Methanotrophic Activity and Diversity in Different Sphagnum Magellanicum Dominated Habitats in the Southernmost Peat Bogs Of

Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | , 6 Discussions Biogeosciences 1 , V. Pancotto 4,5 , L. Bodrossy 4 9358 9357 , Y. Pan , and H. J. M. Op den Camp 1 2 , E.S. Langelaan Sphagnum magellanicum , A. J. P. Smolders 2,3 1 , C. Fritz erent 1 ff This discussion paper is/has been under review for the journal Biogeosciences (BG). Please refer to the corresponding final paper in BG if available. Bioresources Unit, AIT, Austrian Institute ofCSIRO, Technology Marine GmbH, and 2444 Atmospheric Seibersdorf, Research Austria and Wealth from Oceans, National Research CADIC-CONICET, B. Houssay 200, 9410 Ushuaia, Tierra del Fuego, Argentina Radboud University Nijmegen, Institute for Water and Wetland Research (IWWR), Radboud University Nijmegen, Institute for Water and Wetland Research (IWWR), University of Groningen, Centre for Energy and Environmental Studies, Nijenborgh 4, 9747 4 5 Flagship, Hobart Tasmania 70006 Australia Received: 31 August 2011 – Accepted: 9Correspondence September to: 2011 H. – J. Published: M. 19 Op September den 2011 Published Camp by ([email protected]) Copernicus Publications on behalf of the European Geosciences Union. Biogeosciences Discuss., 8, 9357–9380, 2011 www.biogeosciences-discuss.net/8/9357/2011/ doi:10.5194/bgd-8-9357-2011 © Author(s) 2011. CC Attribution 3.0 License. M. S. M. Jetten Methanotrophic activity and diversity in di dominated habitats in the southernmost peat bogs of Patagonia N. Kip 1 Department of Microbiology, Heyendaalseweg2 135, 6525 AJ Nijmegen, The Netherlands Department Aquatic Ecology3 Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands AG, Groningen, The Netherlands Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | A erent pmo ff Sphag- Verrucomi- pore water). Little 1 − l mosses from lawn and 4 ect, but for good predic- ff ) was observed in living 1 Sphagnum 35 µmol CH − , NC10 phylum and the > day litter situated at depths around the wa- 1 A clone library. The methanotrophic di- − mosses are able to act as a methane fil- pmo dominated peat bogs in the Northern Hemi- . This permitted a species-independent com- 9360 9359 gDW mosses, even in those mosses with a low initial 4 Sphagnum Proteobacteria ) was found in pool mosses and could be corre- A based microarray that can be used to analyse the 1 − ) in 1 Sphagnum − pmo day Sphagnum erent habitats of this study and surprisingly comparable to 1 Sphagnum ff mosses and act as a filter for methane, thereby recycling 0.5 µmol CH − day < 1 mosses sampled and a positive correlation was found be- − species in the most active mosses suggests that these might be gDW 4 gDW erent bog microhabitats. Potential methane oxidizing activity was 4 ff Sphagnum magellanicum Sphagnum Sphagnum peatlands are important ecosystems in the methane cycle. Methan- dominated bog microhabitats. In contrast to the Northern Hemisphere X (Murrell and Jetten, 2009). These genes encode subunits of the methane Methylocystis 4 µmol CH (Op den Camp et al., 2009; Conrad, 2009; Ettwig et al., 2010). The proteobac- > mosses from lawns and hummocks. Methane oxidation activity was relatively mmo A microarray and a complementary A microarray data indicated that both alpha- and gammaproteobacterial methan- Methanotrophs occur within the Peat bogs are a harsh environment for microbes to live in because of the low pH terial methanotrophs have beenecosystems intensively (Dedysh, studied 2009). and have Unfortunately, been current detected molecular in techniques peat designed characterized using functional genesand like the methane monooxygenase genes, monooxygenase enzyme, which catalyses theway first step and in can the only methaneversity oxidation be of path- methanotrophs found is in a methanotrophic methanotrophs. community of A an fast ecosystem (Bodrossy screening et tool al., to 2003). crobia study the di- (around 4.5) and the lowhave nutrient been content. studied Microbial insphere and a (Dedysh, methanotrophic 2009; communities few Opeltmunities and can Berg, 2004; be Kip