Greenhouse gas metabolizing prokaryotes in peatlands

Greenhouse gas metabolizing prokaryotes in peatlands Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften Dr. rer. nat. der Fakult¨at f¨ur Biologie, Chemie und Geowissenschaften der Universit¨at Bayreuth vorgelegt von Katharina Palmer Bayreuth, den 09.05.2012 Die vorliegende Arbeit wurde von September 2008 bis Mai 2012 am Lehrstuhl f¨ur ¨ Okologische Mikrobiologie der Universit¨at Bayreuth unter der Leitung von PD Dr. Marcus A. Horn angefertigt. Teile der praktischen Arbeit wurden auf den Forschungsstationen Kevo (University of Turku, Finland) und Oulanka (University of Oulu, Finland) durchgef¨uhrt. Erm¨oglicht wurde die Arbeit durch finanzielle Unterst¨utzung der Deutschen Forschungsgemeinschaft (DFG HO 4020/2-2), aus dem LAPBIAT Projekt (Teil das 6. EU Rahmenprogramms ”Infrastructures”), des Deutschen Akademischen Austauschdienstes (DAAD), der Suomen Akatemia (Academy of Finland) und der Universit¨at Bayreuth. Promotionsgesuch eingereicht am: 09.05.2012 Tag des wissenschaftlichen Kolloquiums: 29.10.2012 Pr¨ufungsausschuss: PD Dr. Marcus A. Horn (Erstgutachter) Prof. Dr. Ortwin Meyer (Zweitgutachter) Prof. Dr. Gerhard Gebauer Prof. Dr. Stefan Peiffer Prof. Dr. Gerhard Rambold (Vorsitzender) IV We know what we are, but not what we may be. William Shakespeare - Hamlet I shall be telling this with a sigh Somewhere ages and ages hence: Two roads diverged in a wood, and I - I took the one less traveled by, And that has made all the difference. Robert Frost - The road not taken A truly happy person is one who can enjoy the scenery while on a detour. Author Unknown V Contents Contents Contents IX List of Tables X List of Figures XIII List of Abbreviations XIV Summary 1 Zusammenfassung 6 Acknowledgements 12 1 General introduction 15 1.1 Greenhouse gases and their impact on earth’s climate . . . . . . . . . 15 1.2 Peatland ecosystems as important sources of greenhouse gases . . . . 18 1.2.1 Peatland formation and peatland types . . . . . . . . . . . . . 18 1.2.2 Permafrost peatlands . . . . . . . . . . . . . . . . . . . . . . . 18 1.3 Processes involved in greenhouse gas production in anoxic peatland soils .................................... 21 1.3.1 Fermentation ........................... 23 1.3.2 Methanogenesis.......................... 25 1.3.2.1 Hydrogenotrophic methanogenesis . . . . . . . . . . 25 1.3.2.2 Acetoclastic methanogenesis . . . . . . . . . . . . . . 27 1.3.2.3 Methylotrophic methanogenesis . . . . . . . . . . . . 28 1.3.2.4 Factors influencing methanogenesis in soils . . . . . . 28 VI Contents 1.3.3 Denitrification........................... 30 1.3.3.1 Denitrifying microorganisms . . . . . . . . . . . . . . 30 1.3.3.2 Enzymes involved in denitrification . . . . . . . . . . 31 1.3.3.3 Factors influencing denitrification in soils . . . . . . . 36 1.3.4 Dissimilatory nitrate reduction . . . . . . . . . . . . . . . . . 37 2 List of publications and manuscripts included in the dissertation 38 2.1 Published articles in peer-reviewed journals . . . . . . . . . . . . . . . 38 2.2 Manuscripts in preparation . . . . . . . . . . . . . . . . . . . . . . . . 38 2.3 Previous peer-reviewed publications not included in the dissertation . 39 2.4 Published abstracts at national and international conferences . . . . . 39 2.5 Additional presentations of parts of the work at international meetings 41 3 Greenhouse gas production in pristine peatlands 42 Contributions.................................. 42 3.1 Hypotheses tested in this work . . . . . . . . . . . . . . . . . . . . . . 44 3.2 Processes leading to formation of CH4................. 46 3.2.1 Fermentative and methanogenic processes in a pH-neutral fen 46 3.2.2 Diversity of prokaryotes putatively associated with fermentations and methanogenesis in a pH-neutral fen . . . . . . . . . 49 3.2.3 Conclusions: Fermentation and methanogenesis in pH-neutral fensoil............................... 53 3.3 Processes involved in turnover of N2O.................. 55 3.3.1 N2O production and consumption in pristine peatlands . . . . 55 3.3.2 Diversity of peatland denitrifiers . . . . . . . . . . . . . . . . . 61 3.3.3 Environmental factors shaping denitrifier communities in peatlands................................ 72 3.3.4 Conclusions: Denitrification in peatland soils . . . . . . . . . . 76 VII Contents 3.4 Effect of water table manipulations on anaerobic processes in a model peatland.................................. 77 3.4.1 Effect of water table manipulations on fermentation potentials inacidicfensoil.......................... 77 3.4.2 Effect of water table manipulations on methanogenesis in an acidicfensoil ........................... 79 3.4.3 Effect of water table manipulations on denitrification in an acidicfen ............................. 85 3.4.4 Conclusions: water table manipulations . . . . . . . . . . . . . 89 3.5 Generalconclusions............................ 91 References 95 4 Manuscripts 125 4.1 Published articles in peer-reviewed journals . . . . . . . . . . . . . . . 125 Genome-Derived Criteria for Assigning Environmental narG and nosZ Sequences to Operational Taxonomic Units of Nitrate Reducers . . . . . 131 Contrasting denitrifier communities relate to contrasting N2O emission patterns from acidic peat soils in arctic tundra . . . . . . . . . . . . . . . 181 Putative Actinobacterial nitrate reducers and Proteobacterial denitrifiers are abundant in permafrost affected N2O metabolizing acidic palsa peatsoil..................................193 4.2 Manuscripts in preparation . . . . . . . . . . . . . . . . . . . . . . . . 193 Denitrification activity of a new and diverse denitrifier community in a pH neutral fen soil in Finnish Lappland is nitrate limited . . . . . . . . . 227 Stability of methanogenic diversity in an acidic fen under the influence of experimentaldrought...........................260 VIII Contents Denitrifier communities in an acidic fen are stable during experimental drought ..................................283 4.3 Previous peer-reviewed publications not included in the dissertation . 283 Association of Novel and Highly Diverse Acid-Tolerant Denitrifiers with N2O Fluxes of an Acidic Fen . . . . . . . . . . . . . . . . . . . . . . . 294 Curriculum vitae 294 IX high diversity of bacterial families was detected in fen soil by barcoded amplicon pyrosequencing of bacterial 16S rRNA genes, including Firmicutes,Alphaand Deltaproteobacteria as well as many novel families. Within the detected families, genera known for syntrophic interactions with methanogens (e.g., Clostridium,Syntrophobacter) were found, indicating that those genera might be important providers for methanogenic substrates. Diversity of methanogens was lower than bacterial diversity, as only Methanomicrobiales and Methanocellales (methanogens of both orders are hydrogenotrophs) were detected by analysis of the structural gene marker mcrA (encoding the methyl-coenzyme M reductase), likewise indicating that hydrogenotrophic methanogens are dominating in pH-neutral fen soil. Both process data and molecular data suggest that (i) hydrogenotrophic methanogenesis is the main process of CH4formation in pH-neutral fen soil, and (ii) a high diversity of bacterial families occur in pH-neutral fen soil that are likely involved in diverse fermentations, thus providing substrates for fen methanogens. Denitrification is considered to be the main source of N2O in waterlogged soils such as peatland soils, as high water saturation promotes anoxia in the soil. As N2O is an intermediate in the denitrification process, denitrifiers can be producers as well as consumers of N2O. Despite their important role in the global N2O budget, factors controlling denitrification in pristine peatlands and the associated diversity of the denitrifier community are virtually unknown. Thus, denitrification and N2O consumption potentials as well as denitrifier community composition were assessed in all five northern peatlands. In situ N2O emissions range from <0.01 mg N2O·m−2·d−1 in pH-neutral fen soil to approximately 10 mg N2O·m−2·d−1in cryoturbated peat circle soil, thus cryoturbated peat soil is an important N2O source, emitting in the same range as tropical or agricultural soils. In situ N2O emissions were positively and negatively correlated with soil nitrate and ammonia contents, respectively. All 2 soils produced and consumed N2O in anoxic microcosms without apparent delay. N2O production capacities and apparent affinities (vmax/KM) for nitrate were likewise positively correlated with soil nitrate content. N2O production capacities were especially high in acidic, permafrost-affected cryoturbated peat soil, and co-occurred with high in situ N2O emissions and high nitrate contents. On the other hand, N2O consumption capacities were highest in pH-neutral fen soil, and co-occurred with low in situ emissions and low nitrate content. Phylogenetic analyses of the nitrate reductionand denitrification-associated genes narG,nirK/nirS, and nosZ (encoding nitrate, nitrite, nitrite, and N2O reductases, respectively) indicated that the diversity of the denitifier community was highest in pH-neutral fen soil, and that diversity was positively correlated with pH. Detected nitrate reductase genes affiliated mainly with Betaproteobacterial and Actinobacterial narG.Betaproteobacterial narG dominated in pH-neutral fen soil, while Actinobacterial narG were predominant in all more acidic soils, indicating that