 0.003% of the sequences cor- 1999; Andreoni and Gianfreda, 2007; Kazy et al., 2010; responded to the target gene. In addition, because mcrA Cury et al., 2015). These studies are most frequently con- and 16S rRNA gene phylogenies are congruent, this ducted in surface environments or in shallow aquifers. approach allowed the methanogen community to be Even though the diversity study based on 16S rRNA gene described and revealed higher diversity than previously indicates a predominance of Firmicutes and the water observed with other methods. Indeed, hybridization cap- composition suggests that sulphate reduction could play a ture recovered sequences from the Methanobacteriales major role in this aquifer, the choice was made to be as order, belonging to the rare biosphere, which were not exhaustive as possible regarding the metabolisms detected through direct sample sequencing due to the involved in bioattenuation. For this purpose, four primer sequencing depth, or through PCR amplification, due to pairs designed to target nitrate-reducing bacteria (Brow possible primer bias. This method appeared to be appro- et al., 2013), iron-reducing bacteria (Staats et al., 2011), priate for our constraints given its sensitivity, and the fact sulphate-reducing bacteria belonging to the Proteobacte- that it does not need amplification step, which is often ria or Clostridiales (Winderl et al., 2007; von Netzer et al., unfruitful in this type of study. The SHS probes 2013) were tested as part of this study (Table S1). The (Table S1) were designed from bssA sequences of sul- bssA gene was not amplified by PCR in the DNA phate-reducing bacteria belonging to the Deltaproteobac- extracted from FW samples whether supplemented or not teria and the Firmicutes, as well as the three sequences supplemented with concentrated biomass, although the previously amplified with the primers of Winderl et al. degradation results obtained subsequently in the micro- (2007) in enrichments with mono-aromatic hydrocarbons cosms demonstrated the capacity to degrade the Tm, pX. (KX576575, KX576576, KX576577). After analysis of Our team has been trying to demonstrate the presence of reads (Table S2), 498 500 reads proved close to bssA this gene directly in FW for some years without success. and bssA-like genes and were grouped into three bssA Three times only this gene was amplified by the primers of homologous contigs. The contig sequence_10944 alone Winderl et al. (2007) in microcosms from three different includes 98% of the bssA reads indicating that such aquifers (aquifers 1, 2 and 3), after incubation periods of bssA gene dominates in the ecosystem. Sequence cap- more than 100 days (data not shown). It should be noted ture by hybridization gives quantitative results close to that the amplicons corresponding to the expected size that obtained by qPCR as demonstrates by Denonfoux had to be systematically purified on an agarose gel in et al. (2013). The three contigs obtained by sequence order to eliminate numerous non-specific amplifications capture and the three bssA amplicons obtained in this before sequencing. Although this amplification approach study were compared with sequences from pure strains has proven its value in surface environments and shallow and environmental samples deposited in international aquifers, it did not seem appropriate for the constraints of databases after having been translated into AA our study sites (trace mono-aromatic hydrocarbons, low sequences. The results are presented as a phylogenetic biomass and impossibility of recovering biofilms in this tree constructed from the comparison of a 90AA region. industrial context). Therefore, we had to develop a new The main contig, contig sequence_10944 (aquifer 1) and molecular biological approach enabling detection of the the amplicons obtained from the sulphate-reducing bssA gene directly in FW while conducting time-consuming enrichments from FWs (aquifers 2 and 3) are closed to biodegradation assays. bssA sequences defined as sensu stricto by Acosta- Gonzalez et al. (2013). The dominant bssA gene obtained by SHS is close to the BF clone obtained from BssA detection assay using sequence capture by an enrichment described as degrading benzene and hybridization approach dominated by Peptococcaceae-related Gram-positive In 2013, Denonfoux and co-workers successfully com- microorganisms (87% identity, 276AA). In our current bined a SHS method with next-generation sequencing state of knowledge, the reason for the presence of this for the first time, in order to capture a biomarker gene in gene in this enrichment described by Abu Laban et al. a complex metagenome. The methodology was used to (2010) cannot be explained since the initial benzene