investigatedgenes et with (Stackebrandt al., molecular and 2011a). tools Goebel, The based 1994), microbial on while com- the methanotrophic bacterial communities 16S can rRNA be Microorganisms play an important role inand the the biogeochemical knowledge cycles of about thesecarbon their peatlands and diversity nutrient can turnover. help Withincarbon peat to ecosystems source improve methane our for serves understanding as methane an2005; of oxidizing important Kip the bacteria; et al., methanotrophs 2010; (Raghoebarsingon Larmola et et and al., al., inside 2010). Methanotrophsbenthic were carbon shown and to be reducing present al., methane 2010). emissions (Raghoebarsing et al., 2005; Kip et Carbon dioxide and methane are importantare greenhouse rising gases rapidly and since their industrial concentrations timeside (Forster emissions et from al., peatlands 2007). contribute Methanetion to and models carbon the more diox- greenhouse information e is needed about the carbon cycle in these ecosystems. 1 Introduction responsible for the bulk of methane oxidation. pmo versity was similar in thethe di methanotrophic diversity found in peatpmo mosses from the Northern Hemisphere.otrophs The were present in all methane oxidation activity. Prolonged incubationhummock of with methane revealed thatable and the showed methanotrophic an community increased activityotrophic present within was 15 vi- days. The high abundance of methan- methanotrophic activity ( num high ( ter levels and richrRNA in gene methane. sequencing The and total bacterial the community methanotrophic was communities studied were using 16S studied using a Sphagnum peat ecosystems themoss temperate species; South Americanparison peat of bogs the arefound di dominated in by all tween one activity andactivity in (23 µmol situ CH methanelated concentrations. with higher in Substantial situ methane methane concentrations oxidation ( Sphagnum otrophs living inter and and on thereby the centrations reduce and methane the emissions. corresponding activity and We diversity investigated of in methanotrophs situ in methane di con- Abstract 5 5 25 15 20 10 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | , ” ◦ – C 4 ◦ Sphag- Donatia sampled Rostkovia (Brid) with ” bog (54 erent habi- , which use 4 ., ff C at 100 cm species were Methylocella (Grootjans et ◦ erent ff S. cuspidatum Methylocystis ( A and 16S rRNA) Sphagnum magellan- Sphagnum magellan- pmo erent micro habitats in ff S. magellanicum ssp. Nothofagus ssp Methylocapsa magellanicum ., . S Alphaproteobacteria and (Forster f.) Gaudich and bog, called “high CH Sphagnum magellanicum Sphagnum falcatulum Tetroncium A microarray and ( like mosses from the di Empetrum ssp , frequently dominates these di Sphagnum pmo 9362 9361 C at 5 cm below surface to 4–8 Methylocystis Astelia pumila ◦ erent hydrological niches, such as pools (wet), ff ssp. and co-exist only in pools (Kleinebecker et al., 2007). In ected by anthropogenic alteration such as drainage, Sphagna can be found next to (Vorobev et al., 2010; Dedysh, 2009; Conrad, 2009). ff erent peat bogs in Tierra del Fuego, Argentina. Methan- C. July is usually the coldest month with mean tempera- ff ◦ S. magellanicum bog was dominated by mosses occupy margins of pools and form also small lawns (few W, 40 m a.s.l.). Annual average daily air temperatures are 5–6 0 erent sites: a pure Methyloferula ff 44 Marsipospermum ◦ bog. Pore water reflected acid conditions in both bogs (pH 3.5–4.5). Sphagnum magellanicum and genera and the acidophilic methanotrophs of the genera W, 200 m a.s.l.) and a mixed-cushion bog peatland, called “low CH species were the first facultative methanotrophs described (Dedysh et al., Forster and Forster covering more than 70 %. Also the above mentioned Sphagnum 0 ssp., S; 66 0 Sphagnum Sphagnum cuspidatum Sphagnum fimbriatum 20 C. At both bogs soil temperature is relatively low and stable through out the ◦ ◦ 58 ◦ occupies all hydrological niches from pool to hummocks rising up to 1 m above erent peat habitats. species occur that cover the di Carex . The ff Sphagnum , S; 68 0 The present study describes methane oxidation activity in Most studies on methanotrophy in peat lands have been performed in the Northern agricultural use or elevated atmospheric nutrient deposition. 