Actinobacterial nitrate reducers might have a higher tolerance to acidity. The number and the identity of observed operational taxonomic units (OTUs) of nirK,nirS, and nosZ in pH-neutral fen soil was clearly distinct from those of the more acidic soils, and indicated Alpha-,Beta-, and Gammaproteobacterial denitrifiers in all peatlands. These results were confirmed by canonical correspondence analysis (CCA) of relative OTU abundances in amplicon libraries, suggesting that the denitrifier community of the pH neutral fen was unique among the analyzed peatlands. Permafrost-affected soils mainly clustered together in the CCA plots based on all analyzed genes. pH was the most important factor determining the community composition of nitrate reducers (as indicated by narG) and denitrifiers (as indicated by all analyzed genes). Significant influences of soil carbon content (on narG,nirK), precipitation (on narG), or temperature (on nosZ) were also detected. The ratio of narG/nosZ copy numbers was positively correlated 3 with N2O emissions, as was the occurrence of certain OTUs of nirK and nirS. The collective data indicate that (i) denitrification is an ongoing processes in different types of pristine peatland soils, (ii) source and sink function of peatland denitrifiers for N2O are influenced by soil nitrate content as well as denitrifier community composition, and (iii) nitrate reducer and denitrifier community composition are affected by pH, temperature, precipitation, and soil carbon content. Global warming is predicted to increase the frequency of extreme weather events, causing periods of prolonged drought or excess rainfall. Those events affect the water table level in peatlands and might thus affect microbial communities involved in the production of CH4and N2O. Thus, the influence of short-term water table manipulations including application of artificial drought conditions or excessive flooding was assessed in the acidic fen as a model system. Fermentative, methanogenic and denitrifying potentials that were assessed in anoxic microcosm studies with fen soil taken at different timepoints of the water table manipulation revealed that the potential activity of methanogens and denitrifiers was affected by changing water tables (i.e., methanogenic activity based on instantaneous CH4production potentials was lowered by drought and increased by flooding, denitrifying activity was increased by flooding), whereas the potential activity of fermenters was largely unaffected. Changes in the copy numbers of mcrA,narG, and nosZ detected by quantitative PCR were rather small when compared to the observed changes in potential activity, indicating that the community size of methanogens, nitrate reducers, and N2O reducers, respectively, is rather unaffected by short-term water table manipulations. Community composition of methanogens, nitrate reducers and denitrifiers (as assessed by TRFLP-fingerprinting of mcrA,narG, and nosZ, respectively) was similar at all sampled timepoints of the manipulation experiments, indicating that the microbial community composition is not affected by enhanced water table fluctuations. 4 The collective data indicate a stable microbial community in fen soil that is able to adapt its activity to the changing conditions quite rapidly. The collective observations underline the importance of peatland ecosystems for greenhouse gas fluxes. CH4is produced in a model peatland, and the studied peatlands can be sources as well as sinks for N2O, and thus might contribute significantly to the global N2O budget. Changing water tables affected the potential activity of methanogens and denitrifiers, even though the community composition of methanogens and denitrifiers were quite stable. Thus, greenhouse gas metabolizing microorganisms in peatland ecosystems are prone to react sensitively to global change, which might in turn affect the source and sink strengths of peatland ecosystems for CH4and N2O and as a consequence change their contribution to the global budget of those greenhouse gases. 5 Zusammenfassung Moorgebiete der n¨ordlichen Hemisph¨are speichern betr¨achtliche Mengen Kohlenstoff und Stickstoff, und es wird vorhergesagt, dass sie empfindlich auf die globale Erw¨armung reagieren. Die meisten Moorgebiete sind Quellen des Treibhausgases Methan (CH4), welches von Methanogenen im Moorboden in trophischer Interaktion mit G¨arern gebildet wird. Das Treibhausgas Distickstoffmonoxid (N2O) kann im Moorboden von Denitrifikanten produziert und verbraucht werden. Quellenund Senkenfunktionen f¨ur CH4und N2O variieren stark zwischen verschiedenen Moortypen. Das Hauptziel der Arbeit waren daher die Untersuchung der mikrobiellen Prozesse, welche an Treibhausgasfl¨ussen aus Moorgebieten beteiligt sind, und zwar G¨arungen und Methanogenese als wichtige mitwirkende Prozesse an CH4Fl¨ussen und Denitrifikation als mitwirkender Prozess an N2O-Fl¨ussen. F¨unf BeispielMoore mit unterschiedlichen Eigenschaften (zwei Niedermoore (pH 7 und pH 5), Hochlandtundra (pH 4), ein Palsamoor (pH 4,5) und cryoturbierte Torfkreise (pH 4). Diese unterschiedlichen Moorgebiete differierten in ihren in situ CH4und N2OEmissionen sowie z.B. im Nitratgehalt, der Jahresdurchschnittstemperatur und/oder im pH. Prozesse, die an der CH4-Bildung beteiligt sind, wurden am Beispiel des pHneutralen Niedermoores untersucht. Das Niedermoor produzierte CH4in situ sowie in unsupplementierten anoxischen Mikrokosmeninkubationen mit Niedermoorboden. Supplementierung mit N-Acetylglucosamin (NAG) stimulierte die Bildung von G¨arungsprodukten sowie CH4und resultierte in der verst¨arkten Akkumulation von G¨arungsprodukten (haupts¨achlich Acetat, H2/CO2, sowie geringe Mengen Ethanol, Formiat und Propionat) als die Methanogenese durch Bromoethansulfonat (BES) inhibiert wurde, was darauf hindeutet, dass Methanogene G¨arungsprodukte als Substrate verwenden und somit trophisch mit den G¨arern verkn¨upft sind. Supplemen6 tierung mit H2/CO2und Formiat stimulierte die Methanogenese stark, w¨ahrend die Stimulierung der Methanogenese mit Acetat und Methanol deutlich geringer war. Dies deutet darauf hin, dass hydrogenotrophe Methanogenese ein wichtiger CH4-bildender Prozess im pH-neutralen Niedermoorboden ist. Mittels kodierter Amplicon-Pyrosequenzierung von bakteriellen 16S rRNA Genen wurde eine große Diversit¨at bakterieller Familien im Niedermoorboden detektiert, dazu geh¨orten Familien der Firmicutes,Alphaund Deltaproteobacteria sowie viele neue Familien. Innerhalb der detektierten Familien wurden Gattungen, welche f¨ur syntrophe Interaktionen mit hydrogenotrophen Methanogenen bekannt sind (z.B. Clostridium,Syntrophobacter), gefunden. Dies weist darauf hin, dass diese Gattungen wichtige Lieferanten f¨ur methanogene Substrate darstellen k¨onnen. Die Diversit¨at der Methanogenen war niedriger als die der Bakterien, da nur Methanomicrobiales und Methanocellales (Methanogene beider Ordnungen sind Hydrogenotrophe) durch Analyse des strukturellen Genmarkers mcrA (kodierend f¨ur die Methyl-Coenzym M Reduktase) detektiert wurden. Auch dies deutet auf eine Dominanz der hydrogenotrophen Methanogenen im pH-neutralen Niedermoor hin. Sowohl Prozessals auch molekulare Daten deuten darauf hin, dass (i) hydrogenotrophe Methanogenese der Hauptprozess der CH4-Bildung im pH-neutralen Niedermoor ist, und (ii) eine hohe Diversit¨at bakterieller Familien im pH-neutralen Niedermoor vorkommt, welche wahrscheinlich an diversen G¨arungsprozessen beteiligt sind und somit Substrate f¨ur die Methanogenen bereitstellen. Denitrifikation wird als Hauptquelle von N2O in staunassen B¨oden wie z.B. Moorb¨oden angesehen, da hohe Wassers¨attigung Anoxia im Boden f¨ordert. Da N2O ein Intermediat im Denitrifikationsprozess ist, k¨onnen Denitrifikanten sowohl N2OProduzenten als auch -Konsumenten sein. Obwohl sie eine wichtige Rolle f¨ur den globalen N2O-Haushalt darstellen, sind Faktoren, welche die Denitrifikation in un7 ber¨uhrten Moorgebieten kontrollieren, sowie die Diversit¨at der assoziierten Denitrifikantengemeinschaft weitgehend unbekannt. Daher wurden Denitrifikationsund N2O-Verbrauchspotentiale sowie die Zusammmensetzung der Denitrifikantengemeinschaft in allen f¨unf n¨ordlichen Moorgebieten untersucht. In situ N2O-Emissionen bewegen sich im Bereich von <0,01 mg N2O·m−2·d−1in pH-neutralem Niedermoorboden bis zu ungef¨ahr 10 mg N2O·m−2·d−1in cryoturbiertem Torfkreisboden, daher stellt cryoturbierter Moorboden eine wichtige N2O-Quelle dar und emittiert in der selben Gr¨oßenordnung wie tropische B¨oden oder Ackerb¨oden. In situ N2OEmissionen waren positiv bzw. negativ mit dem Nitratbzw. Ammoniumgehalt des Bodens korreliert. Alle B¨oden produzierten und verbrauchten N2O in anoxischen Mikrokosmen-Inkubationen ohne erkennbare Verz¨ogerung. N2O-Produktionskapazit¨aten und apparente Affinit¨aten (vmax/KM) f¨ur Nitrat waren ebenfalls positiv mit dem Nitratgehalt des Bodens korreliert. N2O-Produktionskapazit¨aten waren in sauren, Permafrost-beeinflussten cryoturbierten Torfkreisb¨oden besonders hoch und traten gemeinsam mit hohen in situ N2O-Emissionen und hohen Nitratgehalten auf. N2O-Verbrauchskapazit¨aten waren dagegen am h¨ochsten in pH-neutralem Niedermoorboden und traten zusammen mit niedrigen in situ Emissionen und niedrigem Nitratgehalt auf. Phylogenetische Analysen der Nitratreduktionsund Denitrifikations-assoziierten Genmarker narG,nirK/nirS und nosZ (kodierend f¨ur Nitrat-, Nitrit-, Nitritund N2O-Reduktasen) deuteten darauf