Molecular hydrogen in seawater supports growth of diverse marine bacteria

Molecular hydrogen (H2) is an abundant and readily accessible energy source in marine systems, but it remains unknown whether marine microbial communities consume this gas. Here we use a suite of approaches to show that marine bacteria consume H2 to support growth. Genes for H2-uptake hydrogenases are prevalent in global ocean metagenomes, highly expressed in metatranscriptomes and found across eight bacterial phyla. Capacity for H2 oxidation increases with depth and decreases with oxygen concentration, suggesting that H2 is important in environments with low primary production. Biogeochemical measurements of tropical, temperate and subantarctic waters, and axenic cultures show that marine microbes consume H2 supplied at environmentally relevant concentrations, yielding enough cell-specific power to support growth in bacteria with low energy requirements. Conversely, our results indicate that oxidation of carbon monoxide (CO) primarily supports survival. Altogether, H2 is a notable energy source for marine bacteria and may influence oceanic ecology and biogeochemistry.

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Study description
This study describes the significance of marine microorganisms utilising hydrogen as an alternate energy source and reveals new mechanisms by which trace gas oxidising marine microorganisms influence the biogeochemistry of global oceans. This study combines culture-dependent and culture-independent based analyses, including genome-resolved metagenomics and thermodynamic modelling to show that diverse marine bacteria consume hydrogen to support growth. Specifically, we demonstrated that microbial communities spanning tropical, temperate and subantarctic waters consume H2 at rates sufficient to support the growth of bacteria with low energy requirements and at environmentally relevant concentrations. Using axenic cultures of the ultramicrobium Sphingopyxis alaskensis, we provide the first demonstration of atmospheric H2 oxidation by a marine bacterium. Concomitantly, using the Tara Oceans dataset, we revealed that the capacity for H2 oxidation as well as carbon monoxide oxidation is

March 2021
widespread amongst marine bacteria, and of increasing importance in the deeper ocean and in subantarctic waters, with H2 likely supporting mixotrophic growth, and CO supporting long-term bacterial survival.
Metagenomes (n = 14), ex-situ activity (n = 14) and dissolved H2 and CO (n = 14) measurements were prepared using subsamples of homogenised surface water and surface microlayer samples collected from each of the sampling locations (Munida

Sampling strategy
For seawater samples, 1 L water samples were obtained from approximately 0 to 20 cm below the surface using sterile Schott bottles or using a manual glass plate sampler. These samples were then homogenised prior to aliquoting into serum vials in triplicate for exsitu analysis of dissolved H2 and CO, and microcosm activity experiments. For each sampling location, one set of triplicate samples were autoclaved and used as a control. In addition, a portion of these samples were also used for DNA extraction for metagenomic analysis. For axenic culture experiments, cultures were also grown and analysed in triplicate for each condition. For qRT-PCR experiments, all biological triplicate samples, standards and negative controls were run in technical duplicate. A replication level of three is the standard sample size for environmental microbiological studies, and the results indicate consistency between replicates, with error bars included on all plots to illustrate any variation between replicates No statistical methods were used to predetermine sample size.

Data collection
Samples were collected by different authors as described within the author contribution statement (copied below). All data was recorded digitally and was analysed computationally as follows: -Concentrations of H2 and CO gas for both microcosm and axenic culture experiments were measured using a Valco TGA-6791-W-4U-2 Trace Gas Analyzer -DNA for shotgun metagenomic sequencing was extracted using a DNeasy PowerSoil kit and sample libraries were prepared using a Nextera XT DNA Sample Preparation Kit.
-Shotgun metagenomic sequencing was performed on an Illumina NextSeq500 platform at the Australian Centre for Ecogenomics.
-Metabolic annotations were completed using DIAMOND and normalized using the single copy ribosomal proteins available via SingleM.
-Phylogenetic analysis was carried out using ClustalW in MEGA11 -Environmental driver analysis was carried out in R using the package randomForest -Growth of Sphingopyxis alaskensis was determined using optical density using an Eppendorf BioSpectrophotometer and analysed using GraphPad Prism 9 -Quantitative RT-PCR was conducted using a QuantStudio 7 Flex Real-Time PCR system and analysed using GraphPad Prism 9 Different authors were responsible for field sample collection (G.S., P. Growth curve and gas chromatography measurements for Sphingopyxis alaskensis, Robiginitalea biformata and Marinovum algicola were collected between 18/11/2019 and 23/12/2020; note extended timescale was due to the impact of COVID-19 related lockdowns in Melbourne, Australia. Quantitative RT-PCR analysis on S. alaskensis was carried out between 28/09/2020 and 29/10/2020.

Data exclusions
The metagenomes were filtered to remove reads corresponding to major organisms detected in the extraction control. One timepoint in the ex-situ gas chromatography measurements was removed due to machine error.

Reproducibility
All experiments and measurements undertaken were successful, and replicates were consistent within so we did not attempt to repeat the experiments beyond the technical and biological replications used within each experiment.

Randomization
Samples that were obtained in the field were returned to the laboratory, homogenized and sub-sampled for the various treatments for the ex situ microcosm and gas chromatography experiments. For axenic culture experiments, bacterial cultures were independently inoculated into triplicate flasks for growth, gas chromatography and qRT-PCR experiments, leading to three biologically distinct populations of bacterial cells per treatment condition.

Blinding
Blinding was not relevant to our study as it relates to solely microbial processes and experimental data was collected electronically and analysed digitally.
Did the study involve field work? Access & import/export Suitable footwear, gloves and equipment, including pre-sterilised Schott bottles, were used during the sampling process to minimize anthropogenic effects for all samples collected. Water samples were collected in compliance with Australian biosecurity regulations, with those from the Munida Transect also subject to New Zealand biosecurity laws.

Disturbance
The study was minimally disruptive to the sampling locations as water was only collected from the surface and surface microlayer using Schott bottles and manual glass plate samplers, respectively. In total, <20 L of water was collected from the Munida Transect, <15 L of water was collected from Port Phillip Bay and <10 L of water was collected from Heron Island. Disturbance was negligible as small volumes of samples were collected from each sampling location in a non-invasive manner.
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