2004). Dominating cushion plants were fasciculares vascular plants were present inis the densely mixed-cushion packed bog. with Theof tap soil roots 120 below cm cushion (1–2 mm causing plants 2011). diameter) thorough and Lawns methane fine of roots oxidationthe mixed-cushion exceeding (Grootjans bog depths et hosted abundantly al.,Peatlands vascular studied 2010; plants remained Fritz compared una et to al., the summer water table.coll.) and Other poor fens al., 2010). Inicum contrast, the mixed-cushion bogsquare consists meters) of embedded in little a matrix of evergreen cushion plants (Roig and Collado, bog (54 with cold summers around 9 ture of 2 growing season decreasing fromdepth. 8–12 The less than 1 % cover ofssp vascular plants like icum 2.1 Study sites description We studied two45 di 2 Materials and methods clone libraries. lawns (intermediate) andmoss hummocks species, (dry).tats. In The South presence American ofof only peatlands di one only moss one species enables species-independent analyses from various habitats in two di otrophic activity was studiedotrophic communities along of a gradientone peat of bog methane were availability. investigated using The a methan- Hemisphere (Dedysh, 2009) andreceived hardly peatlands any in attention. the South Americanman Southern peat influence lands Hemisphere and are have remote have areas soBlanco many without far hu- and ecological interesting de features lanum (Grootjans Balze, et 2004). al., 2010; In the Northern Hemisphere many di Methylocapsa Methylocella 2000), but recently also other facultative isolated (Im et al.,methanotrophs 2010; have Dunfield been et shown al., toin 2010; be the able Belova presence to et of survive al., acetate,al., a an 2011). 2011). important long These period carbon facultative source without in methane peat ecosystems (Belova et methanotrophs yet. Gammaproteobacterial methane-oxidizingtype bacteria I belong methanotrophs, to whichhyde the use fixation. the The type ribulose IIthe monophosphate methanotrophs serine belong pathway to for pathway the for formalde- Methylosinus formaldehyde fixation. This group includes the for conventional methanotrophs are not able to detect the verrucomicrobial and NC10 5 5 25 15 20 10 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | C, in - and ◦ pmoA lpha gene was performed with A microarray and mmoX analy- pmoA pmo Therefore we also designed a new primer SolV resulted in a product of the expected 9364 9363 2 of all three verrucomicrobial methanotrophs pmoA A microarray data are available upon request. Verrucomicrobia. pmo 1 and , primers and were not expected to hybridize with the C. Genomic DNA isolation, ◦ mosses were thoroughly washed and incubated in 120 ml bottles gene primers: A682R and A189F (Holmes et al., 1995). All PCRs pmoA 20 ). Methane production tests were performed in 60 ml bottles containing amples were taken in December 2007, March 2008 and April 2008. − 1 − pmoA mosses were washed with sterile demineralized water after sampling and Methyloacidiphilum fumarolicum Sphagnum gene primers used were specific only for known methanotrophic A methane emissions 6). Despite the low number of measurements models exhibited well-spread resid- = For the clone library the PCR amplification of the Details on protocols to estimateSphagnum methane s emissions are given in Fritz et al., 2011. n size and other proteobacterial cultures,al. all (2011b) isolated were tested, methanotrophs did described not in result Kip in et a PCR product. (Qiagen). DNA sequencingpmoA was performed withGammaproteobacteria the primers usedgene of in methane-oxidizing the PCR.set The based on[10]: the VpmoA216: (reverse).5’-GTTTCnACCATnCGnATrTAyTCAGG-3’ Initial 5’-GGAAAGAymGrATGTGGTGGCC-3’ (forward) validation using and a VpmoA622: pureture cul- of sis was performed asBodrossy described et before al., 2003). (Stralis-Pavese et All al., 2011; Kiptwo et general al., 2010; were performed in abined. gradient from All 50 