hin, dass die Diversit¨at der Denitrifikantengemeinschaft in pH-neutralem Niedermoorboden am h¨ochsten war und dass die Diversit¨at positiv mit dem pH korreliert war. Detektierte NitratReduktasegene waren haupst¨achlich mit narG der Betaproteobacteria und Actinobacteria verwandt. Betaproteobacteria-verwandte narG dominierten in pH-neutralem Niedermoorboden, w¨ahren Actinobacteria-verwandte narG in allen saureren B¨oden dominierten, was darauf hindeutet, dass Actinobacteria-verwandte Nitratre8 duzierer eine h¨ohere S¨auretoleranz aufweisen k¨onnten. Die Zahl und Identit¨at der beobachteten operativen taxonomischen Einheiten (operational taxonomic units, OTUs) of nirK,nirS und nosZ unterschieden sich klar von denen der saureren B¨oden, und deuteten auf Alpha-,Betaund Gammaproteobacteria-verwandte Denitrifikanten in allen Moorgebieten hin. Diese Ergebnisse wurden durch kanonische Korrespondenzanalysen (canonical correspondence analysis, CCA) der relativen OTU-H¨aufigkeiten in den Amplicon-Bibliotheken best¨atigt, was darauf hindeutet, dass die Denitrifikantengemeinschaft des pH-neutralen Niedermoores unter den untersuchten Moorgebieten einzigartig war. Permafrost-beeinflusste B¨oden fielen in den CCA-Ordinationen aller analysierten Genmarker meist zusammen. pH war der wichtigste die Gemeinschaftszusammmensetzung der Nitratreduzierer (abgeleitet von narG) und Denitrifikanten (abgeleitet von den anderen Genmarkern) bestimmende Faktor. Signifikante Einfl¨usse des Kohlenstoffgehaltes des Bodens (auf narG, nirK) der Niederschlagsmenge (auf narG) oder der Temperatur (auf nosZ ) wurden ebenfalls detektiert. Das Verh¨altnis der narG/nosZ Kopienzahlen und das Auftreten gewisser OTUs von nirK und nirS waren positiv mit den N2O-Emissionen korreliert. Die gesammelten Daten weisen darauf hin, dass (i) Denitrifikation in verschiedenen Arten unber¨uhrter Moorgebiete abl¨auft, (ii) N2O Quellenund Senkenfunktionen der Moordenitrifikanten von dem Nitratgehalt des Bodens sowie der Gemeinschaftzusammensetzung der Denitrifikanten beeinflusst werden und (iii) die Gemeinschaftszusammensetzung der Nitratreduzierer und Denitrifikanten von pH, Temperatur, Niederschlagsmenge und Kohlenstoffgehalt des Bodens beeinflusst wird. Aufgrund der globalen Erw¨armung werden erh¨ohte H¨aufigkeiten von ExtremwetterEreignissen erwartet, welche l¨angere D¨urreperioden oder verst¨arkte Niederschl¨age bewirken. Diese Ereignisse wirken sich auf den Wasserstand in Moorgebieten aus und k¨onnten daher die mikrobiellen Gemeinschaften, welche an der Bildung von CH4 9 und N2O beteiligt sind, beeinflussen. Daher wurde der Einfluss von kurzzeitigen Manipulationen des Wasserstands, welche k¨unstliche D¨urre und verst¨arkte ¨ Uberflutung beinhalteten, am Beispiel des sauren Niedermoores untersucht. G¨arungs-, CH4Bildungsund Denitrifikationspotentiale, welche in anoxischen Mikrokosmenstudien mit zu verschiedenen Zeitpunkten der Manipulationsexperimente genommenen Niedermoorbodenproben untersucht wurden, zeigten, dass die potentielle Aktivit¨at der Methanogenen und Denitrifikanten von durch die ¨ Anderung des Wasserstands beeinflusst wurden (d.h. methanogene Aktivit¨at basierend auf spontanen CH4Bildungspotentialen wurde durch die D¨urre erniedrigt und durch die ¨ Uberflutung erh¨oht, denitrifizierende Aktivit¨at wurde durch die ¨ Uberflutung erh¨oht), wohingegen die potentielle Aktivit¨at der G¨arer kaum beeinflusst wurde. ¨ Anderungen in der Kopienzahl von mcrA,narG und nosZ, welche mittels quantitativer PCR bestimmt wurden, waren eher gering im Vergleich zu den beobachteten ¨ Anderungen der potentiellen Aktivit¨aten, was darauf hindeutet, dass die Gr¨oße der Methanogenen- , Nitratreduziererund N2O-Reduzierer-Gemeinschaft kaum durch die kurzzeitigen Manipulationen des Wasserstands beeinflusst wird. Die Gemeinschaftszusammensetzung der Methanogenen, Nitratund N2O-Reduzierer (untersucht mittels TRFLP-Fingerprinting von mcrA,narG bzw. nosZ) war zu allen beprobten Zeitpunkten der Manipulationsexperimente ¨ahnlich, was darauf hindeutet, dass die mikrobielle Gemeinschaftzusammensetzung nicht durch verst¨arkte Wasserstandsschwankungen beeinflusst wird. Die gesammelten Daten weisen auf eine stabile mikrobielle Gemeinschaft im Niedermoorboden hin, welche ihre Aktivit¨at relativ schnell an sich ¨andernde Bedingungen anpassen kann. Die gesammelten Beobachtungen unterstreichen die Bedeutung von Moor-¨ Okosystemen f¨ur Treibhausgasfl¨usse. CH4wird in einem Modell-Moorgebiet gebildet, und die untersuchten Moorgebiete k¨onnen Quellen sowie Senken f¨ur N2O sein und somit 10 signifikant zum globalen N2O-Haushalt beitragen. Sich ¨andernde Wasserst¨ande beeinflussten die potentielle Aktivit¨at der Methanogenen und Denitrifikanten, obwohl die Gemeinschaftszusammensetzung der Methanogenen und Denitrifikanten relativ stabil war. Treibhausgas-metabolisierende Mikroorganismen sind daher geneigt empfindlich auf globale ¨ Anderungen zu reagieren, was wiederum die CH4und N2O Quellenund Senkenst¨arken der Moor-¨ Okosysteme beeinflussen k¨onnte und als Konsequenz daraus ihren Beitrag zum globalen Treibhausgas-Haushalt. 11 1 General introduction 1.2 Peatland ecosystems as important sources of greenhouse gases 1.2.1 Peatland formation and peatland types Northern wetlands store substantial amounts of carbon and nitrogen and are thus important players in the carbon and nitrogen cycles [133, 167]. Wetland soils are either temporarily or permanently waterlogged which provides anoxic conditions in most parts of the soil [23, 181]. Peatlands are caracterized by the accumulation of peat which forms when assimilation exceeds decomposition under oxygen limited conditions [22, 153]. Peatlands develop when organic material accumulates in a lake or pond (Figure 2). The initial state of a peatland is a fen peat which is rich in inorganic nutrients (minerotrophic) as it receives nutrient from groundwater, surface runoff and rainwater. Fens are generally pH-neutral to slightly acidic habitats and are vegetated by grasses and mosses [173]. As peat accumulation continues, the peat surfaces is raised above the groundwater table and a bog peat is formed (Figure 2). Bogs receive all nutrients from rainwater and are thus usually nutrient limited. The main vegetation are Sphagnum mosses and the pH is generally more acidic than in fen soils [176, 181]. 1.2.2 Permafrost peatlands Permafrost-affected soils in the northern hemisphere cover about 16% of the global soil surface, and store substantial amounts of carbon and nitrogen [133, 167]. Permafrost-affected peatlands in the continuous and discontinuous permafrost zone of the northern hemisphere include palsa peats and cryoturbated peat circles [138, 150, 179]. Palsas are elevations of peat soil above the ground level due to uplifting of peat layers by a frozen ice lense and are widely distributed in the circumarctic regions (i.e., Canada, USA, Finland, Sweden, Iceland, Russia) [150, 194]. Palsa 18 1.2 Peatland ecosystems as important sources of greenhouse gases Figure 2: Peatland development. Accumulation of organic matter in a pond (A, B), fen (C), raised bog peat (D); based on [176]. Brown=mineral substratum, blue=water, dark green=fen peat, light brown=bog peat, light green=trees. Vegetation cover changes with time due to changes in ground properties. development is affected by various environmental factors including depth of snow cover, ground water table depth, vegetation cover and degree of wind erosion [150]. Palsa development includes many freeze-thaw cycles leading to accumulation of ice in the active layer and subsequent upheaval of the peat soil [150]. However, as palsas mature the ice lenses will eventually melt and the palsa collapses, forming a thermokarst lake [150]. On the other hand, cryoturbated peat circles are bare surface peat areas in arctic tundra in which vegetation has been removed due to mixing by frost action [138, 177]. Vegetation cover is absent from approximately 12% of the arctic soil surfaces, including cryoturbated peat circles [177]. Cryoturbation occurs mainly in soils with poor soil drainage and frequent freeze-thaw cycles, leading to formation of patterned ground [11, 128, 179]. Cryoturbated soils contain high amounts of incompletely degraded soil organic matter [85]. Organic carbon stored in permafrost soil is redistributed to the active layer (i.e., the layer that thaws in 19 1 General introduction summer months) by cryoturbation, and cryoturbation can increase the amount of organic carbon stored in permafrost soil [11, 63]. Wetlands are the most important single CH4source, as their emissions account for 25% of the observed global CH4emissions [182]. In northern peatlands, about 20% of the assimilated CO2are released into the atmosphere as CH4[139]. However, total CH4production in wetland soils is even higher, as about 20 to 40% of the produced CH4is oxidized on root surfaces or in more oxic surface layers [182]. N2O emission from northern peatlands have been studied to a much lesser extent than CH4emissions, and most studies have focused on N2O emissions from managed peatlands [93, 98, 99]. Pristine northern peatlands can be net sources of N2O, even though their emission rates are generally low [93, 161]. On the other hand, northern peatlands can act as permanent or temporary sinks for N2O [16, 43, 117, 100, 161]. Permafrost-affected peatlands like certain palsa peats and especially cryoturbated peat circles are large point sources of N2O with emission rates comparable to those of tropical and agricultural soils [100, 138]. N2O emissions from palsa peat soils are highly variable [100]. The ability of permafrost-affected peatlands to emit N2O is increased by a low degree of plant cover (resulting in reduced competition for Nsources), high nitrate content, low C:N ratios and high gross mineralization activities [20, 100, 138]. Peatland ecosystems in the northern hemisphere and especially permafrost-affected tundra ecosystems are predicted to be severely affected by global warming, as warming occurs at faster than average rates in those systems [4, 38, 150, 174]. Increasing temperatures are likely to lower the water table in northern peatlands and thus increase the amount of CO2, CH4and N2O released from peatland soils [4, 99, 100]. Moreover, permafrost thawing may cause high emissions of CO2, CH4and N2O from stored carbon and nitrogen [32, 147]. 