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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 To cite this version: 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 HAL Id: hal-01658765 https://hal.archives-ouvertes.fr/hal-01658765 Submitted on 8 Dec 2017 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License bs_bs_banner 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- À À [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 À indeed impossible, to assess the degradation potential of Conductivity at 25°C(lScm 1) 6000 À 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 lÀ1) 16.0 previous studies, amplifications of the bssA gene using Constituentsa Carbonates (mg lÀ1) < 20 the primers cited previously have unfortunately often À Sulphates (mg l 1) 2186.5 À proved to be unsuccessful, as a result of the few targets Ammonium (mg l 1) 1.75 À available and problems of non-specific amplification Calcium (mg l 1) 25.1 À1 (Aullo€ et al., 2016). Next-generation sequencing (NGS) Magnesium (mg l ) 15.9 Sodium (mg lÀ1) 1400 techniques allow a deeper analysis of genetic diversity Potassium (mg lÀ1) 34.0 À and thus offer the possibility of dispensing with culture- Chloride (mg l 1) 260 À1 Silicates (mg SiO2 l ) 15.8 based techniques. However, diversity analyses are very À Phosphorus (mg l 1) < 0.05 À often unable to identify microbial populations with low- Nitrates (mg l 1) < 2 level representation. The sequence capture by hybridiza- Fluoride (mg lÀ1) 1.51 Barium (mg lÀ1) 0.013 tion (Gasc et al., 2016), therefore, constitutes an alterna- À Total iron (lgl 1) 3200 fi À tive for the ef cient detection of rare or unknown Ferrous iron (lgl 1) < 100 À sequences in metagenomic samples (Denonfoux et al., Manganese (lgl 1)44 À1 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
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