Growth and activity of ANME clades with different sulfate and sulfide concentrations in presence of methane. Growth and activity of ANME subclusters

Extensive geochemical data showed that significant methane oxidation activity exists in marine sediments. The organisms responsible for this activity are anaerobic methane-oxidizing archaea (ANME) that occur in consortia with sulfate-reducing bacteria. A distinct zonation of different clades of ANME (ANME-1, ANME-2a/b and ANME-2c) exists in marine sediments, which could be related to the localized concentrations of methane, sulfate and sulfide. In order to test this hypothesis we performed long-term incubation of marine sediments under defined conditions with methane as a headspace gas: low or high sulfate (4 and 21 mM, respectively) in combination with low or high sulfide (0.1 and 4 mM, respectively) concentrations. Control incubations were also performed, with only methane, high sulfate or high sulfide. Methane oxidation was monitored and growth of subtypes ANME-1, ANME-2a/b, and ANME-2c assessed using qPCR analysis. A preliminary archaeal community analysis was performed to gain insight into the ecological and taxonomic diversity. Almost all of the incubations with methane had methane oxidation activity, with the exception of the incubations with combined low sulfate and high sulfide concentrations. Sulfide inhibition occurred only with low sulfate concentrations, which could be due to the lower Gibbs free energy available as well as sulfide toxicity. ANME-2a/b appear to mainly grow in incubations which had high sulfate levels and methane oxidation activity, whereas ANME-1 did not show this distinction. ANME-2c only grew in incubations with only sulfate addition. These findings are consistent with previously published in situ profiling analysis of ANME subclusters in different marine sediments. Interestingly, since all ANME subtypes also grew in incubations with only methane or sulfate addition, ANME may also be able to perform anaerobic methane oxidation under substrate limited conditions or alternatively perform additional metabolic processes.

大量地球化学数据表明，海洋沉积物中存在显著的甲烷氧化活性。介导该活性的功能微生物为厌氧甲烷氧化古菌（anaerobic methane-oxidizing archaea，ANME），其可与硫酸盐还原菌形成共生聚集体。海洋沉积物中不同演化支的ANME类群（ANME-1、ANME-2a/b及ANME-2c）存在明显的分带现象，该分带或与甲烷、硫酸盐和硫化物的局部浓度密切相关。为验证上述假说，本研究以甲烷作为顶空气体，在可控条件下对海洋沉积物开展了长期孵育实验：设置低/高硫酸盐浓度（分别为4 mM和21 mM），并分别搭配低/高硫化物浓度（分别为0.1 mM和4 mM）；同时设置仅添加甲烷、高浓度硫酸盐或高浓度硫化物的对照孵育组。通过定量PCR（qPCR）分析监测甲烷氧化情况，并对ANME-1、ANME-2a/b及ANME-2c亚型的生长水平进行评估；此外还开展了初步的古菌群落分析，以解析其生态与分类学多样性。几乎所有添加甲烷的孵育组均表现出甲烷氧化活性，仅低硫酸盐与高硫化物联合处理的孵育组除外。硫化物抑制效应仅在低硫酸盐浓度条件下出现，这可能与体系可利用的吉布斯自由能较低以及硫化物的毒性作用相关。ANME-2a/b亚型主要在高硫酸盐水平且存在甲烷氧化活性的孵育组中增殖，而ANME-1则未表现出这一特异性。ANME-2c仅在仅添加硫酸盐的孵育组中得以生长。上述研究结果与此前已发表的不同海洋沉积物中ANME亚簇原位群落谱分析结果一致。值得注意的是，由于所有ANME亚型在仅添加甲烷或硫酸盐的孵育组中同样实现了生长，这表明ANME或可在底物受限条件下开展厌氧甲烷氧化，抑或具备其他额外的代谢途径。