20 1.3 Processes involved in greenhouse gas production in anoxic peatland soils 1.3 Processes involved in greenhouse gas production in anoxic peatland soils Peatland soils are oxygen limited due to the high degree of water-saturation, and organic matter is thus degraded mainly anaerobically to CO2and CH4(Figure 3, Figure 4). Complex structural polymers like cellulose, chitin or lignin are first degraded to monomers (e.g., glucose, xylose, N-acetyl-gucosamine, or aromatic compounds) which are further degraded by fermentation (producing alcohols, short chain fatty acids, CO2, and H2) [30, 82, 192]. Monomers as well as fermentation products are electron donors in respiratory processes that use alternative electron acceptors like nitrate, manganese, iron, sulfate or CO2(Table 2) [30, 192]. Primary fermentation products such as alcohols and short chain fatty acids are utilized by syntrophic secondary fermenters which produce acetate, CO2and H2[30, 82]. Primary and secondary fermentation products are finally converted to CH4by methanogens [30, 82]. Figure 3: Schematic overview of the main processes in the biological carbon (A) and nitrogen (B) cylcles. DNRA=dissimilatory nitrate reduction to ammonium, Anammox=anaerobic ammonium oxidation (based on [96, 59]). Methanogenesis competes with other anaerobic processes under anoxic conditions [30]. The reduction of alternative electron acceptors like nitrate or iron is thermodynamically more favorable than methanogenesis, and methanogens are thus often out21 1 General introduction competed (Table 2) [92, 94]. However, concentrations of alternative electron acceptors in wetlands are spacially and temporally variable and often low [1, 13, 125, 149], and methanogenesis can although occur simultaneously with those processes, given a sufficently high concentration of H2[23]. Figure 4: Schematic overview of anaerobic processes in wetland soils that lead to the degradation of organic material and the production of CH4. C1=one-carbon organic compounds (excluding CH4) such as formate, methanol, and methylamine. Based on [30]. Table 2: Redoxpotentials of different half-cell reactions under standard conditions (E0’), based on [89, 96, 168]. Half-cell reaction ne− 1E0’ (V) 2Process O2/H2O 2 0.82 Aerobic respiration NO− 3/0.5 N25 0.74 Denitrification NO− 3/NO− 22 0.43 Nitrate reduction Mn+ 4/Mn+ 22 0.39 Mn(IV) reduction Fe3+/Fe2+ 1 0.20 Fe(III) reduction SO2− 4/S2−8 -0.22 Sulfate reduction CO2/CH48 -0.24 Methanogenesis CO2/Acetate 8 -0.29 Acetogenesis 1Number of transfered electrons 2Redoxpotentials under standard conditions (Temperature: 25 ◦C, Pressure: 101.3 kPA, pH 7.0). 22 1.3 Processes involved in greenhouse gas production in anoxic peatland soils 1.3.1 Fermentation In fermentative processes, electrons obtained by oxidation of a substrate are transfered to a partly oxidized intermediate [168]. Substrates include carbohydrates, amino acids, alcohols, and carbonic acids [168]. Energy is conserved in form of ATP via substrate-level phosphorylation by kinase-reactions from activated compounds such as acetyl-coenzyme A (Figure 5). Those activated compounds are formed in previous steps via lyaseor dehydrogenase-reactions [168] (Figure 5). Figure 5: Examples for ATP generation by substrate-level phosphorylation (SLP). Acetate kinase forms ATP from acetyl-CoA after an initial lyase- (A) or dehydrogenase-reaction (B). Based on [168]. Table 3: Examples of primary fermentations, based on [96, 168]. Fermentation type Reaction 1∆G0’ (kJ/mol) 2Example organisms Alcoholic fermentation Hexoses →2 EtOH + 2 CO2-218 Saccharomyces sp. Lactic acid fermentation Hexoses →2 Lactat + 2 H+(homofermentative) -198 Lactobacillus sp., Enterococcus sp. Hexoses →Lactate + EtOH + 2 H++ CO2(heterofermentative) -208 Leuconostoc sp., Lactobacillus sp. Butyric acid fermentation Hexoses →Butyrate + Acetate + H2+ 2 CO2-247 Clostridium sp. Mixed acid fermentation Hexoses →EtOH + 2,3-Butanediol + Succinate + Lactate + Acetate + Formate + H2+ CO2-200 to -260 Escherichia sp., Enterobacter sp. 1Equations are not stoiciometrically balanced. 2Gibbs free energy under standard conditions (Temperature: 25 ◦C, Pressure: 101.3 kPA, pH 7.0; given for glucose utilization). Primary fermentation products include ethanol, acetate, H2and CO2(Table 3). Alcohols (e.g. ethanol, propanol), organic acids (e.g. acetate, succinate, butyrate) or aromatic compounds (e.g. benzoate) are subjected to secondary fermentations by syntrophic organisms (Table 4) [145]. As those secondary fermentations are endergonic under standard conditions, syntrophic partners (e.g. methanogens) are 23 1 General introduction required to remove products such as H2or CO2and thus drive the reaction [96, 145, 168]. The ability to conserve energy via fermention is widespread in bacteria, Archaea, and Eukarya, and organisms can be facultative or obligate fermenters [168]. Table 4: Examples of secondary fermentations (syntrophic processes coupled to methanogenesis), based on [96, 145]. Substrate Reaction 1∆G0’ (kJ) 2Example organisms 3 Butyrate 2 CH3CH2CH2COO−+ 2 H++ 2H2O→5 CH4+ 3 CO2-177 Syntrophomonas sp. Propionate 4 CH3CH2COO−+ 4 H++ 2 H2O→7 CH4+ 5 CO2-249 Syntrophobacter sp. Ethanol 2 CH3CH2OH + CO2→2 CH3COO−+ 2 H++ CH4-112 Desulfovibrio vulgaris Acetate CH3COO−+ H+→CH4+ CO2-36 Clostridium sp. 1Chemical equation of the combined syntrophic reaction including secondary fermentation and methanogenesis. 2Gibbs free energy under standard conditions (Temperature: 25 ◦C, Pressure: 101.3 kPA, pH 7.0). 3Examples of secondary fermenters involved in this type of syntrophic reaction. 24 1.3 Processes involved in greenhouse gas production in anoxic peatland soils 1.3.2 Methanogenesis Methanogenesis is an anaerobic respiration process catalyzed by strictly anaerobic obligately methanogenic Archaea [6, 92]. Methanogens belong to the Euryarchaeota [184] and form 6 orders (Methanobacteriales,Methanocellales,Methanococcales,Methanomicrobiales,Methanopyrales,Methanosarcinales) including 32 genera [51, 144, 169]. Methanogens have a limited substrate range and produce CH4hydrogenotropically, acetoclastically or methylotrophically [51, 92] (Figure 6, Table 5). Hydrogenotrophic methanogens reduce CO2with H2to form CH4[6, 92]. Many hydrogenotrophic methanogens also utilize formate, while some can utilize secondary alcohols like 2-propanol or 2-butanol, ethanol, or CO [92]. Hydrogenotrophic methanogenesis is widespread and occurs in all methanogenic orders [6]. Acetoclastic methanogens belong to the order Methanosarcinales (genera Methanosarcina and Methanosaeta) and utilize acetate by oxidizing the carboxyl-group to CO2and reducing the methyl-group to CH4[92]. While Methanosarcina produce CH4hydrogenotrophically, methylotrophically and acetoclastically, Methanosaeta are strictly acetoclastic [51, 92]. Methylotrophic methanogens utilize methylated compounds such as methanol, methylamines, and methylated sulfides and occur only within the Methanosarcinales (with exception of Methanosaeta) and within the genus Methanosphaera (Methanobacteriales) [6, 92]. The methyl-coenzyme M reductases Mcr and Mrt (catalytical subunits encoded by mcrA or mrtA) are central enzymes involved in all types of methanogenic pathways (Figure 6; [34]), and mcrA is frequently used as a structural genemarker to assess the community composition of methanogens in environmental samples [47, 55, 101, 191]. 1.3.2.1 Hydrogenotrophic methanogenesis In hydrogenotrophic methanogenesis, CO2is reduced with H2or formate as a pri25 1 General introduction Figure 6: Schematic overview of the three methanogenic pathways. MethylCoenzyme M reductase functions as a key enzyme in all three methanogenic pathways. Further enzymes, cofactors and compounds are not shown. Dashed lines indicate that more than one step is needed for the conversion. R=e.g., -SH, -OH, or NH2. Mcr=Methyl coenzyme-M reductase I; Mrt=Methyl coenzymeM reductase II. Based on [34]. Table 5: Types of methanogenesis. Type Reaction ∆G0’ (kJ) 1Organisms 2 Hydrogenotrophic 4 H2+ CO2→CH4+ 2 H2O -35 Most methanogens Acetoclastic CH3COOH →CH4+ CO2-33 Methanosarcina,Methanosaeta Methylotrophic 4 CH3OH →3 CH4+ CO2+ H2O -105 Methanosarcina and others 1Gibbs free energy under standard conditions (Temperature: 25 ◦C, Pressure: 101.3 kPA, pH 7.0). 