to PCR 60 products degrees were and purified PCR products using were the com- QIAquick PCR Purifications Kit sequently simplified until thedeletion). least We adequate only model included was( methane reached oxidation (stepwise rates found backward inuals. living Sphagnum stands 2.5 Bacterial and methanotrophicSphagnum community analysis kept frozen at Regression analyses (linear model)velopment were Core performed Team, intransformed 2010) R potential followed software methane packages byregressions (R oxidation model were De- rates carried justification out before procedures. starting regression with analysis. all We environmental factors log- Multiple (Table 1) and sub- 2.4 Statistical analysis 2.3 Methane oxidation and production tests Whole with 1 ml of methane.chromatograph Methane equipped was with measured on(80 a 100 a mesh flame-ionization Hewlett-Packard model detector 5890only and gas dinitrogen a gas. Porapakthe Methane Q dark. oxidation column tests were performed at 10, 15 and 20 Litter samples (dead plant2008. material For forming all peat)the the were day samples taken of the in sampling depth was Aprilfor measured. below the and The the depth December depth of water belowsubmerged the table surface samples. sample and served below below as mean the the summer reference surface water at table. Negative depths depict Equipment ®, Giesbeek, the Netherlands), connectedafter to sampling vacuum infusion 150 flasks (40 ml ml) samples to we exclude measured internal methane concentrations stagnantValues of at sampler pH a were water. depth determined after of Forhout, collection 30 using cm deeper Belgium) a below (litter) handheld and water (Consort a level. ®Additional standard C933, water Turn- pH samples electrode were drawn (SP10T, with Consort 60 ®, ml-syringes. Turnhout, Belgium). Pore water was sampledmoss at and depths litter of samples 5–10bic cm for peat from incubation water pools (more samples and details were lawns see taking prior Fritz using collecting et 5 cm al., ceramic 2011). cups (Eijkelkamp Anaero- Agrisearch 2.2 Pore water methane concentration, pH measurements and 5 5 25 15 20 10 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ” ) 4 1 − pmoA pmoA day ). The 1 39.0; d.f. − TM = erent peat 11.8; d.f. 4) erent micro ff ff = gDW 4 C, resulting in a 01; F ” bog coinciding . ◦ ). Low methane , respectively for ected by anthro- 4 4 0 1 05; F ff . − 0 ). In the “low CH 4 p < day ) compared to the lawn p < 2 2 . Ecological relevant pa- − − ” bog the surface methane m 4 4 2 µM CH < A gene clones were deposited in ) in the “high CH 1 mosses from the two di − pmo Plasmid Miniprep Kit (EZNA day 2 TM − ). Two measurements even revealed a net 2 mosses from aerated habitats like hummock m − and 252 µmol CH 9366 9365 4 1 Sphagnum − . The combined variation of methane porewater Sphagnum magellanicum C were twice as high as rates at 10 day ◦ 2 − Sphagnum erence might be explained by methanotrophic consump- m 7.6 mg CH ff 4 > ) and comparable to the drier habitats like lawns and hum- ” bog. At this site the average water table was 5.5 cm deeper 1 4 − ’ sites. In the pool of the “high CH 4 day 1 − ” bog (500–800 shoots m 4 gDW 4 Sphagnum magellanicum ” and ‘low CH 4 cells, constructed as described by Inoue et al. (1990). pDNA with ligated gene was isolated with the E.Z.N.A. gene sequences were sequenced with M13 forward and reverse primers (In- The pGEM-T Easy Vector System Kit (Promega) was used for ligation of the tration in the pore(Fig. water 1). and Methane pore the water initial concentrationof could methane the explain 68 oxidation variation % found rates ( in ofhabitats the the of potential tested methane oxidation mosses ratesconcentration along together the with di height of94 % the of mosses variation above found the3). in water Methane the table oxidation potential accounted rates methane at for oxidationQ10 20 rates of ( around 2. oxidizing activity was found in and lawns thatbog were the typically pool depleted(0.5 in was µmol CH depleted methane in ( mocks. methane and The the results show methane a oxidizing clear activity positive was correlation