Mitigation of Methane Emissions from Peatlands - a Role for Micro-propagated Sphagnum-Associated Methanotrophs

泥炭地是大气甲烷最大的天然来源，储存了30%以上的陆地碳（Rodhe, 1990; Gorham, 1991; Hein et al., 1997），大部分碳来自泥炭藓类和互利共生的甲烷营养细菌群落（图1）（Clymo and Hayward, 1982; Raghoebarsing et al. 2005;）。然而，由于人类活动和气候变暖，全球超过15%的泥炭地已经被破坏。特别是在英国，只有不到20%的泥炭地存活了下来（Bain et al., 2011）。英国提出了旨在重新湿润泥炭地和在受损地区重新种植泥炭藓的恢复项目。先前的研究表明，当使用beadomess可持续种植的Sphagnum moss （BeadaHumok）来恢复天然泥炭地时，泥炭地的甲烷排放量显着减少（Keightley, 2020），然而，尚未证明甲烷营养细菌是否与微繁殖的Sphagnum有关。在我的硕士论文项目中，我证明了BeadaHumok在自然泥炭地的适应性进化之前就能直接从温室中主动降解甲烷，并且与生长在自然泥炭地的Sphagnum苔藓相比，显示出相似的降解甲烷的能力。鉴定了一些相关的甲烷营养细菌，它们以Methylocystis属、Methylosinus属、Methylocapsa属和Methylocella属为主。该项目的总体目标是描述泥炭地恢复过程中与可持续生长的泥炭藓相关的甲烷化养菌的结构和生态生理学，重点是温室生长系统中甲烷化养菌的维持，以及恢复前后甲烷化养菌种群的变化。本项目的目标如下：利用培养依赖（分离）和独立方法（功能遗传标记和元组学方法）确定温室内可持续培养的不同Sphagnum藓类的甲烷氧化电位并鉴定相关的甲烷化养菌。研究Sphagnum相关的甲烷化养菌的生态生理学，评估其利用非甲烷基质（兼性甲烷化）的能力甲烷养菌与苔藓的相互作用（甲烷养菌降解甲烷的能力；泥炭藓的光合能力）。制定“质量控制”程序，以便在恢复项目中使用苔藓之前确定甲烷氧化菌的存在和活性。评估恢复项目中泥炭藓建立后沼泽中是否维持了与苔藓相关的甲烷氧化菌。我们将设计一个泥炭地生态系统来模拟泥炭藓的自然生长环境（如Kox等人（2021）所述），并通过气相色谱法测量甲烷的降解率。Sphagnum相关甲烷氧化菌的多样性和活性将使用靶向DNA、RNA和蛋白质的分子方法来确定，包括定量PCR和宏基因组学/元转录组学/宏蛋白质组学。用13C标记底物的稳定同位素孵育将用于确定潜在兼性甲烷氧化菌存在的底物范围。还将进行甲烷氧化菌的分离。我们将利用通量测序的方法来检测甲烷氧化菌在泥炭苔藓中的定殖和分布，并将这些方法应用于泥炭苔藓移植前后的泥炭地恢复项目。

As the largest natural source of atmospheric methane, peatlands store over 30% of terrestrial carbon (Rodhe, 1990; Gorham, 1991; Hein et al., 1997), most of the carbon comes from Sphagnum mosses and mutually beneficial symbiotic methanotrophic bacteria community (Figure 1) (Clymo and Hayward, 1982, Raghoebarsing et al. 2005;). However, more than 15% of peatlands have been destroyed globally due to human activities and climate warming. Especially in the UK, less than 20% of peatlands survived (Bain et al., 2011). Restoration projects aiming to rewet peatland and replant Sphagnum moss in damaged areas was presented in the UK. Prior research has shown that methane emissions from peatlands are significantly reduced when Beadamoss sustainably grown Sphagnum moss (BeadaHumok) is used to restore natural peatlands (Keightley, 2020), however, it has not been shown whether methanotrophic bacteria are associated with micropropagated Sphagnum. In my MSc dissertation project, I demonstrated that BeadaHumok actively degrades methane straight from the greenhouse even before adaptive evolution in natural peatland and showed similar ability to degrade methane compared to Sphagnum mosses grown in natural peatland. I identified some of the associated methanotrophic bacteria, they were dominated by the genera Methylocystis, Methylosinus, Methylocapsa and Methylocella.The overall aim of the project is to characterise the structure and ecophysiology of methanotrophs associated with sustainably grown Sphagnum moss during peatland restoration, with emphasis on the maintenance of methanotrophs in the greenhouse growing system, and changes in methanotroph populations before and after restoration. The project has the following objectives:Determine methane oxidation potential and identify associated methanotrophs of different Sphagnum mosses grown sustainably by micropropagation in the greenhouse using cultivation-dependent (isolation) and -independent approaches (functional genetic markers and meta-omics approaches)Study the ecophysiology of Sphagnum-associated methanotrophs to evaluate their capability for using non-methane substrates (facultative methanotrophy)Investigate the interaction of methanotrophs and mosses (ability of methanotrophs to degrade methane; photosynthetic capacity of Sphagnum mosses).Development of a 'quality control' procedure to determine presence and activity of methanotrophs prior to use of mosses in restoration projects.Assess whether moss-associated methanotrophs are maintained in the bogs post establishment of Sphagnum moss in restoration projects.We will design a peatland mesocosm for simulating the natural growth environment of Sphagnum moss (as by Kox et al. (2021) and measure the rate of degradation of methane by gas chromatography.The diversity and activity of Sphagnum associated methanotrophs will be determined using molecular methods targeting DNA, RNA and protein, including quantitative PCR, and metagenomic/metatranscriptomics/metaproteomics. Stable isotope incubations with 13C labelled substrates will be used to determine the substrate range of potentially facultative methanotrophs present. Isolation of methanotrophs will also be carried out.We will use throughput sequencing to develop methods to detect the colonisation and distribution of methanotrophs in Sphagnum moss and apply these to peatland restoration projects before and after transplantation of Sphagnum mosses.