2Examples of secondary fermenters involved in this type of syntrophic reaction. [92] mary electron donor to CH4[92]. When formate is used as an electron donor, 4 molecule of formate are oxidized to CO2and the obtained reduction equivalents are used to reduced 1 molecule of CO2[92]. In a first step, CO2binds to methanofuran (MF) and is reduced by ferredoxin to a formyl-group, the ferrodoxin in turn is reduced by H2(Figure 7; [169]). The formyl-group is transfered to tetrahydromethanopterin (H4MPT), dehydrated to a methenyl-group and subsequently reduced to methylene-H4MPT and methyl-H4MPT by reduced F420 [92, 169]. The methyl-group is then transfered to coenzyme-M (CoM) and reduced to CH4in a final step by the methy CoM reductase [92, 169]. 26 1.3 Processes involved in greenhouse gas production in anoxic peatland soils Figure 7: Reactions involved in hydrogenotrophic methanogenesis. F420=coenzyme F420; Fd=ferredoxin; MF=methanofuran; H4MPT=tetrahydromethanopterin; HS-CoB=coenzyme B; HSCoM=coenzyme M; based on [92, 169] 1.3.2.2 Acetoclastic methanogenesis In acetoclastic methanogenesis, acetate is split, the carboxyl-group is oxidized to CO2, while the methyl-group is reduced to CH4[92]. Acetate is first activated with ATP and transfered to coenzyme A by acetate kinase-phospotransacetylase, forming acetyl-CoA. Acetyl-CoA is cleaved to methyl-H4MPT and CO-CoA by the CO 27 1 General introduction logues of the bacterial NosZ. In contrast to their bacterial conterparts these N2O reductases are membrane-bound and receive electrons from menaquinol [83, 197]. However, there are archaeal denitrifiers capable of N2O reduction whose genomes do not harbor nosZ homologues, thus there is likely a second archaeal type of N2O reductase [197]. Some denitrifiers lack N2O reductases, thus N2O is the endproduct of denitrification in those organisms [61, 197]. On the other hand, there are organisms like Wolinella succinogenes that reduce N2O using a slightly modified N2O reductase without being capable of complete denitrification [197]. W. succinogenes reduces nitrate to nitrite and N2O to N2, but the intermediate steps are missing [197]. In fungal denitrification, the reductive enzymes are located in the mitochondria and are coupled to the mitochondrial electron transport chain to produce ATP [74]. The membrane-bound fungal nitrate reductase (Nar) reduces nitrate utilizing ubiquinol as electron donor [107, 165]. Properties of fungal Nar resemble those of the bacterial conterpart. However, no orthologues of the bacterial nar genes have so far been detected in fungal genomes, indicating that fungal Nar is evolutionary distinct from bacterial Nar [165]. In contrast, the fungal nitrite reductase gene nirK detected in the genomes of many denitrifying fungi is an orthologue to the bacterial nirK [68]. Fungal NirK is a copper-containing nitrite reductase loctaed in the mitochondrion [75, 107]. Fungal NO reductases are clearly distinct from prokaryotic NO reductases. Fungal Nor is a member of the cytochrome P450 superfamily and is thus called P450nor [107, 165]. The reduction of NO is very different from the oxidation reactions of other P450 proteins, as those require further electron donating proteins [165]. P450nor is a soluble enzyme that utilizes NADH or NADPH to reduce NO to N2O [165]. N2O is the major end product in fungal denitrification, even though some fungi are also capable of N2production [107, 151, 195]. So far, no 34 1.3 Processes involved in greenhouse gas production in anoxic peatland soils N2O reductase has been isolated from fungi or identified in fungal genomes [107]. 35 1 General introduction 1.3.3.3 Factors influencing denitrification in soils Denitrification rates in soils as well as the relative contribution of N2O to the total emitted N gases depend the composition of the denitrifier community as well as on a variety of environmental factors. Soil pH is one of the most important parameters affecting denitrification activities in soil [152]. In general, low pH impairs denitrification and increases the contribution of N2O to total N2gases [25, 152]. However denitrification rates of denitrifier communities from acidic soils are higher at in situ pH than at more neutral pH [117, 123]. Acidic pH has a more pronounced effect on N2O reductase than on the other reductases involved in denitrification [8, 91]. This results in higher N2O:N2ratios in acidic soils. The exact mechanism of inhibition is not yet resolved, but it is likely due to post-transcriptional effects during translation or protein assembly [8, 91]. In general, denitrification activities increase with increasing temperatures, e.g. pure cultures of Pseudomonas denitrificans have a temperature optimum of 38◦C [178]. In soils, the temperature optimum of denitrification is often higher than the observed in situ temperatures [97, 117, 143]. Even though total denitrification activity is increased at higher temperatures, N2O emissions can be lower, as mostly complete denitrification to N2occurs at those temperatures [93]. N2O reductases are more sensitive to cold temperatures than the other reductases involved in denitrification, thus the N2O:N2ratios are higher at colder temperatures, and winter-time denitrification activities significantly impact on the annual N2O emissions [53, 93, 110]. However, there are also differences between soil types, and some soils do not show increased N2O:N2ratios at low temperatures, indicating that denitrifiers and N2O reductases can be adapted to cold temperatures [31]. Freeze-thaw cycles lead to enhanced N2O emissions from soil, and N2O emission peaks are often observed in the end of winter [46, 79, 78, 131]. 36 1.3 Processes involved in greenhouse gas production in anoxic peatland soils Apart from soil pH and temperature, denitrification in soils is also affected by soil water content, carbon and nitrate availability, and soil C:N ratios [23, 21]. Soil nitrate content often limits denitrification in soils, while carbon sources are not limiting [5, 50, 7, 175]. Due to the influences of the mentioned environmental parameters, denitrification activities and N2O emissions are highly variable in soils, and even so-called ”hot spots” for denitrification exist [50, 138, 189]. 1.3.4 Dissimilatory nitrate reduction Dissimilatory nitrate reduction to ammonium (DNRA) reduces nitrate via nitrite to ammonium [83]. DNRA competes with denitrification for nitrate and can be the dominant process in some soils [142]. High carbon content and low nitrate availability favor DNRA over denitrification [83, 172]. The process occurs in Bacteria as well as in fungi [83, 164]. In Bacteria, the reduction of nitrate to nitrite is catalyzed by the periplasmic nitrate reductase NapAB (1.3.3.2) [83, 129, 195]. The reduction of nitrite to ammonium is catalyzed by the pentaheme cytochrome cnitrite reductase NrfA [83, 195]. In fungal DNRA (also called ammonia fermentation), nitrate reduction to ammonium is coupled to the oxidation of ethanol to acetate [163, 164, 165]. The NADH-dependent assimilatory nitrate and nitrite reductases encoded by niaD and niiA are utilized for the stepwise reduction of nitrate to ammonium [163, 165]. The electrons for the reduction originate from the oxidation of ethanol to acetate [163, 165]. The oxidation is coupled to ATP-production by acetate kinase [163, 165]. Even though assimilatory enzymes are used for the reduction of nitrate and nitrite, there is no indication that the produced ammonium is actually assimilated by the fungus [165]. 37 2 List of publications and manuscripts included in the dissertation 2 List of publications and manuscripts included in the dissertation 2.1 Published articles in peer-reviewed journals 1. Palmer, K., Drake, H. L., Horn, M. A. (2009). Genome-derived criteria for assigning environmental narG and nosZ sequences to operational taxonomic units of nitrate reducers. Applied and Environmental Microbiology 75: 51705174. Reference number [116]. Own contribution: Concept (50%, together with M. Horn), experimental work (100%), part of the writing (50%). 2. Palmer, K., Biasi, C., Horn, M. A. (2012). Contrasting denitrifier communities relate to contrasting N2O emission patterns from acidic peat soils in arctic tundra. ISME Journal 6: 1058-1077. Reference number [115]. Own contribution: Experimental design (60%, together with M. Horn) and practical work (100%), data analysis (100%), part of the writing (60%). 3. Palmer, K., Horn, M. A. (2012). Putative Actinobacterial nitrate reducers and Proteobacterial denitrifiers are abundant in permafrost affected N2O metabolizing acidic palsa peat soil. Submitted to Applied and Environmental Microbiology. Reference number [119]. Own contribution: Experimental design (70%, together with M. Horn), practical work (100%), data analysis (100%), part of the writing (60%). This manuscript was under revision when the thesis was handed in. 2.2 Manuscripts in preparation 1. Palmer, K., Horn, M. A. (2012). Denitrification activity of a new and diverse denitrifier community in a pH neutral fen soil in Finnish Lapland is nitrate lim38 2.3 Previous peer-reviewed publications not included in the dissertation ited. Reference number [120]. Own contribution: Experimental design (75%, together with M. Horn) and practical work (100%), data analysis (100%), part of the writing (70%). 2. Palmer, K., Drake, H. L., Horn, M. A. (2012). Denitrifier communities in an acidic fen are stable during experimental drought. Reference number [118]. Own contribution: Practical work (100%), data analysis (100%), part of the writing (70%). 3. Palmer, K., Schulz, K., Mundinger, A., Mertel, R., Horn, M. A., Drake, H. L. (2012). Stability of methanogenic diversity in an acidic fen under the influence of experimental drought. Reference number [121]. Own contribution: Practical work (40%, together with K. Schulz, A. Mundinger, R. Mertel), data analysis (90%), part of the writing (70%). 