low between the methane concen- only 4.5 times higher. Thistion, di thereby significantly reducingmosses methane were emissions tested to for methane the oxidizing atmosphere. activity. Therefore 3.2 Methane oxidizing activity tests Initial methane oxidizing activitiesecosystems were of determined (Table 1).was Highest found activity in (23.5 µmol mosses CH collected from methane rich pools (35 µM CH and vascular plant cover was extremelyin low (50–120 the shoots “low m CH consumption of 5.1 µmol CH “high CH concentration was approximately 30 times higher than in the lawn, but emissions were lower when surface peat water wasin depleted the in lawn methane. of Lowest the emissions “high were CH found moss species present wererameters mainly that have been determined in situ are represented3.1 in Table 1. Methane emission Methane emissions from both peatlands wereto generally low. be Emission the rates were highest found with in high pools methane ( pore water concentrations (Table 1). Emissions were substantially 3 Results Samples were takenpogenic from alteration Patagonian such bogs as drainage,deposition. that agricultural have They use showed not or the elevated been typical atmospheric a pool, nutrient lawn and hummock micro habitats and the vitrogen™), targeting vector sequencessequencing adjacent was to performed by thejmegen. the multiple Clone sequencing library cloning sequences facility and site. ofversion their closes 4 the relatives pDNA were (Tamura UMC analyzed et using Sintalignment al., MEGA Radboud, 2007). tool Ni- of AllNeighbor-joining MEGA4 sequences method. (ClustalW). were The aligned Phylogenetic sequences automatically ofthe trees the Genbank using were database the under calculated accession numbers using JF907375-JF907390. the gene amplifications. Ligation was performedgene as prescribed ligation by mix the manufacturer. wasE. transformed coli by heatpmoA shock exposure to XL-1pmoA Blue competent 5 5 25 15 20 10 25 20 15 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | . erent repre- ff Methy- Sphag- Alphapro- and 10 cm below the C at 55 cm. Sub- − ◦ Betaproteobacteria spp. were present in Proteobacteria ) and type II ( at 10 1 - and litter were substantial (3.8 − (13 %) were present in high A microarray (Bodrossy et day 1 Alpha pmo − erent periods of the year. In the Methylosinus ff mosses. The erent habitats in the Southern Hemi- ff gDW Sphagnum 4 Methylocystis-Methylosinus mosses. erent samples (Fig. 3). The microarray re- spp. and Verrucomicrobia ff 9368 9367 Gammaproteobacteria ” bog, see Fig. 2 and the Supplementary Ma- C). ◦ 4 Sphagnum A gene sequences of cultured and non-cultured at 10 mosses showed highest methane oxidizing activity, 1 pmo Sphagnum ” bog were hybridized onto the microarray. All samples − ). Even at greater depths (55 cm below the water table) (28 %) and 4 1 − day Methylocystis ” bog and tested them for methane oxidation and production 1 erent 4 − day ff 55 cm below the water level) and hummock ( 1 − − A PCR products from the pool and hummock samples and the Sphagnum litter from the hummock showed a rather low methane production gDW 4 gDW pmo 4 Acidobacteria mosses and peat litter from the di ” bog lawn mosses showed a threefold increase within 2 weeks and the 4 A, coding for a subunit of the methane monooxygenase gene was used. A mosses showed a higher hybridization with the type II probes over type I. litter and were around 0.5 µmol CH mosses from lawn and hummock sites showed low methane oxidation rates ) methanotrophs and a high methanotrophic biodiversity. The submerged species (Supplement Fig. S1). For a more complete methanotrophic di- Sphagnum pmo litter (dead plant material forming peat) below the water level from the lawn and A clone library showed the presence of ” lawn mosses needed almost one month to show a threefold increase in methane Sphagnum 4 In order to investigate the presence of methanotrophs the unique functional gene Since submerged phagnum similar methanotrophic community at theII surface probes and showed below that the both water table. The type litter samples of lawn ( water level) from the “highshowed