Supplementary Texts, Imachi Et Al

Supplementary Texts, Imachi et al.

Supplementary Text 1,

The reason for why these organic substances were used for cultivation of subseafloor methanogenic community. The substrates for the enrichment of subseafloor methanogenic microbial communities are important. In this study, we provided glucose, yeast extract, acetate and propionate as potential energy and carbon sources for the enrichment in the DHS reactor. Other than acetate, methanogens cannot directly use these substances, but these substrates are more realistic energy and carbon sources for the in situ subseafloorsedimentary habitat. Most of the methanogens can grow on H2 and carbon dioxide as methanogenic substrates(Liu and Whitman, 2008). In many natural anaerobic habitats, including the subseafloor sediments, methanogens should thrive by receiving H2 that is provided by heterotrophic H2-producing bacteria, which catalyze the oxidation of a variety of organic substances(Liu and Whitman, 2008; Stams and Plugge, 2009). The methanogens utilize the H2 produced by these heterotrophic bacteria, and in return, the bacteria benefit from the removal of excess H2 that would otherwise inhibit their growth. This relationship is commonly referred to as interspecies H2 transfer (Schink, 1997; Stams and Plugge, 2009). In syntrophic associations of methanogens and heterotrophic bacteria via interspecies H2 transfer, H2 is continuously provided at a low concentration from heterotrophic bacteria to H2-utilizing methanogens (less than 30 Pa in the case of propionate) (Imachi et al., 2000; Sakai et al., 2007). Furthermore, organic substances, particularly fatty acids, are generally converted to H2 at a slow rate by heterotrophic bacteria because bacteria that live in syntrophy with H2-methanogens are generally exhibit slow growth rates (approximate 5 day doubling time in the case of syntrophic propionate-oxidizing bacteria [Harmsen et al., 1998; Imachi et al., 2007]). Therefore, we focused on interspecies H2 transfer, and we have proposed a new method for cultivating H2-utilizing methanogens;the co-culture method(Sakai et al., 2007; Sakai et al., 2009). Previously, we successfully cultivated phylogenetically diverse methanogens in serum vials containing a defined medium supplemented with ethanol or fatty acids as indirect precursor substrates that are converted to H2 by heterotrophic bacteria, and we eventually isolated novel methanogens in pure culture (Imachi et al., 2008; Sakai et al., 2008). Therefore we assumed that the organic substances used in the current study might also be one of the appropriate substrates for the cultivation of marine subsurface methanogens that have adaptedwell to an extremely slow H2 flux and might form syntrophic associations with heterotrophic H2-producing bacteria in their natural setting.

References

Harmsen HJM, Van Kuijk BLM, Plugge CM, Akkermans ADL, De Vos WM, Stams AJM. (1998). Syntrophobacter fumaroxidans sp. nov., a syntrophic propionate-degrading sulfate-reducing bacterium. Int J Syst Bacteriol48: 1383-1387.

Imachi H, Sakai S, Ohashi A, Harada H, Hanada S, Kamagata Y et al. (2007). Pelotomaculum propionicicum sp. nov., an anaerobic, mesophilic, obligately syntrophic, propionate-oxidizing bacterium. Int J Syst Evol Microbiol57: 1487-1492.

Imachi H, Sakai S, Sekiguchi Y, Hanada S, Kamagata Y, Ohashi A et al. (2008). Methanolinea tarda gen. nov., sp. nov., a methane-producing archaeon isolated from a methanogenic digester sludge. Int J Syst Evol Microbiol58: 294-301.

Imachi H, Sekiguchi Y, Kamagata Y, Ohashi A, Harada H. (2000). Cultivation and in situ detection of a thermophilic bacterium capable of oxidizing propionate in syntrophic association with hydrogenotrophic methanogens in a thermophilic methanogenic granular sludge. Appl Environ Microbiol66: 3608-3615.

Liu Y, Whitman WB. (2008). Metabolic, phylogenetic, and ecological diversity of the methanogenic Archaea. Ann N Y Acad Sci1125: 171-189.

Sakai S, Imachi H, Hanada S, Ohashi A, Harada H, Kamagata Y. (2008). Methanocella paludicola gen. nov., sp. nov., a methane-producing archaeon, the first isolate of the lineage 'Rice Cluster I', and proposal of the new archaeal order Methanocellales ord. nov. Int J Syst Evol Microbiol58: 929-936.