2.3 Previous peer-reviewed publications not included in the dissertation 1. Palmer, K., Drake, H. L., Horn, M. A. (2010). Association of novel and highly diverse acid-tolerant denitrifiers with N2O fluxes of an acidic fen. Applied and Environmental Microbiology 76: 1125-1134. Reference number [117]. 2.4 Published abstracts at national and international conferences 1. Palmer, Horn, M.A. 2012. Unknown denitrifier diversity in a pH neutral fen soil in Finnish Lapland. Annual Meeting Vereinigung f¨ur Allgemeine und Angewandte Mikrobiologie, BioSpectrum. Abstract SMV003, p. 210. 39 2 List of publications and manuscripts included in the dissertation 2. Palmer, K., Horn, M.A. 2012. Palsa peats represent hitherto underappreciated reservoirs of new denitrifier diversity associated with N2O fluxes. International Polar Year 2012 Conference. Abstract online. 3. Palmer, K., Biasi, C., Drake, H.L., Horn, M.A. 2011. Cryoturbation affects denitrifier communities in N2O-emitting arctic permafrost peat soil. Annual Meeting Vereinigung f¨ur Allgemeine und Angewandte Mikrobiologie, BioSpectrum. Abstract EMP104, p. 116. 4. Palmer, K., Biasi, C., Drake, H.L., Horn, M.A. 2011. Impact of cryoturbation on denitrifier community structure and activity in N2O-emitting arctic permafrost peat soil. Ecology of Soil Microorganisms. Abstract 99. 5. Palmer, K., Schulz, K., Horn, M.A., Drake, H.L. 2010. Stability of the methanogenic community in an acidic fen to experimental drought. Annual Meeting Vereinigung f¨ur Allgemeine und Angewandte Mikrobiologie, BioSpectrum. Abstract ECV02, p. 81. 6. Palmer, K., Schulz, K., Horn, M.A., Drake, H.L. 2010. Effects of enhanced drought on the diversity of methanogens in an acidic fen. Bayreuth Center of Ecology and Environmental Research (BayCEER) Workshop 2010, Abstract O 1.5. 7. Palmer, K., Schulz, K., Horn, M.A., Drake, H.L. 2010. Impact of artificial drought on diversity, abundance, and gene expression of methanogens in an acidic fen. 13th International Symposium on Microbial Ecology (ISME-13), Abstract on disk. 40 2.5 Additional presentations of parts of the work at international meetings 2.5 Additional presentations of parts of the work at international meetings 1. Palmer, K., Biasi, C., Horn, M.A. 2011. Denitrifier communitiy composition impacts N2O emission patterns in acidic tundra permafrost soils. Nordic Network for Stable Isotope Research (NordSIR) Meeting. 2. Palmer, K., Drake, H.L., Horn, M.A. 2011. Effect of high latitude on denitrificationdependent N2O-fluxes and denitrifier community structure in peatlands. Gordon Research Conference on Applied and Environmental Microbiology. 41 3 Greenhouse gas production in pristine peatlands 3 Greenhouse gas production in pristine peatlands Contributions to the synopsis Data presented in this section are partly represented in the manuscripts (see references in bold face). Additional data not represented in the manuscripts are derived from the bachelor thesis of Sonja Perras (part of the work on fermentation and methanogenesis in pH-neutral Puukkosuo fen soil) or from own research (part of the work on fermentation and methanogenesis in pH-neutral Puukkosuo fen soil, work on the comparison of denitrifier communities, manipulation experiments). 42 Wetlands including pristine peatlands are sources and sinks of the greenhouse gases CH4and N2O [18, 37, 76, 100, 161]. However, greenhouse gas fluxes from peatlands vary between peatlands and are dependent on many environmental factors such as temperature and pH [19, 23, 161]. N2O emissions from pristine peatlands have been less intensively studied than CH4emissions, and environmental parameters affecting source or sink strenght of peatlands for N2O are largely unresolved. Thus, five peatlands belonging to different peatland types were selected as model systems to assess methanogenesis as well as denitrification-associated N2O turnover and the possible influence of environmental parameters (Table 6). The five peatlands differed in both CH4and N2O emissions as well as in environmental parameters such as nitrate, ammonium or water content, pH, mean annual temperature or mean annual precipitation (Table 6, Figure 9; more detailed information about sampling sites is given in [115, 117, 120, 119]). Some peatlands emitted large amounts of CH4(fen soils), some peatlands emitted large amounts of N2O (Schl¨oppnerbrunnen fen, cryoturbated peat circles), and some peatlands showed only low emissions of either gas (Peat plateau permafrost tundra, Skalluvaara palsa peat; Figure 9). Table 6: Peatlands used in the studies. Site name Abbreviation Peatland type pH 1water nitrate 1ammonium 1MAT 2MAP 3 content (%) content (µM) content (µM) (◦C) (mm) Puukkosuo PS Fen 6.8 90 50-150 77 −0.4 772 Schl¨oppnerbrunnen SB Fen 5.0 85 0 −500 10 5.3 1162 Permafrost tundra PT Peat plateau permafrost tundra 4.0 80 <1 300 −5.6 505 Skalluvaara SV Palsa peat 4.5 73 10 125 −1.6 415 Peat circles PC Cryoturbated permafrost peat 4.0 70 1500 77 −5.6 505 1Determined in watery extracts. 2Mean annual temperature. 3Mean annual precipitation. 43 3 Greenhouse gas production in pristine peatlands Figure 12: Effect of supplemental substrates on methanogenic potentials in anoxic microcosms with Puukkosuo fen soil. Microcosms were preincubated for 120 days before substrate supplementation. Argon was used as headspace gas. 1 mM of acetate, formate, or methanol or 8% H2/2% CO2 (v/v) in the atmosphere. Mean values of three replicate microcosms and standard errors are displayed. ity filtered (i.e., pyrosequencing and PCR-based errors were removed by denoising with PyroNoise and SeqNoise algorithms; method described in [119]) and clustered at 87% (family level) and 84% (species level) similarity for 16S rRNA genes and mcrA, respectively, using the Needleman Wunsch algorithm (method described in [119]). Phylogenetic trees were generated to assign detected sequences to phyla. In total, 159 and 352 family-level bacterial 16S rRNA OTUs were detected in amplicon libraries from forward and reverse reads, respectively. OTUs mainly affiliated to Firmicutes and Proteobacteria (approximately 60% of all sequences, Figure 13). Among the Proteobacteria,Alphaand Delta-Proteobacterial sequences were most 50 3.2 Processes leading to formation of CH4 abundant. Additionally, families affiliated to Actinobacteria,Acidobacteria,Bacteriodetes,Chloroflexi,Cyanobacteria, and Nitrospiraceae were detected (Figure 13). 15% of all sequences (i.e., 66 families) were not closely related to known bacterial families and might thus represent novel families. In acidic bog and fen soil, Proteobacteria,Firmicutes,Acidobacteria and Actinobacteria are also frequently detected [27, 48, 55, 62, 106, 122, 185]. Sequences and isolates of bacteria in pHneutral permafrost soil consisted mainly of Actinobacteria,Firmicutes and Alphaand Delta-Proteobacteria [157]. Some Delta-Proteobacteria are important sulfate reducers, indicating that pH-neutral Puukkosuo fen soil harbors the potential for sulfate reduction. Additionally, many genera within the Delta-Proteobacteria and Firmicutes such as Syntrophobacter spp. or Clostridium spp. are capable of syntrophic interactions with hydrogenotrophic methanogens and are frequently detected in wetlands [30, 48, 145, 146, 156, 185], indicating that those syntrophic organisms might provide substrates to hydrogenotrophic methanogens in Puukkosuo fen soil. mcrA sequences from Puukkosuo fen were assigned to 9 and 9 species-level OTUs based on forward and reverse reads, respectively. OTU distributions obtained with sequences from forward and reverse reads were similar, thus only results from forward reads are described in more detail. Three species-level OTUs had a relative abundance of >1% of all mcrA sequences. OTU 1, 2, and 3 accounted for 48.3%, 48.2%, and 1.1% of detected mcrA sequences and were related to uncultured mcrA as well as to mcrA of Methanoregula spp., Methanocella paludicola, and Methanoregula spp., respectively (Figure 14), indicating that both Methanomicrobiales and Methanocellales are important players in the CH4production of Puukkosuo fen. Methanogens of both groups produce CH4hydrogenotrophically [6, 92], and higher stimulation with substrates useable by hydrogenotrophic methanogens was observed in microcosm experiments (Figure 12). None of the detected mcrA affiliated with mcrA of 51 3 Greenhouse gas production in pristine peatlands Figure 13: Bacterial families detected in pH-neutral fen soil by amplicon pyrosequencing of bacterial 16S rRNA genes. In total, 2 375 quality filtered sequences were derived from reverse reads, which were assigned to 352 OTUs based on a family-level threshold similarity of 87 %. Results obtained with forward read sequences were similar and are thus not shown. Affiliation of families to major groups and number of detected families per group (in parentheses) are displayed. the Methanosarcinaceae, indicating that this group and thus acetoclastic methanogenesis is of minor importance in Puukkosuo fen soil. Indeed, the stimulatory effect of acetate on methanogenic potentials in fen soil microcosms was much lower than of H2/CO2(Figure 12). 