CH a rather similar methanotrophiction community, as patterns revealed by on a the similarsults hybridiza- showed microarray abundance of of the bothteobacteria type di I ( S The pool mosses and litter samples showed a similar hybridization pattern indicating a pmo lomonas versity analysis aal., microarray 2003) was represents performed.methanotrophs. di This micro-habitats was compared. A 16Smosses rRNA clone from library the analysis pool wasterial performed in using for the more “high details. CH of The bacteria 16S to rRNA be genesented present clone the inside libraries biggest or showed phylum on aNext (42 to very %), that diverse with the set anumbers majority within the of Patagonian marker So far bacterial diversity was onlytried studied in to peatlands get in the an Northernthe insight Hemisphere. into We the diversity ofsphere. bacteria Furthermore, the and diversity especially between the of same methanotrophs moss in species growing in di 3.4 Bacterial and methanotrophic community analysis Patagonian hummocks mosses grow upNevertheless, to upon 1 m prolonged above incubation the ofidation water lawn increased level and and exponentially hummock pointing are mosses, very to methaneThe dry. growth ox- “high of CH methanotrophs (datahummock not sample shown). needed atCH least three weeks ofoxidation. incubation with methane. The “low 3.3 Induced methane oxidationSphagnum in lawn and hummock in comparison to poolthe mosses. lawns Water and levels parts and methane can concentrations be fluctuate inundated in or dry in di ples. The potential methaneto 10.5 oxidation µmol CH rates of potential methanotrophic activity wasnot found. representative in Measured sitution. methane rates, Potential oxidation due methane rates production to rates are Sphagnum the were high higher at oxygen 55 concentration cmmerged during than at incuba- 5 cm inrate the (0.01 µmol lawn CH correlating with the highestnum methane pore waterhummock in concentration, the we “high CH sampled (Table 1). These subsurfacetion samples and showed a both high methane pore oxidation water and methane methane concentra- production were found in these sam- 5 5 25 15 20 10 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | A X- .A and pmo mmo A clone A genes pmo pmo Methylothermus Methylomicrobium Methyloferula stellata , mosses showed a much X-based PCR on the two and mmo was detected. The -related sequences. The specific Sphagnum Methylococcus Methylobacter , 9370 9369 Methylobacter Methylomonas and moss was found to be dependent on the availability of ” bog, overall methanotrophic activity is low and inde- 4 Methylomonas spp. by screening with a Sphagnum Methylomonas bogs. High rates of methane oxidation coincided with substantially mosses, although the hummock as well as environmental clades) showed a weaker signal, but still indi- spp., which are commonly found in Northern peatlands (Dedysh et al., Methylocella (Larmola et al., 2010), but our results suggest the methane pore water litter before it reaches the living parts. However, if methane concentrations Sphagnum Sphagnum Sphagnum ¨ Living mosses collected from lawns and hummocks showed a low methane oxidation The PCR primers used thus far are not able to amplify verrucomicrobial Methylocella uke et al., 2010). Potential methane oxidation has also been reported in Northern (L rate, but litter (dead parts) sampledrate. at In and addition, below the also waterproduction methane level and showed production a consumption was much activities found higher atcontrasting in the these micro-sites. same samples. depths The The indicatewater activity methane the level of co-existence are of influenced methanotrophs by and fluctuatingwhich water methanogens makes levels over around the the litter the year sometimes inconditions a microbes anoxic peat take and their ecosystem, sometimes opportunities oxic andaerobic-anaerobic (Lai, this interface shows 2009). that restricting Under there the is both not microbial a real activities, fixed like e.g. in a rice field oxidation with methane porehas water been concentration hypothesized rather to thanin be water the