ª 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology, Microbial Biotechnology, 10, 469–479 Sequence capture by hybridization applied for bssA 475 biodegradation step does not seem to involve the addi- enrichment culture with FW from the aquifer 1 at the end tion of a fumarate molecule (enabled by the benzylsucci- of degradation (Fig. 2) is phylogenetically located in the nate synthase) but a carboxylation as described by the OX39-homologues cluster as described by von Netzer authors. In our case, the procurement of a bssA gene et al. (2013). The genes present in this cluster could be associated with the Peptococcaceae family is consistent involved in the degradation of xylenes (Herrmann et al., with the diversity data obtained in this study (Fig. 1) and 2009; Bragalini et al., 2014). This sequence is close to tends to confirm the supposed role of Peptococcaceae the Desulfotomaculum sp. Ox39 bssA-like gene (74% in the degradation of Tm, pX in this deep aquifer. In similarity, 227AA) and to environmental sequences referring to the broad dominance of Firmicutes over obtained from enrichments derived from contaminated Deltaproteobacteria, we can hypothesize that these are aquifers (Herrmann et al., 2009; von Netzer et al., the principal sulphate-reducing bacteria in this deep 2013). Finally, the two last contigs that represent only aquifer. Several studies have provided evidence for a 2% of the bssA homologous sequences obtained by positive correlation between the dominance of sulphate- SHS (Table S2), contig sequence_48572 and contig reducing bacteria affiliated to Peptococcaceae in subsur- sequence_31410, form a separate cluster located face environments and depth (Moser et al., 2005; Chi- between the assA and the bssA genes. The existence of vian et al., 2008; Itavaara€ et al., 2011; Guan et al., deeply branching bacteria was also found in the DNA- 2013). These microorganisms play a major role in the SIP study performed on a sample from the Testfeld Sud€ carbon cycle in deep environments via the recycling of aquifer contaminated by hydrocarbons (Winderl et al., organic material, which is, in our context, the degrada- 2010), suggesting that a large part of the diversity of tion of mono-aromatic hydrocarbons. To date, only two bssA sequences sensu stricto and sensu lato, and bssA strains of Peptococcaceae (Desulfotomaculum sp. Ox39 homologues (assA, nmsA, hbsA) is still to be discovered. and Desulfosporosinus meridiei) have been described as The contig sequence_48572 is close to the Desulfobac- being able to degrade toluene and/or xylenes (Liu and ula toluolica bssA sequence (42% similarity, 115AA; Garcia-Dominguez, 2004; Morasch et al., 2004). How- ABM92935). As regard the contig sequence_31410 is ever, several studies using non culture-based close to a Desulfotomaculum sp. 46_20 alkylsuccinate approaches tend to demonstrate that these microorgan- synthase obtained from an oil reservoir in Alaska (46%/ isms are often playing a key role in the degradation of 138AA; KUK63464), but also to a pyruvate-formate lyase mono-aromatic hydrocarbons. In the case of a gas con- derived from a strain affiliated to Peptococcaceae from a densate-contaminated aquifer, Desulfosporosinus sp. Opalinus Clay rock porewater BRC-3 borehole (39%/ was shown to initiate toluene degradation (Fowler et al., 142AA; KJS47223) and to a glycyl radical enzyme of the 2012, 2014). The key role of the Peptococcaceae has strain Desulfosporosinus sp. BRH_c37, also obtained also been demonstrated in other environments and in from the BRC-3 site (38%/143AA; KUO70645). sulphate-reducing conditions and/or methanogenesis (Abu Laban et al., 2009; Winderl et al., 2010; Pilloni Conclusions et al., 2011; Sun and Cupples, 2012; Sun et al., 2014; Abu Laban et al., 2015; Tan et al., 2015). It is interesting The study of the potential for TX degradation in olig- to note that the bssA amplicon obtained for the otrophic environments such as deep aquifers is very

(A)Enrichment with formation water (FW + TX) (B) Enrichment with formation water supplemented with concentrated biomass (FWCB + TX) 100 100 90 90 80 80 70 70 o-xylene 60 60 toluene 50 50 p-xylene 40 40 m-xylene 30 30 20 20 10 10 0 0

Relative BTEX abundance (%) 0 200 400 600 800 1000 0 200 400 600 800 1000 Days Days

Fig. 2. Degradation of mono-aromatic hydrocarbons (toluene, m- and p-xylenes) during incubation of formation water collected anoxically to protect autochthonous microbiota, FW (A) or with the formation water supplemented with concentrated biomass, FWCB (B). Filled circles: o-xylene, filled triangles: toluene, filled diamonds: p-xylene, filled squares: m-xylene. Arrow indicates at day 447 the addition of toluene, m- and p-xylenes (10 ppm). Start levels of mono-aromatic hydrocarbons were 10 ppm.

ª 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology, Microbial Biotechnology, 10, 469–479 476 M. Ranchou-Peyruse et al.

81 Desulfosporosinus sp. BRH_c37 / Opalinus Clay rock porewater BRC-3 borehole (KUO70645) 54 Peptococcaceae / Opalinus Clay rock porewater BRC-3 borehole (KJS47223) Geobacter toluenoxydans JCM15764 / Tar-oil-contaminated aquifer (ABM92938) Desulfobacula toluolica Tol2 / Contaminated Testfeld Sued aquifer sediment (ABM92935) Desulfotomaculum sp. 46_296 / Oil reservoir (KUK52067)

100 Sulfate-reducing bacterium PRTOL1 / Aviation fuel-contaminated soil (ACI45753) Sulfate-reducing bacterium TRM1 / Contaminated Testfeld Sued aquifer sediment (ABM92939) 50 Magnetospirillum sp. TS-6 / Soils (BAD42366)

