1
Supplementary Texts, Imachi et al.
2
3
Supplementary Text 1,
4
The reason for why these organic substances were used for cultivation of subseafloor
5
methanogenic community. The substrates for the enrichment of subseafloor methanogenic
6
microbial communities are important. In this study, we provided glucose, yeast extract,
7
acetate and propionate as potential energy and carbon sources for the enrichment in the DHS
8
reactor. Other than acetate, methanogens cannot directly use these substances, but these
9
substrates are more realistic energy and carbon sources for the in situ subseafloor sedimentary
10
habitat. Most of the methanogens can grow on H2 and carbon dioxide as methanogenic
11
substrates (Liu and Whitman, 2008). In many natural anaerobic habitats, including the
12
subseafloor sediments, methanogens should thrive by receiving H2 that is provided by
13
heterotrophic H2-producing bacteria, which catalyze the oxidation of a variety of organic
14
substances (Liu and Whitman, 2008; Stams and Plugge, 2009). The methanogens utilize the
15
H2 produced by these heterotrophic bacteria, and in return, the bacteria benefit from the
16
removal of excess H2 that would otherwise inhibit their growth. This relationship is
17
commonly referred to as interspecies H2 transfer (Schink, 1997; Stams and Plugge, 2009). In
18
syntrophic associations of methanogens and heterotrophic bacteria via interspecies H2 transfer,
19
H2 is continuously provided at a low concentration from heterotrophic bacteria to H2-utilizing
20
methanogens (less than 30 Pa in the case of propionate) (Imachi et al., 2000; Sakai et al.,
21
2007). Furthermore, organic substances, particularly fatty acids, are generally converted to H2
22
at a slow rate by heterotrophic bacteria because bacteria that live in syntrophy with
23
H2-methanogens are generally exhibit slow growth rates (approximate 5 day doubling time in
24
the case of syntrophic propionate-oxidizing bacteria [Harmsen et al., 1998; Imachi et al.,
25
2007]). Therefore, we focused on interspecies H2 transfer, and we have proposed a new
1
1
method for cultivating H2-utilizing methanogens; the co-culture method (Sakai et al., 2007;
2
Sakai et al., 2009). Previously, we successfully cultivated phylogenetically diverse
3
methanogens in serum vials containing a defined medium supplemented with ethanol or fatty
4
acids as indirect precursor substrates that are converted to H2 by heterotrophic bacteria, and
5
we eventually isolated novel methanogens in pure culture (Imachi et al., 2008; Sakai et al.,
6
2008). Therefore we assumed that the organic substances used in the current study might also
7
be one of the appropriate substrates for the cultivation of marine subsurface methanogens that
8
have adapted well to an extremely slow H2 flux and might form syntrophic associations with
9
heterotrophic H2-producing bacteria in their natural setting.
10
References
11
Harmsen HJM, Van Kuijk BLM, Plugge CM, Akkermans ADL, De Vos WM, Stams AJM.
12
(1998). Syntrophobacter fumaroxidans sp. nov., a syntrophic propionate-degrading
13
sulfate-reducing bacterium. Int J Syst Bacteriol 48: 1383-1387.
14
Imachi H, Sakai S, Ohashi A, Harada H, Hanada S, Kamagata Y et al. (2007). Pelotomaculum
15
propionicicum sp. nov., an anaerobic, mesophilic, obligately syntrophic, propionate-oxidizing
16
bacterium. Int J Syst Evol Microbiol 57: 1487-1492.
17
Imachi H, Sakai S, Sekiguchi Y, Hanada S, Kamagata Y, Ohashi A et al. (2008). Methanolinea
18
tarda gen. nov., sp. nov., a methane-producing archaeon isolated from a methanogenic
19
digester sludge. Int J Syst Evol Microbiol 58: 294-301.
20
Imachi H, Sekiguchi Y, Kamagata Y, Ohashi A, Harada H. (2000). Cultivation and in situ
21
detection of a thermophilic bacterium capable of oxidizing propionate in syntrophic
22
association with hydrogenotrophic methanogens in a thermophilic methanogenic granular
23
sludge. Appl Environ Microbiol 66: 3608-3615.
24
Liu Y, Whitman WB. (2008). Metabolic, phylogenetic, and ecological diversity of the
25
methanogenic Archaea. Ann N Y Acad Sci 1125: 171-189.