level. key Water environmentalconcentration level factor to regulating be methanotrophy theecosystems main (Basiliko et driver, al., as 2007). was also suggested in other studies on peat living parts of the methane. This availability determines thethe presence water and level activity is ofconcentrations methanotrophs an of and important methane regulator canter as be level, expected. only methane under will If already waterSphagnum the have logged mosses been conditions grow oxidized high high byare above bacteria the low, attached wa- to like thependent in aerobic of the the “low water CH level. Statistical analyses also show a correlation of methane This study revealed aonian high activity and diversityreduced of methane methanotrophic emissions especially bacteria at inof low Patag- the water methanotrophs levels, reflecting in an reducing important emissions role at these sites. Methane oxidation by the 4 Discussion using these primers. uble methane monooxygenase enzymebe from also which be used theSearching for encoding for phylogenetic gene analysis (mmoX) (Aumanpool can et samples al., did 2000; notpossessing Miguez result methanotrophs et are in not al., any abundantly 1997). presentsuitable. PCR or product that the (Kip PCR et primers al., are not 2010), indicating (Pol et al., 2007). We designedbial new sequences primers (see based Materials on and currently Methods) available but verrucomicro- were not able to obtain a PCR product to perform microarray analysis. 2004; Dedysh et al.,(Vorobev et 2000, al., 2004) 2010) andgene are the and the are recently therefore only discovered not known detected methanotrophs in that this do microarray. not These genera have do a have a sol- probes, which target the typeand Ib thermophilic (type gammaproteobacterial X) genera methanotrophsMethylocaldum (including the thermotolerant cated a diverse community acrossotrophs the inside analyzed hummock samples. and Thein lawn viability the mosses of methane was the consumption obvious methan- the after experiments presence longer (see of incubation above). methanotrophs times in Themethane the microarray oxidation. hummock also Unfortunately mosses confirms which the showed lawn very mosses low did initial not yield enough PCR product lower abundance and alsosamples. a The microarray lower also methane showedprobes a oxidation targeting strong activity the signal genera compared withbroad the to gamma-proteobacterial diversity all of other library (see above) also revealed two all the 5 5 25 15 20 10 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | . erent ff Methy- Sphag- mosses Sphagnum Methylocys- Methylocys- Methylocystis sp. and erent. Verrucomicrobia ff Sphagnum dominated peatland and A diversity of the ´ ebron et al., 2007) and rice Methylocystis pmo Sphagnum Proteobacteria mosses, although the mosses from the 9372 9371 Sphagnum mosses from a Dutch peat bog (Kip et al., 2011a). Recently , which were the two families that were found to be abundant erence. Nardy Kip is supported by a grant from the Darwin Centre for Biogeo- ff mosses from other peat bogs around the world (Kip et al., 2010). Here A clone library and microarray both showed the presence of Sphagnum Methylomonas sp. were present in all the pmo mosses was very high compared to other studies using the same microarray on and spp. in the most active mosses indicates they are probably the key players in this Sphagnum methanotrophic isolates from freshwater lake5266, sediment, 2000. Appl. Environ. Microbiol., 66, 5259– The type II probes of the microarray showed that both Despite the low methane oxidation rates of the dry hummock, the microarray analysis So far, acidophilic methanotrophs belong to the The The microbial community inside and attached to Patagonian Acknowledgements. capable of the above mentionedmethanotrophic traits players of it the is system. necessary to isolate andSupplementary test material the related important to thishttp://www.biogeosciences-discuss.net/8/9357/2011/bgd-8-9357-2011-supplement.pdf article is available online at: oxidation activity compared totis all other samples. Theecosystem. high