16S rRNA gene sequences and mcrA sequences affiliated with hydrogenotrophic methanogenic groups are frequently observed as the predominant groups in peatland soils, even though sequences affiliated with Methanosarcinales are also detected [40, 54, 55, 191],[121].Methanosarcinales seem to be more abundant in wetter than in drier sites in an acidic mesotrophic fen in Lapland, and were identified as CO2consumers in slightly acidic Schl¨oppnerbrunnen fen soil [55, 191]. 52 3.2 Processes leading to formation of CH4 Figure 14: Phylogenetic tree of representative mcrA sequences (forward reads) from Puukkosuo fen soil. The tree is based on in silico translated amino acid sequences obtained by amplicon-pyrosequencing. OTUs were grouped at species-level phylogenetic similarity of 84% after ampliconnoise qualityfiltering. Values in parenthese represent relative abundances of the OTUs. In total, 1 785 quality filtered sequences were used for OTU calculations. Gray boxes indicate branches where the majority of sequences group into a certain phylogenetic class. The percentage of replicate trees in the bootstrap analysis (10 000 replicates), in which the associated taxa clustered together, are shown next to the branches (values below 50% have been omitted). mcrA of Methanococcus maripaludis S2 was used as outgroup. 3.2.3 Conclusions: Fermentation and methanogenesis in pH-neutral fen soil pH-neutral Puukkosuo fen soil showed the potential to produce acetate, formate, ethanol, and H2/CO2from NAG and thus provide precursors for fen methanogenesis. Methanogenic potentials were stimulated mainly by H2/CO2and formate, indicating a predominance of hydrogenotrophic methanogenesis in pH-neutral fen 53 3 Greenhouse gas production in pristine peatlands soil. This finding was supported by analysis of mcrA as a structural gene marker for methanogens, as all detected sequences affiliated with mcrA of hydrogenotrophic taxa. The bacterial community in Puukkosuo fen soil was dominated by Firmicutes, Alphaand Delta-Proteobacteria including syntrophic genera as well as by novel families, indicating high bacterial diversity and novelty as well as diverse metabolic capacities in pH-neutral fen soil. 54 3.3 Processes involved in turnover of N2O 3.3 Processes involved in turnover of N2O 3.3.1 N2O production and consumption in pristine peatlands N2O-emissions from pristine peatlands are highly variable (Figure 9). Water-saturated fen soils containing minor amounts of nitrate emit only small amounts of N2O and often can display also sink-functions for N2O (Figure 9) [43, 42, 93, 141, 161],[117, 120], while permafrost-affected tundra soils like palsa peats or cryoturbated peat circles can emit high amounts of N2O (Figure 9) [100, 138],[119]. Especially in palsa peats, N2O emissions are highly variable depending e.g., on the thickness of plant cover and nitrate availability, and nitrate-limited palsas also act as N2O sinks [100],[119]. Statistical analyses were conducted to assess the correlation between environmental parameters and in situ N2O emission of five contrasting peatland soils (Table 6, Figure 9). Spearman rank correlation revealed positive (R= 0.9, P= 0.08) and negative (R=−0.8, P= 0.08) correlations between soil nitrate and ammonium content and in situ N2O emission, respectively (Table 7), indicating that denitrification rather than nitrification is the process responsible for observed N2O emission from peatlands (Figure 9). Indeed, peatland soil has a high degree of water-saturation, and denitrification is the main source of N2O in water-saturated and thus mainly anoxic soils [23, 130]. The amount of total carbon in the studied peatland soils decreased with increasing water content and increasing mean annual temperature (R=−0.9 and R=−0.8, respectively), indicating that wet and warm conditions facilitate the mineralization of soil carbon. Interestingly, in situ N2O emissions did not correlate with soil pH (Table 7), indicating that nitrate availability rather than soil pH is the main driver of N2O emissions from peat soil. N2O production in unsupplemented anoxic microcosms with peatland soil ranged from <1 to 1200 nmol·g−1 DW (Figure 15). N2O production was highest in microcosms 55 3 Greenhouse gas production in pristine peatlands Table 7: Correlation of soil parameters and observed N2O emissions. Spearman rank correlations were calculated based on mean values for environmental parameters. Values for N2O emissions were based on Figure 9, values for environmental parameters were taken from site descriptions [100, 119, 120, 185]. Parameter pH water content MAT 1NO− 3NH+ 4Total C 2Total N 3C:N Correlation (R) 0.10 -0.20 0.21 0.87 -0.82 0.10 0.50 -0.60 Significane (P) 0.95 0.78 0.78 0.08 0.08 0.95 0.45 0.35 1Mean annual temperature. 2Total carbon. 3Total nitrogen. with cryoturbated peat soil and lowest in microcosms with fen soil (Figure 15). In microcosms with palsa and cryoturbated peat soil, N2O that was produced within the first 50 to 100 hours of incubation was subsequently consumed (Figure 15), demonstrating the capacity of peatland soils for N2O reduction. N2O production was always higher in microcosms that were amended with acetylene to block N2O reduction [190] than in unamended microcosms (Figure 15), indicating that N2O was not the sole endproduct of denitrification in either of the systems, but that complete denitrification to N2occured. In acetylene-amended microcosms, highest N2O production (1800 nmol·g−1 DW ) was observed in microcosms with cryoturbated peat soil (Figure 15), reflecting the high nitrate content of the soil and the observed high in situ emissions (Figure 9) [100, 138],[115]. N2O production in acetyleneamended microcosms with palsa peat soil and with pH-neutral fen soil was about 10 fold lower than in microcosms with cryoturbated peat soil (approximately 200 nmol·g−1 DW ). Lowest N2O production was observed in acetylene-amended microcosms with permafrost tundra soil (<14 nmol·g−1 DW ) (Figure 15). Spearman rank correlation of environmental parameters and observed N2O to (N2O+N2) in anoxic microcosms with peat soil revealed that the contribution of N2O to total N-gases decreased with increasing pH (R=−0.9, P= 0.08) and 56 3.3 Processes involved in turnover of N2O Figure 15: Production of N2O in unsupplemented anoxic microcosms with peatland soil from 0 to 20 cm depth. Open symbols represent microcosms without addition of acetylene, closed symbols represent microcosms with addition of acetylene to block N2O reductase [190]. The insets in (A), (B), (C), and (D) represent enlargements to allow better visualization of relatively small amounts of N2O produced in those microcosms. A: Puukkosuo fen (pH 6.8), B: Schl¨oppnerbrunnen fen (pH 5.0), C: Permafrost tundra (pH 4.0), D: Skalluvaara palsa peat (pH 4.5), E: Cryoturbated peat circles (pH 4.0). increasing mean annual temperature (R=−1.0, P= 0.02), indicating that under warmer and pH-neutral conditions N2is the favoured endproduct of denitrification. Indeed, the relative contribution of N2O to total N-gases is higher under more acidic conditions in soils and pure cultures as N2O reductase is more severely affected by acidic pH than the other N-reductases involved in denitrification [8, 39, 91, 170, 171]. Moreover, N2O-reduction is more strongly affected by low temperatures than the preceeding reductive steps, thus N2O release from soil is often high also in winter [31, 53, 110]. However, denitrifier communities can be adapted to low pH and low temperatures as well [31, 117]. Supplemental nitrate (0 to 500 µM) stimulated N2O production in nitrate-depleted acetylene-amended anoxic microcosms with peatland soil to different extents (Figure 16). Maximal initial N2O production rates (vmax) were highest in microcosms with Schl¨oppnerbrunnen fen soil (pH 5), while vmax was lowest in microcosms with 57 3 Greenhouse gas production in pristine peatlands Skalluvaara palsa peat soil (pH 4.5) (Figure 16, Table 8). In microcosms with permafrost tundra soil, N2O production rates were highest with 10 µM nitrate and decreased with increasing nitrate concentrations, indicating that denitrification in permafrost tundra soil is saturated at low nitrate concentrations. Supplemental nitrite (0 to 500 µM) likewise stimulated N2O production in nitrate-depleted acetylene-amended anoxic microcosms with peatland soil (Figure 16). vmax were highest in microcosms with Puukkosuo fen soil (52 nmol·g−1 DW ·h−1), intermediate in microcosms with Schl¨oppnerbrunnen fen and cryoturbated peat soil (32 −33 nmol·g−1 DW ·h−1), and lowest in permafrost tundra and Skalluvaara palsa peat soil (18 and 15 nmol·g−1 DW ·h−1, respectively) (Table 8). vmax values were in the same range in nitrateand nitrite-supplemented microcosms with Schl¨oppnerbrunnen fen soil, while vmax values were 2to 9-fold higher in nitritethan in nitrate-supplemented microcosms with soil from the other peatlands (Table 8). vmax/KM(indicative for a soils nitrate/nitrite affinity) were highest for nitrateand nitrite-dependent denitrification in microcosms with cryoturbated peat soil (Table 8). Nitrate-dependent vmax/KMwere positively correlated with the nitrate content of the soil and in situ N2O emissions as well as negatively correlated with the ammonium content of the soil (Spearman rank correlation: R= 0.9, P= 0.08; R≈1.0, P < 0.0001; R=−0.8, P= 0.08 for nitrate content, in situ emissions, and ammonium content, respectively). Table 8: Parameters of apparent nitrateand nitrite-dependent Michaelis-Menten kinetics in anoxic acetylene-amended microcosms with peatland soil. Nitrate amended Nitrite amended Soil Puukkosuo Schl¨oppnerbrunnen Permafrost Skalluvaara Cryoturbated Puukkosuo Schl¨oppnerbrunnen Permafrost Skalluvaara Cryoturbated fen fen tundra palsa peat peat circles fen fen tundra palsa peat peat circles vmax (nmol·h−1·g−1 DW )118 25 2 3 17 52 33 18 15 32 KM(µM) 129 11 n.a. 217 3 62 7 21 39 6 vmax/KM(10−3·h−1)117 53 n.a. 211 466 22 105 41 25 407 1Kinetic parameters calculated from Figure 16. 