2
1
Sakai S, Imachi H, Hanada S, Ohashi A, Harada H, Kamagata Y. (2008). Methanocella
2
paludicola gen. nov., sp. nov., a methane-producing archaeon, the first isolate of the lineage
3
'Rice Cluster I', and proposal of the new archaeal order Methanocellales ord. nov. Int J Syst
4
Evol Microbiol 58: 929-936.
5
Sakai S, Imachi H, Sekiguchi Y, Ohashi A, Harada H, Kamagata Y. (2007). Isolation of key
6
methanogens for global methane emission from rice paddy fields: a novel isolate affiliated
7
with the clone cluster Rice Cluster I. Appl Environ Microbiol 73: 4326-4331.
8
Sakai S, Imachi H, Sekiguchi Y, Tseng I-C, Ohashi A, Harada H et al. (2009). Cultivation of
9
methanogens under low-hydrogen conditions by using the coculture method. Appl Environ
10
Microbiol 75: 4892-4896.
11
Schink B. (1997). Energetics of syntrophic cooperation in methanogenic degradation.
12
Microbiol Mol Biol Rev 61: 262-280.
13
Stams AJM, Plugge CM. (2009). Electron transfer in syntrophic communities of anaerobic
14
bacteria and archaea. Nat Rev Microbiol 7: 568-577.
15
16
Supplementary Text 2,
17
Chemical analyses. The temperature, pH and oxidation-reduction potential (ORP) of effluent
18
seawater were measured using an InPro3250 pH and redox sensor (Mettler Toledo).
19
Concentrations of acetate, propionate and other organic acids such as succinate, malate,
20
fumarate, butyrate and lactate were measured by HPLC using a Shim-pack SCR-102H
21
column (Shimadzu; mobile phase, 4 mM p-toluenesulfonic acid; column temperature, 45°C).
22
Glucose and ethanol concentrations were determined by HPLC using an SCR101-H column
23
(Shimadzu; eluent, H2O; column temperature, 60°C) and a refractive index detector
24
(Shimadzu RID-10A). The TOC was measured using a TOC analyzer (TNC-6000, Toray
25
Eng.) according to the Japanese Industrial Standards (JIS K0102 22.1). Methane
3
1
concentration was determined by gas chromatography (GC) (GC3200G, GL Science) with a
2
thermal conductivity detector. Measurement of dissolved methane in effluent seawater was
3
performed as previously described (Hatamoto et al., 2010). The stable carbon isotope
4
compositions of CH4 and CO2 in the sampled gas phase were analyzed by Taiyo-Nissan Co.
5
Ltd. using a SerCon ANCA-ORCHID GC isotope ratio mass spectrometer (SerCon). The
6
concentration of monomethylamine was measured by HPLC using a Mightysil RP-18 PA
7
column (Kanto Chemical; eluent, acetonitrile/H2O) and a UV detector (Waters 996
8
Photodiode array detector) at 340 nm, after induction by 2,4,6-trinitrobenzene sulfonic acid.
9
Methanol was analyzed by GC-mass spectrometry (JEOL JMS-AM20) using a DB-WAX
10
column (Agilent Technologies). Trimethylamine was analyzed by GC (Shimadzu GC-2014)
11
using a Chromosorb W column (Shinwa Chemical Industries) and a flame photometric
12
detector. All effluent seawater samples were filtered with a 0.22 µm pore-size
13
polyethersulfone filter unit (Millipore) immediately after sampling and stored at 4°C until
14
measurements.
15
Nucleic acid extraction, PCR and cloning. DNA extraction and PCR amplification were
16
performed as described previously (Miyashita et al., 2009). For PCR amplification, we used
17
the primer pairs Arch21f (DeLong, 1992)/Ar912r (Miyashita et al., 2009) and EUB338F*
18
(Amann et al., 1990; Daims et al., 1999)/1492R (Weisburg et al., 1991) for the construction
19
of 16S rRNA gene-based archaeal and bacterial clone libraries, respectively. For construction
20
of archaeal 16S rRNA gene-based clone libraries from enrichment cultures, Acrh21f-Mvb
21
(5’-TTC TGT TTG ATC CTG GCA GA-3’) was used together with Arch21f as the forward
22
primer (in equal concentrations), in order to cover Methanobrevibacter species 16S rRNA
23
gene sequences (see dialed explanation Supplementary Text 3). We also used primers
24
Arch21F (or Arch21F-Mvb)/1492R or 8f (Weisburg et al., 1991)/1492R to obtain the nearly
25
full-length 16S rRNA gene from archaeal and bacterial isolates, respectively. For PCR
4
1
amplification of the mcrA gene, we used primers Luton-mcrA (Luton et al., 2002) and
2
ME1/ME3 (Hales et al., 1996) for the clone analysis and methanogenic isolates, respectively.
