Magnetite drives microbial community restructuring and stimulates aceticlastic methanogenesis of type II Methanosarcina in mangrove sediments

Abstract

Background: Mangrove wetlands are critical hotspots of methane emissions, yet the role of naturally occurring minerals in shaping their microbial communities and methanogenic processes is poorly understood. Magnetite, a common iron mineral in soils and sediments, has been reported to enhance aceticlastic methanogenesis and facilitate syntrophic methanogenesis. In this study, we integrated multi-omic profiling with cultivation-based approaches to investigate the impact of magnetite on methanogenesis of microbial consortia derived from mangrove sediments, using lactate as a substrate.

Results: Across five serial transfers, mangrove microbial consortia converted lactate to propionate and acetate, which were subsequently degraded into methane. Magnetite addition significantly stimulated methane production, leading to notable changes in community structure, particularly for aceticlastic methanogens, with Methanosarcina predominating in the magnetite-amended cultures and Methanothrix in controls. Four Methanosarcina strains T3, T4, T13, and MeOH were subsequently isolated from magnetite-amended cultures. Combined analyses of metagenome-assembled genomes and the genomes of these isolates revealed that the enriched Methanosarcina in magnetite-amended cultures belonged to type II deficient in hydrogenotrophic methanogenesis pathway. Metatranscriptomic analyses suggested that magnetite addition stimulated aceticlastic methanogenesis of type II Methanosarcina and hydrogenotrophic methanogenesis of Methanomicrobiales in the consortia. Furthermore, pure culture experiments confirmed that magnetite stimulated aceticlastic methanogenesis by Methanosarcina sp. T3, although its gene expression patterns differed from those observed in the microbial consortia. Additionally, Methanofastidiosales, an uncultured archaeal lineage possessing H₂-dependent methylotrophic methanogenesis, was detected in all transfers.

Conclusions: Our findings demonstrate that magnetite alters methanogenic consortia in mangrove sediments, selectively stimulating aceticlastic methanogenesis of type II Methanosarcina and modulating hydrogenotrophic activity in Methanomicrobiales. By integrating multi-omics analyses with pure culture validation, we demonstrate, for the first time, that magnetite directly enhances the aceticlastic methanogenesis of type II non-hydrogenotrophic Methanosarcina. This study provides new insights into the influence of magnetite on complex microbial consortia, offers a deeper understanding of the physiology of type II non-hydrogenotrophic Methanosarcina, and advances knowledge of mineral-mediated regulation of methanogenic networks in anoxic environments. Video Abstract.

Keywords: Methanosarcina; Aceticlastic methanogenesis; Hydrogenotrophic methanogenesis; Magnetite; Mangrove sediments; Metagenome; Metatranscriptome; Methane.

Fig. 1

Effect of magnetite on methane production of mangrove microbial consortia using lactate as substrate at 1st (A–D), 3rd (E–H), and 5th (I–L) generation in the presence (red lines) or absence (black lines) of magnetite. Arrows indicated the sampling timepoints for 16S rRNA gene amplicon sequencing (1st, 3rd, and 5th generation), and for metagenomic and metatranscriptomic sequencing (5th generation). Data represent mean values and standard deviations from three independent cultures. Difference in methane production between the two groups were tested by two-tailed paired t tests, indicated by * when p < 0.05, or **p < 0.01

Fig. 2

Effects of magnetite on relative abundances of archaea (A) and bacteria (B) at 1st, 3rd, and 5th generation. Error bars represent standard deviations from three independent cultures. C control group without magnetite addition, CM magnetite-amended group. M: original inoculum. Only taxa with a relative abundance greater than 1% in at least one group are shown in the figure

Fig. 3

Phylogenetic trees of bacterial and archaeal genomes recovered from metagenome and Methanosarcina isolate T3 based on 120 bacterial and 53 archaeal single-copy marker proteins, and the relative abundances of each genome in metagenomic (CE, CL, CME, CML) and metatranscriptomic (CL-trans, CML-trans) libraries in the absence and presence of magnetite in the 5th generation. CE and CL, stage E and L for the control group; CME and CML, stage E and L for the magnetite-amended group. Archaeal tree is presented in brown

Fig. 4

Aceticlastic methanogenesis pathways and transcript levels of the core genes in Methanosarcina (A, B) and Methanotrichaceae (C, D) in mangrove microbial consortia in the presence or absence of magnetite. For (A), genes that were significantly upregulated (log₂FC > 1, FDR < 0.05) or downregulated (log₂FC < − 1, FDR < 0.05) in magnetite-amended group for Methanosarcina sp. T3, Mes4 and Mes5 MAGs are presented in dark red and dark blue bold, respectively; for (C), genes that were significantly down-regulated in the control group (log₂FC < − 1, FDR < 0.05) for both Methanotrichaceae Mex and Mtr MAGs are presented in dark blue bold; for (B) and (D), if proteins are composed of multiple subunits, values from the most highly expressed subunit are represented. C control group, CM magnetite-amended group. Details regarding the fold differences and FDR values of each gene are provided in Table S4

Fig. 5

Hydrogenotrophic methanogenesis in Methanomicrobiales and syntrophic interactions. A Propionate degradation pathway of represented bacterial MAGs (blue dash line) supporting syntrophic interactions with hydrogenotrophic methanogens. B Hydrogenotrophic methanogenesis pathway in Methanomicrobiales MAGs (Meg and Mem2) during syntrophic growth with propionate-oxidizing bacteria. Genes up-regulated in the magnetite-amended group (log₂FC > 1, FDR < 0.05) in both Meg and Mem2 are highlighted in bold dark red. C Transcript levels of core genes in Methanomicrobiales MAGs (Meg and Mem2). If proteins are composed of multiple subunits, values from the most highly expressed subunit are represented. C control group, CM magnetite-amended group. Fold differences and FDR values for each gene are provided in Table S4

Fig. 6

Effects of magnetite addition on growth (A, B) and transcript activity (C) of Methanosarcina sp. T3 in pure culture with acetate (40 mM) as the substrate. For (A) and (B), data represent mean values and standard deviations from three independent cultures. For (C), genes that were up-regulated and down-regulated in the presence of magnetite are presented in dark red and blue, respectively. Details regarding the fold differences and FDR values of each gene are provided in Table S6. Arrows indicated the sampling timepoints for transcriptomic analysis. Difference in methane production between the two groups were tested by two-tailed paired t tests, indicated by * when p < 0.05, or **p < 0.01