Survival strategies of aerobic methanotrophs under hypoxia in methanogenic lake sediments

Abstract

Background: Microbial methane oxidation, methanotrophy, plays a crucial role in mitigating the release of the potent greenhouse gas methane from aquatic systems. While aerobic methanotrophy is a well-established process in oxygen-rich environments, emerging evidence suggests their activity in hypoxic conditions. However, the adaptability of these methanotrophs to such environments has remained poorly understood. Here, we explored the genetic adaptability of aerobic methanotrophs to hypoxia in the methanogenic sediments of Lake Kinneret (LK). These LK methanogenic sediments, situated below the oxidic and sulfidic zones, were previously characterized by methane oxidation coupled with iron reduction via the involvement of aerobic methanotrophs.

Results: In order to explore the adaptation of the methanotrophs to hypoxia, we conducted two experiments using LK sediments as inoculum: (i) an aerobic "classical" methanotrophic enrichment with ambient air employing DNA stable isotope probing (DNA-SIP) and (ii) hypoxic methanotrophic enrichment with repeated spiking of 1% oxygen. Analysis of 16S rRNA gene amplicons revealed the enrichment of Methylococcales methanotrophs, being up to a third of the enriched community. Methylobacter, Methylogaea, and Methylomonas were prominent in the aerobic experiment, while hypoxic conditions enriched primarily Methylomonas. Using metagenomics sequencing of DNA extracted from these experiments, we curated five Methylococcales metagenome-assembled genomes (MAGs) and evaluated the genetic basis for their survival in hypoxic environments. A comparative analysis with an additional 62 Methylococcales genomes from various environments highlighted several core genetic adaptations to hypoxia found in most examined Methylococcales genomes, including high-affinity cytochrome oxidases, oxygen-binding proteins, fermentation-based methane oxidation, motility, and glycogen use. We also found that some Methylococcales, including LK Methylococcales, may denitrify, while metals and humic substances may also serve as electron acceptors alternative to oxygen. Outer membrane multi-heme cytochromes and riboflavin were identified as potential mediators for the utilization of metals and humic material. These diverse mechanisms suggest the ability of methanotrophs to thrive in ecological niches previously thought inhospitable for their growth.

Conclusions: Our study sheds light on the ability of enriched Methylococcales methanotrophs from methanogenic LK sediments to survive under hypoxia. Genomic analysis revealed a spectrum of genetic capabilities, potentially enabling these methanotrophs to function. The identified mechanisms, such as those enabling the use of alternative electron acceptors, expand our understanding of methanotroph resilience in diverse ecological settings. These findings contribute to the broader knowledge of microbial methane oxidation and have implications for understanding and potential contribution methanotrophs may have in mitigating methane emissions in various environmental conditions.

Keywords: Methylobacter; Methylomonas; Aerobic methanotrophy; Hypoxia; Lake sediment; Methanogenic zone.

Fig. 1

Experimental layout. Experimental design—sediment cores were collected from the methanotrophic zone in Lake Kineret and used to initiate two enrichment experiments. A Experiment 1: Incubated with ambient air, representing a "classical" enrichment approach coupled with DNA-SIP analysis. B Experiment 2: Hypoxic enrichment by repeatedly introducing small amounts (spikes) of oxygen (1%) to maintain low-oxygen conditions. Extracted DNA was used for 16S rRNA amplicon sequencing, and representing samples were also used to assemble and bin Methylococcales MAGs. Genome-based metabolic comparative analyses were performed using different bioinformatic platforms C

Fig. 2

Aerobic enrichment experiment employing DNA-SIP. A The average DNA concentration (ng/μl) for each fraction (n = 3 biological replicates) in the ¹³C-methane-fed cultures (blue) and the ¹²C-methane-fed cultures (colorless). Error bars indicate the standard error (standard deviation/n) of the DNA concentration, while the error bars for density are smaller than the symbol. B Principal Component Analysis (PCoA) based on Bray–Curtis dissimilarity illustrating microbial diversity in labeled DNA fractions (circles), unlabeled DNA fractions (triangles), and time zero samples that were not fractionated (squares) for both ¹³C-methane-fed cultures (in blue) and ¹²C-methane-fed cultures (colorless). C The average relative abundance of dominant Methylococcales in the labeled DNA fractions of the ¹³C-methane-fed cultures

Fig. 3

Hypoxic enrichment experiment. A In vitro monitoring of oxygen levels (%) in bottles exposed to O₂ + CH₄ (blue) and O₂ + N₂ (green). B Principal Component Analysis illustrating microbial diversity exposed to O₂ + CH₄ (circles), O₂ + N₂ (triangles), and time zero samples without enrichment (squares). These are color-coded based on treatment, with blue, green, and gray representing O₂ + CH₄, O₂ + N₂, and time zero, respectively. C The average relative abundance of dominant Methylococcales spp. enriched in the O₂ + CH₄ treatment

Fig. 4

Phylogenomic analysis and metabolic profiling. A phylogenomic tree along with the metabolic presence-absence profile of 67 Methylococcales genomes. A comprehensive list of the proteins is available in Table S7 and predicted OMC proteins are available in Table S8. Additional information regarding presence-absence of kye genes particulate methane monooxygenase pmoCAB operon and pxmABC operon, soluble methane monooxygenase (mmoXYBZDC), lanthanide-dependent methanol dehydrogenases (xoxF), and methanol dehydrogenase (mxaF) is available in Table S9. Notably, the five LK Methylococcales are highlighted in bold. The phylogenetic tree is built on 100 genes (refer to Table S5), and the taxa clustering percentage is based on 100 bootstrap resamples, consistently yielding values of 98 or higher (specific values not shown)