Sakai S, Imachi H, Sekiguchi Y, Ohashi A, Harada H, Kamagata Y. (2007). Isolation of key methanogens for global methane emission from rice paddy fields: a novel isolate affiliated with the clone cluster Rice Cluster I. Appl Environ Microbiol73: 4326-4331.

Sakai S, Imachi H, Sekiguchi Y, Tseng I-C, Ohashi A, Harada H et al. (2009). Cultivation of methanogens under low-hydrogen conditions by using the coculture method. Appl Environ Microbiol75: 4892-4896.

Schink B. (1997). Energetics of syntrophic cooperation in methanogenic degradation. Microbiol Mol Biol Rev61: 262-280.

Stams AJM, Plugge CM. (2009). Electron transfer in syntrophic communities of anaerobic bacteria and archaea. Nat Rev Microbiol7: 568-577.

Supplementary Text 2,

Chemical analyses.The temperature,pH and oxidation-reduction potential (ORP) of effluent seawater were measured using an InPro3250 pH and redox sensor (Mettler Toledo). Concentrations of acetate, propionate and other organic acids such as succinate, malate, fumarate, butyrate and lactate were measured by HPLC using a Shim-pack SCR-102H column (Shimadzu; mobile phase, 4 mM p-toluenesulfonic acid; column temperature, 45°C). Glucose and ethanol concentrations were determined by HPLC using an SCR101-H column (Shimadzu; eluent, H2O; column temperature, 60°C) and a refractive index detector (Shimadzu RID-10A). The TOC was measured using a TOC analyzer (TNC-6000, Toray Eng.) according to the Japanese Industrial Standards (JIS K0102 22.1). Methane concentration was determined by gas chromatography (GC) (GC3200G, GL Science) with a thermal conductivity detector. Measurement of dissolved methane in effluent seawater was performed as previously described (Hatamoto et al., 2010). The stable carbon isotope compositions of CH4 and CO2 in the sampled gas phase were analyzed by Taiyo-Nissan Co. Ltd. using a SerCon ANCA-ORCHID GC isotope ratio mass spectrometer (SerCon). The concentration of monomethylamine was measured by HPLC using a Mightysil RP-18 PA column (Kanto Chemical; eluent, acetonitrile/H2O) and a UV detector (Waters 996 Photodiode array detector) at 340 nm, after induction by 2,4,6-trinitrobenzene sulfonic acid. Methanol was analyzed by GC-mass spectrometry (JEOL JMS-AM20) using a DB-WAX column (Agilent Technologies). Trimethylamine was analyzed by GC (Shimadzu GC-2014) using a Chromosorb W column (Shinwa Chemical Industries) and a flame photometric detector. All effluent seawater samples were filtered with a 0.22 µm pore-size polyethersulfone filter unit (Millipore) immediately after sampling and stored at 4°C until measurements.

Nucleic acid extraction, PCR and cloning. DNA extraction and PCR amplification were performed as described previously (Miyashita et al., 2009). For PCR amplification, we used the primer pairs Arch21f (DeLong, 1992)/Ar912r (Miyashita et al., 2009) and EUB338F* (Amann et al., 1990; Daims et al., 1999)/1492R (Weisburg et al., 1991) for the construction of 16S rRNA gene-based archaeal and bacterial clone libraries, respectively. For construction of archaeal 16S rRNA gene-based clone libraries from enrichment cultures, Acrh21f-Mvb (5’-TTC TGT TTG ATC CTG GCA GA-3’) was used together with Arch21f as the forward primer (in equal concentrations), in order to cover Methanobrevibacter species 16S rRNA gene sequences (see dialed explanation Supplementary Text 3). We also used primers Arch21F (or Arch21F-Mvb)/1492R or 8f (Weisburg et al., 1991)/1492R to obtain the nearly full-length 16S rRNA gene from archaeal and bacterial isolates, respectively. For PCR amplification of the mcrA gene, we used primers Luton-mcrA (Luton et al., 2002) and ME1/ME3 (Hales et al., 1996) for the clone analysis and methanogenic isolates, respectively. The PCR conditions were the following: initial denaturation at 95°C for 10 s, followed by 20 to 35 cycles of denaturation at 95°C for 5 s, annealing at 50°C for 30 s, and extension at 72°C for 90 s. To reduce possible bias caused by PCR amplification, we used PCR products obtained at minimized PCR cycle numbers ranging from 20 to 35 cycles at five-cycle intervals. Clone library construction and sequencing were performed as described previously (Miyashita et al., 2009).

