1 reduction in submerged peat moss Sphagnum cuspidatum by 2 tightly associated methanotrophs. Abstract 21Wetlands present the largest natural sources of methane (CH4) and their potential CH4 emissions 22 greatly vary due to the activity of CH4-oxidizing bacteria associated with wetland plant species. In this 23 study, the association of CH4-oxidizing bacteria with submerged Sphagnum peat mosses was studied, 24 followed by the development of a novel mesocosm set-up. This set-up enabled the precise control of 25 CH4 input and allowed for monitoring the dissolved CH4 in a Sphagnum moss layer while mimicking 26 natural conditions. Two mesocosm set-ups were used in parallel: one containing a Sphagnum moss 27 layer in peat water, and a control only containing peat water. Moss-associated CH 4 oxidizers in the 28 field could reduce net CH4 emission up to 93%, and in the mesocosm set-up up to 31%. Furthermore, 29CH4 oxidation was only associated with Sphagnum, and did not occur in peat water. Especially 30 methanotrophs containing a soluble methane monooxygenase enzyme were significantly enriched 31 during the 32 day mesocosm incubations. Together these findings showed the new mesocosm setup 32 is very suited to study CH4 cycling in submerged Sphagnum moss community under controlled 33 conditions. Furthermore, the tight associated between Sphagnum peat mosses and methanotrophs 34 can significantly reduce CH 4 emissions in submerged peatlands. 35 36 Introduction 39 Methane (CH4) has a 25 times higher Global Warming Potential (GWP) than carbon dioxide (CO2; on a 40 100 year time scale) and is the second most important greenhouse gas (GHG), contributing for about 41 16% to global warming [1, 2]. CH4 in the atmosphere originates from both natural and anthropogenic 42 sources. Wetlands are the largest natural CH4 source, emitting an estimated 167 Tg CH4 yr -1 into the 43 atmosphere [3], indicating an imbalance between CH4 production and CH4 consumption by 44 methanotrophs. Climate change has the potential to further stimulate the emission of CH4 from 45 (especially artic) wetlands [4]. Therefore, it is important to understand sources, sinks and microbial 46 transformations of CH4 in wetland ecosystems. 47 the other methanotroph-containing (sub)phyla Gammaproteobacteria and Verrucomicrobia 90 (Methylacidiphilaceae [33-35]. Within the Alphaproteobacteria especially methanotrophs of the 91 family Methylocystaceae (Methylocystis spp.) and the acidophilic methanotrophs of the family 92Beijerinckiaceae (Methylocella, Methyloferula, Methylocapsa) are often found and several of these 93 have been isolated from peatlands [24-26, 36, 37]. Using Fluorescence in situ Hybridization (FISH) 94 combined with confocal microscopy, Alphaproteobacteria have shown to be localized inside 95Sphagnum mosses, in the dead hyaline cells [38]. Furthermore, Verrucomicrobia including the class 96 containing CH4 oxidizers, Methylacidiphilae, can make up 10% of the total microbial community 97 associated with Sphagnum. However, the Methylacidiphilae found wi...