3
The PCR conditions were the following: initial denaturation at 95°C for 10 s, followed by 20
4
to 35 cycles of denaturation at 95°C for 5 s, annealing at 50°C for 30 s, and extension at 72°C
5
for 90 s. To reduce possible bias caused by PCR amplification, we used PCR products
6
obtained at minimized PCR cycle numbers ranging from 20 to 35 cycles at five-cycle
7
intervals. Clone library construction and sequencing were performed as described previously
8
(Miyashita et al., 2009).
9
Total RNA extraction from the enrichment samples was performed immediately after
10
sampling from the DHS reactor using the method described previously (Sekiguchi et al.,
11
2005). The remaining DNA was digested with RNase-free DNase I (Promega). The absence
12
of contaminating genomic DNA in the RNA extract was confirmed by PCR using the primer
13
pairs EUB338F*/907r (Lane, 1991) and Arch21f/Ar912 with 35 PCR cycles. The
14
concentration of RNA was quantified spectrophotometrically with a Quanti-iT RNA assay kit
15
(Invitrogen). Reverse transcription (RT)-PCR was performed with a commercial RT-PCR kit
16
(SuperScript III One-Step RT-PCR System with Platinum Taq DNA polymerase, Invitrogen)
17
according to the manufacturer’s instructions. The same primer sets were used for the RT-PCR
18
of 16S rRNA and mcrA mRNA as were used for the DNA-based clone analyses described
19
above. The subsequent procedures were also the same as used for the DNA-based clone
20
analyses described above.
21
FISH. The samples were fixed with 2% paraformaldehyde in anaerobic synthesis seawater
22
excluding organic substances for 12 h at 4°C and stored in 50% ethanol with
23
phosphate-buffered saline (PBS; 130 mM NaCl, 10.8 mM Na2HPO4, 4.2 mM NaH2PO4 [pH
24
7.2]) at -20°C. For FISH detection, the fixed samples were diluted in 1 x PBS and sonicated to
25
disperse cells at 20 W for 1 min on ice using an ultrasonic homogenizer (Model UH-50, SMT
5
1
Co. Ltd.). Each sample (approximately 0.5 g dry wt) was transferred to a 1.5 ml tube. Then,
2
FISH was performed according to a previous report (Sekiguchi et al., 1998), modified by the
3
addition of 1% blocking reagent (w/v, Roche Diagnostics) to the hybridization buffer.
4
Catalyzed reporter deposition (CARD)-FISH with the horseradish peroxidase
5
(HRP)-labeled ARC915 probe (Thermo Electron Biopolymer) was performed based on a
6
method described previously (Kubota et al., 2006; Pernthaler and Amann, 2004). For
7
CARD-FISH experiments, the fixed samples were embedded in a MetaPhor low melting point
8
agar (Cambrex), and the following pretreatments for the incremental penetration of the
9
HRP-labeled probe into fixed archaeal cells were performed: (i) lysozyme treatment (10
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)
11
proteinase K treatment (20 µg/ml in 100 mM Tris-HCl [pH 7.5] and 50 mM EDTA [pH 8.0])
12
at 37°C for 1 hour; and (iii) 0.01 M HCl for 10 min at room temperature. To inactivate
13
endogenous peroxidase, the cell samples were incubated with H2O2 solution (final
14
concentration, 0.3% [v/v] in methanol) for 10 min at room temperature. As a negative control,
15
sediment samples were hybridized with an HRP-labeled nonsense-probe, NON338 (Manz et
16
al., 1992), which did not produce a CARD-FISH signal in the samples.
17
References
18
Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA. (1990). Combination
19
of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed
20
microbial populations. Appl Environ Microbiol 56: 1919-1925.
21
Daims H, Brühl A, Amann R, Schleifer KH, Wagner M. (1999). The domain-specific probe
22
EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a
23
more comprehensive probe set. Syst Appl Microbiol 22: 434-444.
24
DeLong EF. (1992). Archaea in coastal marine environments. Proc Natl Acad Sci USA 89:
25
5685-5689.