70 Azoarcus sp. DN11 / Benzene-contaminated underground aquifer (BAF63031) 99 Thauera_sp._DNT-1 / Anaerobic sludge from a wastewater treatment plant (BAC05501) Monoaromatic hydrocarbons-degrading enrichment / Deep aquifer 2 used for natural gas storage ; this study BssA Environmental sequence SL35-bssA / Production water from petroleum reservoir (AEY68269) sensu stricto Contig sequence_10944 / Deep aquifer 1 used for natural gas storage ; this study 86 99 52 Benzene-degrading enrichment – Clostridia clone BF / Soil from a former coal gasification site (ADJ93876) 72 Toluene-degrading methanogenic consortium – clone bssA-2 / Pensacola aquifer - 6m depth (ABO30979)

99 Monoaromatic hydrocarbons-degrading enrichment / Deep aquifer 3 used for natural gas storage ; this study 100 Desulfotomaculum gibsoniae Groll / Black muf from freshwater ditch (WP_006524663)

95 Environmental sequence B49bss_012 / Contaminated Testfeld Süed aquifer sediment (ABM91877) Environmental sequence FI-TR021 / Hydrocarbon polluted marine sediments from Figueiras Beach (AGJ00182) BssA 99 Environmental sequence F4A12 / Tar-oil contaminated Flingern aquifer sediment (AFX62261) 51 sensu lato 96 Environmental sequence Zz-px_01 / Contaminated aquifer (ADA81918)

59 Environmental sequence F3B28 / tar-oil contaminated Flingern aquifer sediment (AFX62308) 100 Environmental sequence BGN06 / Guaymas hydrocarbon seep, dive 4573 (AFX62295) 97 deltaproteobacterium NaphS2 / marine sediment (CAO72219) NmsA Environmental sequence BG2NO2 / Gulf of Mexico hydrocarbon seep, dive 161 (AFX62288)

BssA 85 Monoaromatic hydrocarbons-degrading enrichment / Deep aquifer 1 used for natural gas storage ; this study

91 ADA81912.1|_benzylsuccinate_synthase_alpha_subunit_partial_uncultured_bacterium 90 Environmental sequence BAA23 / Amon mud volcano (AFX62278) 92 Desufotomaculum sp. OX39 (ABM92937) 98 Environmental sequence F3N20 / Tar-oil contaminated Flingern aquifer sediment (AFX62314)

100 Contig sequence_48572 / Deep aquifer 1 used for natural gas storage ; this study AssA 73 Contig sequence_31410 / Deep aquifer 1 used for natural gas storage ; this study Desulfotomaculum sp. 46_80 (KUK63464) 69 Desulfoglaeba alkanedexens ALDC / (ADJ51097)

100 Desulfatibacillum aliphaticivorans CV2803 / Oil polluted marine sediments (CRX77071) 100 Desulfatibacillum alkenivorans PF2803 / Oil polluted marine sediments (CRX77072) Clostridium clostridioforme 2789STDY5834865 (CUP61718)

0.1

Fig. 3. Phylogenetic tree based on partial bssA-like amino acid sequences from deep aquifers used for geological natural gas storage (in bold; this study) compared with sequences from pure strains, enrichment cultures or environments retrieved in the databases. Sequences in bold were obtained by PCR from genomic DNA from mono-aromatic hydrocarbon-degrading enrichment cultures (aquifers 1, 2 and 3). Sequences in bold and italic were obtained by SHS method directly from formation water (aquifer 1). The evolutionary distances were computed using the Poisson correction method. Evolutionary analyses were conducted in MEGA6 with a bootstrap test of 1000 replicates. difficult using conventional cultural approaches, as they bssA genes directly in FW, while enrichments require long incubation periods (several months to sev- subsequently showed that the metabolic potential was eral years) and/or biomass. Biomarker detection allowing present. the evaluation of the biodegradation potential of an Direct sequencing of metagenomic samples in recent ecosystem or the monitoring of bioremediation opera- years has allowed for increased precision in microbial tions is necessary tools for environmental engineering. diversity analyses but only dominant taxa could be Currently, the bssA and bssA-like genes represent excel- revealed. The sequence capture by hybridization lent biomarkers for the degradation of some mono-aro- approach used in this study proved its efficiency for the matic hydrocarbons. However, no primer sets tested in specific capture of targeted bssA sequences. Indeed, the study enabled demonstration of the presence of this was the only method that enabled bssA and

ª 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology, Microbial Biotechnology, 10, 469–479 Sequence capture by hybridization applied for bssA 477 bssA-like sequences to be obtained directly from FW. Beller, H.R., Kane, S.R., Legler, T.C., and Alvarez, P.J.J. Therefore, this method constitutes a major asset in (2002) A real-time polymerase chain reaction method for developing a clearer understanding of ecosystems and monitoring anaerobic, hydrocarbon-degrading bacteria based on a catabolic gene. Environ Sci Technol 36: in monitoring bioattenuation phenomena in the context of 3977–3984. mature environmental engineering. The bssA sequences Beller, H.R., Kane, S.R., Legler, T.C., McKelvie, J.R., Lollar, and the diversity analyses based on the 16S rDNA B.S., Pearson, F., et al. 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