Total RNA extraction from the enrichment samples was performed immediately after sampling from the DHS reactor using the method described previously (Sekiguchi et al., 2005). The remaining DNA was digested with RNase-free DNase I (Promega). The absence of contaminating genomic DNA in the RNA extract was confirmed by PCR using the primer pairs EUB338F*/907r (Lane, 1991) and Arch21f/Ar912 with 35 PCR cycles. The concentration of RNA was quantified spectrophotometrically with a Quanti-iT RNA assay kit (Invitrogen). Reverse transcription (RT)-PCR was performed with a commercial RT-PCR kit (SuperScript III One-Step RT-PCR System with Platinum Taq DNA polymerase, Invitrogen) according to the manufacturer’s instructions. The same primer sets were used for the RT-PCR of 16S rRNA and mcrA mRNA as were used for the DNA-based clone analyses described above. The subsequent procedures were also the same as used for the DNA-based clone analyses described above.

FISH.The samples were fixed with 2% paraformaldehyde in anaerobic synthesis seawater excluding organic substances for 12 h at 4°C and stored in 50% ethanol with phosphate-buffered saline (PBS; 130 mM NaCl, 10.8 mM Na2HPO4, 4.2 mM NaH2PO4 [pH 7.2]) at -20°C. For FISH detection, the fixed samples were diluted in 1 x PBS and sonicated to disperse cells at 20 W for 1 min on ice using an ultrasonic homogenizer (Model UH-50, SMT Co. Ltd.). Each sample (approximately 0.5 g dry wt) was transferred to a 1.5 ml tube. Then, FISH was performed according to a previous report (Sekiguchi et al., 1998), modified by the addition of 1% blocking reagent (w/v, Roche Diagnostics) to the hybridization buffer.

Catalyzed reporter deposition (CARD)-FISH with the horseradish peroxidase (HRP)-labeled ARC915 probe (Thermo Electron Biopolymer) was performed based on a method described previously (Kubota et al., 2006; Pernthaler and Amann, 2004). For CARD-FISH experiments, the fixed samples were embedded in a MetaPhor low melting point agar (Cambrex), and the following pretreatments for the incremental penetration of the HRP-labeled probe into fixed archaeal cells were performed: (i) lysozyme treatment (10 mg/ml in 100 mM Tris-HCl [pH 7.5] and 50 mM EDTA [pH 8.0]) at 37°C for 30 min; (ii) proteinase K treatment (20 µg/ml in 100 mM Tris-HCl [pH 7.5] and 50 mM EDTA [pH 8.0]) at 37°C for 1 hour; and (iii) 0.01 M HCl for 10 min at room temperature. To inactivate endogenous peroxidase, the cell samples were incubated with H2O2 solution (final concentration, 0.3% [v/v] in methanol) for 10 min at room temperature. As a negative control, sediment samples were hybridized with an HRP-labeled nonsense-probe, NON338 (Manz et al., 1992), which did not produce a CARD-FISH signal in the samples.

References

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Daims H, Brühl A, Amann R, Schleifer KH, Wagner M. (1999). The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. Syst Appl Microbiol22: 434-444.

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Supplementary Text 3.

An explanation for the use of archaral primer Arch21f-Mvb. In our clone analyses for enrichment samples from the DHS reactor, an mcrA phylotype related to the genus Methanobrevibacter (357D_mcrA1) was detected in the clone libraries, while a 16S rRNA gene phylotype belonging to Methanobrevibacter was not detected. During the clone analysis, we realized that the 16S rRNA gene sequence of Methanobrevibacter probably had mismatches with the Arch21f primer because the complete genome sequence of Methanobrevibacter smithii (GenBank accession number NC_009515) contains 4 mismatches with the Arch21f primer. This primer mismatch presumably caused the Methanobrevibacter phylotype to be missed in the 16S rRNA gene clone libraries. Indeed, after addition of the modified primer Arch21f-Mvb to the PCR amplification, a 16S rRNA phylotype that was very closely related to Methanobrevibacter members was successfully detected from the batch-type cultures (Table S8). Thus, we should pay close attention to select or update/revise PCR primers when archaeal diversity is estimated, because some archaeal PCR primers have high mismatch frequencies in particular marine subsurface archaeal lineages (Teske and Sørensen, 2008).

Reference

Teske A, Sørensen KB. (2008). Uncultured archaea in deep marine subsurface sediments: have we caught them all? ISME J2: 3-18.An explanation for the use of archaral primer Arch21f-Mvb.

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