Microorganisms as bio-filters to mitigate greenhouse gas emissions from high-altitude permafrost revealed by nanopore-based metagenomics

Abstract

The distinct climatic and geographical conditions make high-altitude permafrost on the Tibetan Plateau suffer more severe degradation than polar permafrost. However, the microbial responses associated with greenhouse gas production in thawing permafrost remain obscured. Here we applied nanopore-based long-read metagenomics and high-throughput RNA-seq to explore microbial functional activities within the freeze-thaw cycle in the active layers of permafrost at the Qilian Mountain. A bioinformatic framework was established to facilitate phylogenetic and functional annotation of the unassembled nanopore metagenome. By deploying this strategy, 42% more genera could be detected and 58% more genes were annotated to nitrogen and methane cycle. With the aid of such enlarged resolution, we observed vigorous aerobic methane oxidation by Methylomonas, which could serve as a bio-filter to mitigate CH₄ emissions from permafrost. Such filtering effect could be further consolidated by both on-site gas phase measurement and incubation experiment that CO₂ was the major form of carbon released from permafrost. Despite the increased transcriptional activities of aceticlastic methanogenesis pathways in the thawed permafrost active layer, CH₄ generated during the thawing process could be effectively consumed by the microbiome. Additionally, the nitrogen metabolism in permafrost tends to be a closed cycle and active N₂O consumption by the topsoil community was detected in the near-surface gas phase. Our findings reveal that although the increased thawed state facilitated the heterotrophic nitrogen and methane metabolism, effective microbial methane oxidation in the active layer could serve as a bio-filter to relieve the overall warming potentials of greenhouse gas emitted from thawed permafrost.

Keywords: frame‐shift correction; global warming; high‐altitude permafrost; metatranscriptome; nanopore sequencing.

Figure 1

(A) The correction‐based annotation workflow of FUNpore. (B) The t‐distributed stochastic neighbor embedding (t‐SNE) analysis based on five‐nucleotide frequency of the nanopore reads, Illumina‐assembly (CLC genomic workbench 12.0), and hybrid‐assembly (OPERA‐MS) contigs. The histogram shows the distribution of length of nanopore reads, Illumina‐assembly, and hybrid‐assembly contigs. (C) Precision and recall of FUNpore functional annotation evaluated by the real nanopore sequencing data (highlighted upmost plot) and mock nanopore data set. The real data was the nanopore whole genome sequence of 20 isolated strains. The mock data was downloaded from the NCBI genome database and randomly introduced a 5% error base.

Figure 2

(A) Ternary plot depicts different relative abundances of the genus (>0.5‰) across three altitude samples. Each point represents one genus. The position of each point is determined by the contribution of the indicated compartment to the total relative abundance, and its size represents the average relative abundance across all the three altitude samples. The points are colored according to Phylum affiliation. The significantly enriched genus at different altitude samples was labeled with its taxonomy (namely Flavobacterium, Delftia, Stenotrophomonas, and Oscillatoria). (B) The study area and sampling sites in the Qilian Mountain. The figure was modified from the previous study by Chen et al. [69]. The thawed active layer of permafrost soil samples was collected at HP3000, HP3500, and HP4000. The red triangle represents the frozen permafrost samples collected in HP3500.

Figure 3

The transcriptional activity of nitrogen metabolism in thawed and frozen permafrost soil at HP3500. (A) The transcriptional activity of different microorganisms involved in nitrogen metabolism. The left and right side of the figure respectively represents activities in thawed and frozen permafrost soil. The values in parentheses represent the relative abundance of microorganisms in the community. Table on the top left corner summarizes the total transcriptional activities of different pathways: ammonia => nitrite: N1 + N2, nitrite => nitrate: N3, nitrate => nitrite: N3 + N10, nitrite => nitrogen: N4 + N5 + N6, nitrite => ammonia: N11. (B) Nitrogen metabolism pathway and key genes. The different colors of key genes are correlated with that in (A). Genes only detected in the unclassified genus are colored in “gray,” while genes undetected are colored in “white.” (C) The N₂O emission rate at different temperatures in incubation experiments as validation for the field measurement.

Figure 4

The transcriptional activity of methane metabolism in thawed and frozen permafrost soil at HP3500. (A) The transcriptional activity of different microorganisms involved in methane metabolism. The left and right side of the figure respectively represents activities in thawed and frozen permafrost soil. The values in parentheses represent the relative abundance of microorganisms in the community. The table on the top right corner summarizes the total transcription activities of major methane metabolizing pathways: methanogenesis: the sum of M12 in methanogens, methane oxidation (anaerobic): the sum of M12 in anaerobic archaeal methanotrophs (order Methanosarcinales [ANME‐2 and ANME‐3] and “Ca. Methanophagales” (ANME‐1), methane oxidation (aerobic): the sum of O1–O9, sulfate reduction: the sum of S1–S3. (B) Pathways and key genes of methane metabolism. The different colors of key genes are correlated with that in (A). Genes undetected are colored in “white.” The hollow arrows indicate the direction of anaerobic methane oxidation. (C) The CO₂ and CH₄ emission rates at different temperatures in incubation experiments as validation for the field measurement.