Aerobic and Anaerobic Methanotrophic Communities Associated with Methane Hydrates Exposed on the Seafloor: A High-Pressure Sampling and Stable Isotope-Incubation Experiment

Abstract

High-pressure (HP) environments represent the largest volumetric majority of habitable space for microorganisms on the planet, including the deep-sea and subsurface biosphere. However, the importance of pressure as an environmental variable affecting deep microbial life and their biogeochemical functions in carbon cycling still remains poorly understood. Here, we designed a new high-volume HP-sediment core sampler that is deployable on the payload of a remotely operated vehicle and can maintain in situ HP conditions throughout multi-month enrichment incubations including daily amendments with liquid media and gases and daily effluent sampling for geochemical or microbiological analysis. Using the HP core device, we incubated sediment and overlying water associated with methane hydrate-exposed on the seafloor of the Joetsu Knoll, Japan, at 10 MPa and 4°C for 45 days in the laboratory. Diversity analyses based on 16S rRNA and methane-related functional genes, as well as carbon isotopic analysis of methane and bicarbonate, indicated the stimulation of both aerobic and anaerobic methanotrophy driven by members of the Methylococcales, and ANME, respectively: i.e., aerobic methanotrophy was observed upon addition of oxygen whereas anaerobic processes subsequently occurred after oxygen consumption. These laboratory-measured rates at 10 MPa were generally in agreement with previously reported rates of methane oxidation in other oceanographic locations.

Keywords: high pressure incubation; marine sediment; methane hydrate; methanotrophs; stable isotope probing.

Figure 1

Contextualization of study site. (a) Map of central Japan, including the Joetsu Knoll study site. (b) Image capture from the ROV Hyper-Dolphin Dive 1555, demonstrating the sampling location along a several meter-sized wall of methane hydrate and bacterial mats. Samples were taken from roughly in the area of the black circle. (c) Sediment capture using the HP-Core sampler. (d) Sediment capture using the M-Core sampler.

Figure 2

(a,c) Photographs and (b,d) schematics of the HP-Core. An aluminum chassis (a,c) holds the stainless-steel cylinders comprising the HP-Core. This chassis can be attached to the payload of a remotely operated vehicle (e.g., ROV Hyper-Dolphin) for deployment (a). This does not preclude the simultaneous deployment of other sampling equipment, including push cores and collection boxes (a). After recovery of the HP-Core onboard ship or onshore, the HP-Core (still supported by the aluminum chassis) is connected to pressure and temperature loggers, an HPLC pump for influent media, and an outflow hose for effluent sampling. The influent line [2] enters at the bottom of the HP-Core [1], while the effluent line [3] exits from the top of the HP-Core [1]. The HP-Core is ~0.5 m in height.

Figure 3

Log of HP-Core temperature and pressure over the duration of incubation. Temperature was maintained at ~4.5°C by storing the HP-Core in a walk-in refrigerator throughout the experiment. Pressure was maintained at ~10 MPa (chosen to match the environmental pressure at the sampling depth of 985 mbsl) by injection of sterile artificial seawater via modified HPLC pump. Spikes in the pressure log record the daily effluent sampling for δ¹³C_DIC, during which time pressure fluctuated as the effluent port was opened. Over the course of >40 days, user technique improved and the fluctuations in pressure decreased in magnitude.

Figure 4

Time-resolved record of HP-Core incubation. Daily δ¹³C_DIC measurements are given in black circles. Colored circles represent sampling or amendments (see legend). Gray diamonds are the calculated methane oxidation rate between each day and the day prior. Inset shows the same data on a smaller y-axis in order to better resolve trends within the first 40 days of the experiment.

Figure 5

Heat map of major OTUs identified in the 16S rRNA gene iTag-sequence dataset. OTUs were only selected for presentation if they were present at >2% relative abundance in the M-Core, HP-Core-effluent (T11 and T25), or HP-Core-sediment (T45) samples. M-Core samples are characterized by their richness in Candidate Division JS1 bacteria. T11 and T25 effluent samples host a wide diversity of Delta-, Epsilon-, and Gammaproteobacteria, but notably differ from the T45 samples which are rich in a Methylococcales-associated OTU. The full table of 16S rRNA gene data is provided in the Supplementary Data.

Figure 6

Nonmetric multidimensional scaling (NMDS) plot of 16S rRNA iTag-sequence data from this study. The microbial communities in the samples naturally break into three categories: M-Core sediments, HP-Core effluent, and HP-Core sediments. Among the HP-Core sediments, DNA extraction method accounts for a measurable but small difference in recovered microbial community composition.

Figure 7

Maximum likelihood tree of pmoA gene sequences generated in the MISA assay (trimmed to amino acids 5–49 of M. capsulatas Bath) from the M-Core and HP-Core T45 incubation. Sequences from this study are defined as “Pattern_x” according to unique Hae III and Rsa I-digested RFLP profiles. Sequences from cultured organisms and sequenced genomes of methane- and ammonia-oxidizers are given with their species name. Labels denote the phylogenetic groupings and predicted oxidation metabolisms. Multiple sequence alignments were generated in MUSCLE and the tree was generated in RAxML with 100 bootstraps. Black circles represent relative abundance of pmoA sequences in the M-Core water and T45.1-MoBio samples. The largest contrast between the two samples is seen in the abundance of different methane-oxidizing Gammaproteobaceria-affiliated pmoA patterns.

Figure 8

Comparison of ambient methane oxidation rate measurements between this study and previous studies. Two values are given for this study: calculated methane oxidation rates for the period of putative aerobic conditions (T0–T10 and T29–T45; in gray) and putative anaerobic conditions (T11–T28; in black). The majority of these experiments were conducted near atmospheric pressure with the exception of Nauhaus et al. (2002), which reported a notable increase in the rate of anaerobic methanotrophy with 1.1 MPa.