The abundance capacity of to useand acetate to as consume an methane additional at carbonspp. both source, high might and to represent low fix concentrations an nitrogen byotrophs important several in part peat of lands themight (Belova survival explain et strategy why al., of they 2011; are thisand Buckley found kind other et in of ecosystems. abundance al., methan- However in to 2008; this test Im if et the al., species present 2010). in this This system are indeed sciences (142.16.1061) and Mike Jetten by an ERC grant (nr. 232937). References Auman, A. J., Stolyar, S., Costello, A. M., and Lidstrom, M. E.: Molecular characterization of probably due to the in situlocated fluctuations closed in to methane the availability mean which water are level, bigger than in in lawns hummocks. losinus hummock showed a much lower abundance, which coincides with a lower methane faster upon prolonged incubation compared to those from hummocks, which is most building up an active population. Methanotrophs present in the lawn mosses reacted peat soils (Chen etfields al., (Vishwakarma 2008), et peat al., based 2009),in upland but quite soils similar (C tohowever the we methanotrophic have communities shown that allrable the methanotrophic microhabitats community were composition, showing considering amicro the surprisingly habitats origin compa- where of methane all and the oxygen di concentrations are very di showed that methanotrophs werelow present. densities, They but were viable, most since probably they present respond in to very increased availability of methane by tis in and on several representatives of both familiesmosses were and isolated characterized (Kip in etnum pure al., culture 2011b). from The total acidic peat bogs. Thislands points to from the both presence the ofbig Northern similar geographical di bacterial and communities Southern in Hemisphere peat- and implies that(Op there den is Camp et no al.,abundance 2009; in Conrad, the 2009; 16S Dedysh, rRNAclosely 2009). clone related Both to library, but groups currently none known were methanotrophs. of found in the 16S rRNA sequences were 1998). is comparable to(Kulichevskaya et previously al., 2007; investigated Dedysh peat etsequence al., soils 2006). similarity Most from of to the the 16S isolates rRNA Northern clones or showed Hemisphere environmental samples originating from Siberian peat bogs in depth samples that were anoxic under field conditions (Edwards et al., 5 5 15 10 20 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | -DNA-stable isotope 2 N 15 ciency transformation of Escherichia coli with ffi 9374 9373 ´ on de su biodiversidad, Wetland International, Buenos Aires, 2004. methane monooxygenase andFEMS Microbiol. ammonia Lett., monooxygenase 132, 203–208, may 1995. be evolutionarilyultative Methylocystis related, species capable ofMicrobiol. growth Rep., on 3, methane, 174–181, acetate 2010. and ethanol, Environ. plasmids, Gene, 96, 23–28, 1990. some contrasting mires in Tierra del Fuego, Argentina, Mires and Peat, 6, 1–15, 2010. 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S. 5 5 25 20 15 30 25 10 20 15 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | 4 1 C − ◦ 00 20 01 . . . 0 0 0 day ± ± ± ” bog. 1 − mol CH 4 01 54 02 µ . . . gDW 4 1 4 0 6 0 9 0 C rate at 10 . . . − ◦ 10 24 43 03 10 19 33 0 2 . 1 ...... 0 0 0 0 0 7 ± ± ± day 39 ± ± ± ± ± ± 0 5 1 1 . . . ± − mol CH 14 44 43 04 15 10 µ ...... 70 . gDW 1 4 4 7 9 10 8 13 2 18 C rate at 20 . . . . − ◦ erent temperatures: 38 0 53 0 25 0 20 46 0 1 2 . . . 3 005 0 ± 0 0 . 0 ff 0006 0 ± ± ± ± day . 0 5 ± ± ± 8 2 5 6 . 0 1 . . . . ± − mol CH ± 25 47 22 . . . µ 01 4 . 04 . ” bog and “low CH . gDW 5 4 3 1 4 M 7 3.4–4 0 9 3.6–4 0 6 3.5–4.2 0 5 4–4.3 23 . . . . − µ 4 5 1 3

± ± ± ± n day 6 1 7 6 2 . . . . mg CH io − t m a r t 1 3 ND 3.4–3.6 0 8 5 0 5 3 ND 3.5–4.2 0 6 1 3 ND 4–4.3 8 6 7 n ...... − 2 6 3 3 1 0 1 e erent methane pore water concentrations c ± ± ± ± ± ± ± 5 3 4 5 5 0 7 n ﬀ ...... o c 4 . r 9 4 24 2 13 e . . % g l t 47 45 29 28 16 19 15 26 36 5 . . . . 0 . 0 a C C 0 0 0 0 0 ± ± ° ° w ± ± ± ± ±

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