Geochemical and Microbial Analyses of Post-Earthquake Deep-Sea Environment

The Japan Trench is located on the northwestern margin of the Pacific Plate that is subducting beneath the northeastern Japan Arc (Supplementary Fig. S1). Multi-channel seismic reflection data at the epicentral region were acquired by the R/V Kairei (Japan Agency for Marine-Earth Science and Technology: JAMSTEC) in 1999 (Line MY102 of KR99-08, analyzed in the reference7 ) (Fig. 1). Vertical hydrocasts of the CTD-CMS (Conductivity Temperature Depth profiler with Carousel Multiple Sampling system) were conducted 36 days after the M9.0 event during the MR11-03 cruise of R/V Mirai (JAMSTEC) at the epicentral region (Fig. 1). Stations N1 (38°10.6′N, 143°33.0′E, depth: 3502 m), N2 (38°08.7′N, 143°19.0′E, depth: 2954 m), and N3 (38°06.8′N, 143°05.0′E depth: 1948 m) are located on the western direction of the landward slope of a ridge associated with displacement along an outstanding normal fault that was considered to be the potentially largest slip of the Tohoku Earthquake7 . Station R (38°12.5′N, 143°47.2′E, depth: 5715 m) is above a branch reverse fault (Fig. 1). The stations N2 and R were revisited during the YK11-E04 cruise of R/V Yokosuka (JAMSTEC) with another CTD-CMS. The station JKEO (38°00′N, 146°30′E, depth: 5381 m) is the site of JAMSTEC Kuroshio Extension Observatory located in the abyssal plain of the Pacific plate. The Ocean Drilling Project (ODP) Sites 1150 (39°11′N, 143°20′E) and 1151 (38°45′N, 143°20′E) were the drilled sites during the expedition conducted in 1998 and the pore-water chemistry was already reported31 .
The wired CTD-CMS system of the MR11-03 cruise consisted of a CTD (SBE9 Plus, Sea-Bird Electronics), a CMS (SBE32, Sea-Bird Electronics), 36 Niskin-X bottles (12-liter type, General Oceanics), a dissolved oxygen sensor (RINKO-III, JFE Advantech), and a light transmissometer (C-star 25-cm light-path type, WET Lab). The Light Transmission Anomaly (LTA), calculated from the difference between the in-situ light transmission value (Tr: %) and the value of the transparent layer at intermediate depth for each hydrocast, is used to describe deep-sea water turbidity. The CTD-CMS system of the YK11-E04 cruise consisted of a CTD (SBE11 Plus, Sea-Bird Electronics), a CMS (SBE32, Sea-Bird Electronics), and 12 Niskin-(12-liter type, General Oceanics).
The seawater samples taken by the Niskin bottles were immediately subsampled into several optimized bottles for various geochemical and microbiological analyses10 11 (link)12 . For the analysis of manganese concentration10 , the subsampled seawater in an acid-washed plastic bottle was filtered using a 0.22-µm pore-size PTFE filter and acidified with nitric acid (TAMA Chemical) and analyzed by the luminol-H₂O₂ chemiluminescence detection method. For the analysis of methane11 (link), sample seawater was subsampled into a 120 ml glass vial capped by a butyl-rubber septum after the addition of 0.5 ml HgCl₂-saturated solution for poisoning. The methane concentration and carbon isotope ratio were simultaneously determined with a combination of purge and trap techniques and continuous-flow isotope ratio mass spectrometry. The stable carbon isotope ratio is presented in general delta notation on a permillage scale with respect to the Vienna PDB. For the analysis of molecular hydrogen concentration12 , the subsampling was conducted in a manner similar to that used for methane, except that a Teflon-coated butyl-gum septum was used rather than the untreated butyl-gum septum used for methane. The molecular hydrogen concentration was analyzed onboard using the headspace method with a gas chromatograph equipped with a trace-reduced gas detector (TRD-1: Round Science Inc., Japan)12 within six hours after the subsampling to avoid sample alteration during storage. If a hydrogen sulfide-like smell was detected in a sample, the seawater was subsampled and placed in a Nalgene bottle to quantify the H₂S concentration using methylene blue colorimetry.
Microbial cell counts were determined by a DAPI-staining direct count (Supporting Information). For extraction of microbial DNA, a portion (3 L) of deep-sea water was filtered with a 0.22-µm-pore-size cellulose acetate filter (Advantec, Tokyo, Japan) and was preserved onboard at −80°C. DNA was extracted by using the Ultra Clean Mega Soil DNA Isolation kit (MO Bio Laboratory, Solana Beach, CA, USA). Quantitative PCR of archaeal and entire prokaryotic 16S rRNA genes was performed using 7500 Real Time PCR System13 (link)14 (link). Prokaryotic 16S rRNA gene clone analysis was conducted with another PCR experiment15 (link) (also see SI for details). To assess difference in phylogenetic context of the post-earthquake deep-sea microbial communities in the bottommost deep-sea water, an online tool, UniFrac16 (link), was used for the principal coordinates analysis (PCoA).