6
1
Hales BA, Edwards C, Ritchie DA, Hall G, Pickup RW, Saunders JR. (1996). Isolation and
2
identification of methanogen-specific DNA from blanket bog peat by PCR amplification and
3
sequence analysis. Appl Environ Microbiol 62: 668-675.
4
Hatamoto M, Yamamoto H, Kindaichi T, Ozaki N, Ohashi A. (2010). Biological oxidation of
5
dissolved methane in effluents from anaerobic reactors using a down-flow hanging sponge
6
reactor. Water Res 44: 1409-1418.
7
Kubota K, Ohashi A, Imachi H, Harada H. (2006). Visualization of mcr mRNA in a
8
methanogen by fluorescence in situ hybridization with an oligonucleotide probe and two-pass
9
tyramide signal amplification (two-pass TSA-FISH). J Microbiol Methods 66: 521-528.
10
Lane DJ. (1991). 16S/23S rRNA sequencing. In: Stackebrandt E and Goodfellow M (eds).
11
Nucleic Acid Techniques in Bacterial Systematics. John Wiley & Sons: Chichester, United
12
Kingdom, pp 115-175.
13
Luton PE, Wayne JM, Sharp RJ, Riley PW. (2002). The mcrA gene as an alternative to 16S
14
rRNA in the phylogenetic analysis of methanogen populations in landfill. Microbiology 148:
15
3521-3530.
16
Manz W, Amann R, Ludwig W, Wagner M, Schleifer K-H. (1992). Phylogenetic
17
oligodeoxynucleotide probes for the major subclasses of Proteobacteria: problems and
18
solutions. Syst Appl Microbiol 15: 593-600.
19
Miyashita A, Mochimaru H, Kazama H, Ohashi A, Yamaguchi T, Nunoura T et al. (2009).
20
Development of 16S rRNA gene-targeted primers for detection of archaeal anaerobic
21
methanotrophs (ANMEs). FEMS Microbiol Lett 297: 31-37.
22
Pernthaler A, Amann R. (2004). Simultaneous fluorescence in situ hybridization of mRNA
23
and rRNA in environmental bacteria. Appl Environ Microbiol 70: 5426-5433.
24
Sekiguchi Y, Kamagata Y, Syutsubo K, Ohashi A, Harada H, Nakamura K. (1998).
25
Phylogenetic diversity of mesophilic and thermophilic granular sludges determined by 16S
7
1
rRNA gene analysis. Microbiology 144: 2655-2665.
2
Sekiguchi Y, Uyeno Y, Sunaga A, Yoshida H, Kamagata Y. (2005). Sequence-specific
3
cleavage of 16S rRNA for rapid and quantitative detection of particular groups of anaerobes
4
in bioreactors. Wat Sci Technol 52: 107-113.
5
Weisburg WG, Barns SM, Pelletier DA, Lane DJ. (1991). 16S ribosomal DNA amplification
6
for phylogenetic study. J Bacteriol 173: 697-703.
7
8
Supplementary Text 3.
9
An explanation for the use of archaral primer Arch21f-Mvb. In our clone analyses for
10
enrichment samples from the DHS reactor, an mcrA phylotype related to the genus
11
Methanobrevibacter (357D_mcrA1) was detected in the clone libraries, while a 16S rRNA
12
gene phylotype belonging to Methanobrevibacter was not detected. During the clone analysis,
13
we realized that the 16S rRNA gene sequence of Methanobrevibacter probably had
14
mismatches with the Arch21f primer because the complete genome sequence of
15
Methanobrevibacter smithii (GenBank accession number NC_009515) contains 4 mismatches
16
with the Arch21f primer. This primer mismatch presumably caused the Methanobrevibacter
17
phylotype to be missed in the 16S rRNA gene clone libraries. Indeed, after addition of the
18
modified primer Arch21f-Mvb to the PCR amplification, a 16S rRNA phylotype that was very
19
closely related to Methanobrevibacter members was successfully detected from the batch-type
20
cultures (Table S8). Thus, we should pay close attention to select or update/revise PCR
21
primers when archaeal diversity is estimated, because some archaeal PCR primers have high
22
mismatch frequencies in particular marine subsurface archaeal lineages (Teske and Sørensen,
23
2008).
24
Reference
25
Teske A, Sørensen KB. (2008). Uncultured archaea in deep marine subsurface sediments:
8
1
have we caught them all? ISME J 2: 3-18. An explanation for the use of archaral primer
2
Arch21f-Mvb.
9