2Not applicable. The ratio of N2O to total N-gases (i.e., the amount of N2O produced in the absence to the amount of N2O produced in the presence of acetylene) at different 58 3.3 Processes involved in turnover of N2O Figure 16: Apparent Michaelis-Menten kinetics of nitrate- (A) and nitritedependent (B) denitrification in acetylene-amended anoxic microcosms with peatland soil from 0 to 20 cm depth. Blue squares: Puukkosuo fen (pH 6.8), Red circles: Schl¨oppnerbrunnen fen (pH 5.0), Green upward triangles: Permafrost tundra (pH 4.0), Black diamonds: Skalluvaara palsa peat (pH 4.5), Orange downward triangles: Cryoturbated peat circles (pH 4.0). Individual values of duplicate microcosms (cryoturbated peat circles) or mean values and standard errors of three replicate microcosms (all other soils) are displayed. Michaelis-Menten curves were fitted to the data where applicable (solid lines). concentrations of supplemented nitrate and nitrite differed between the soils (Figure 17). In pH-neutral fen soil, N2O/(N2O+N2) was below 10% when 10 µM nitrate or nitrite were supplied and did not exceed 30% at 100 µM supplied nitrate/nitrite. In contrast, N2O/(N2O+N2) was 100% at all supplied concentrations of nitrate or nitrite in microcosms with permafrost tundra soil. Indeed, N2O/(N2O+N2) was negatively correlated with soil pH (Spearman rank correlation: R=−0.9, P= 0.08 for 100 µM supplied nitrate). Moreover, there was a positive correlation between N2O/(N2O+N2) when nitrate and when nitrite were supplied (Spearman rank correlation: R≈1.0, P < 0.0001 for 10 µM supplied nitrate/nitrite). Nitrate concentrations generally influence the ratios of N2O/N2+N2O produced from denitrification 59 3 Greenhouse gas production in pristine peatlands and Methylobacterium sp., was detected almost exclusively in pH-neutral fen soil from Puukkosuo, where it accounted for >60% of detected nirK, indicating that this OTU might be adapted to neutral pH and thus unable to tolerate higher levels of acidity (Figure 18 B). OTU 4 was only detected in slightly acidic fen soil from Schl¨oppnerbrunnen and was only distantly related to known nirK (Figure 18 B, Figure 20). Thus, there might be novel and specialized NirK-type denitrifiers in this fen, that can tolerate slightly acidic conditions but are more sensible to soil freezing. Detected and estimated species-level OTU numbers were highest in Puukkosuo and Schl¨oppnerbrunnen fen soils and lowest in permafrost-affected palsa peat and peat circle soil (Table 9). Shannon diversity and Species Evenness indices were similar in all soils except for cryoturbated peat circle soil, which showed very low Shannon diversity and Species Evenness (Table 9). nirS sequences likewise grouped into 10 major species-level OTUs (i.e., OTUs with a relative abundance of >3% in at least one of the soils). Three different types of nirS communities were detected: The first type was detected in the pHneutral fen Puukkosuo and consisted mainly of OTUs 3 and 5 (approximately 80% of all sequences), which affiliated with uncultured soil and sediment bacteria in the phylogenetic tree (Figure 18 C, Figure 21). Thus, this community type is likely dominated by hitherto unknown NirS-type denitrifiers. The second nirS community type was detected in Schl¨oppnerbrunnen fen and cryoturbated peat circle soil. This community type was dominated by OTU 1 (Figure 18 C), which affiliated with nirS of Bradyrhizobium sp., Rhodanobacter sp., and Magnetospirillum magneticum in the phylogenetic tree (Figure 21). The third type was detected in unturbated permafrost tundra and Skalluvaara palsa peat soil and was dominated by OTUs 2, 3, and 4 (Figure 18 C). OTUs 2 and 3 were related to nirS of uncultured sediment bacteria, while OTU 4 was related to nirK of Azoarcus tolulyticus (Figure 21). Species-level 66 3.3 Processes involved in turnover of N2O nirS diversity as expressed by number of observed and estimated OTUs as well as by Shannon diversity and Species Evenness indices was high in Puukkosuo fen soil and low in cryoturbated peat circle soil, while it was at a similar level in all other studied soils (Table 9). nosZ sequences grouped into 8 major species-level OTUs (i.e., OTUs with a relative abundance of >1% in at least one of the soils). nosZ OTU distribution differed between the 5 soils (Figure 18 D). 7 major OTUs were detected in Puukkosuo fen soil, OTUs 1, 4, 5, and 6 were most prominent with relative abundances of 45%, 26%, 13%, and 10%, respectively (Figure 18 D). OTU 1 was also detected in the 4 other soils, it dominated detected nosZ in Schl¨oppnerbrunnen fen soil (87%), but had a very low abundance in permafrost tundra and cryoturbated peat circle soil (2% and 3% of detected nosZ, respectively). OTU 2 was almost exclusively detected in permafrost tundra and cryoturbated peat circle soil, where it accounted for 97% and 94% of detected nosZ, respectively (Figure 18 D). OTU 3 was the predominant OTU in Skalluvaara palsa peat soil (52%) and was also abundant in Schl¨oppnerbrunnen fen soil (12%). OTUs 1, 2, 3, 4, 5, and 6 were affiliated with nosZ of Azospirillum lipoferum,Mesorhizobium sp., Bradyrhizobium japonicum,Bosea sp., Azospirillum largimobile, and Herbaspirillum spp., respectively (Figure 22), indicating that detected peatland nosZ are diverse and dominated by Alphaand Beta-Proteobacterial nosZ.Beta-Proteobacterial nosZ were almost exclusively detected in Puukkosuo fen soil (Figure 22), indicating that Beta-Proteobacteria might be more severely affected by the more acidic pH in the other soil types, confirming the effect observed for narG (Figure 18 D, Figure 19). Species-level nosZ diversity as given by the number of observed and estimated OTUs and Shannon diversity and Species Evenness indices was highest in Puukkosuo fen soil (Table 9). This finding is in line with the results obtained with the other gene markers. 67 3 Greenhouse gas production in pristine peatlands The collective data indicate that (i) Actinobacterial,Alphaand Beta-Proteobacterial nitrate reducers and denitrifiers are common in peatlands, (ii) some species occur in all peatlands and might thus contribute to a core denitrifier community in peatlands, (iii) nitrate reducer and denitrifier community compositions differ between different peatland types, and (iii) highest diversity is associated with pH-neutral fen soil. 68 3.3 Processes involved in turnover of N2O Figure 20: Phylogenetic tree of representative nirK sequences from different peatland soils. The tree is based on in silico translated amino acid sequences. Sequences were obtained via amplicon-pyrosequencing from peatland soils. OTUs were grouped at species-level phylogenetic similarity of 83% after ampliconnoise quality-filtering. Values in parenthese represent relative abundances of the OTUs in Puukkosuo fen, Schl¨oppnerbrunnen fen, permafrost tundra, Skalluvaara palsa peat, and cryoturbated peat circles. In total, 19 292 quality filtered sequences were used for OTU calculations. Gray boxes indicate branches where the majority of sequences group into a certain phylogenetic class. The percentage of replicate trees in the bootstrap analysis (10 000 replicates), in which the associated taxa clustered together, are shown next to the branches (values below 50% have been omitted). nirK of Haloarcula marismortui ATCC 43049 was used as outgroup. 69 3 Greenhouse gas production in pristine peatlands Figure 21: Phylogenetic tree of representative nirS sequences (forward reads) from different peatland soils. The tree is based on in silico translated amino acid sequences. Sequences were obtained via amplicon-pyrosequencing from peatland soils. OTUs were grouped at species-level phylogenetic similarity of 82% after ampliconnoise quality-filtering. Values in parenthese represent relative abundances of the OTUs in Puukkosuo fen, Schl¨oppnerbrunnen fen, permafrost tundra, Skalluvaara palsa peat, and cryoturbated peat circles. In total, 5 218 quality filtered sequences were used for OTU calculations. Gray boxes indicate branches where the majority of sequences group into a certain phylogenetic class. The percentage of replicate trees in the bootstrap analysis (10 000 replicates), in which the associated taxa clustered together, are shown next to the branches (values below 50% have been omitted). nirS of Rhodothermus marinus DSM 4252 was used as outgroup. 70 3.3 Processes involved in turnover of N2O Figure 22: Phylogenetic tree of representative nosZ sequences (forward reads) from different peatland soils. The tree is based on in silico translated amino acid sequences. Sequences were obtained via amplicon-pyrosequencing from peatland soils. OTUs were grouped at species-level phylogenetic similarity of 80% after ampliconnoise quality-filtering. Values in parenthese represent relative abundances of the OTUs in Puukkosuo fen, Schl¨oppnerbrunnen fen, permafrost tundra, Skalluvaara palsa peat, and cryoturbated peat circles. In total, 5 920 quality filtered sequences were used for OTU calculations. Gray boxes indicate branches where the majority of sequences group into a certain phylogenetic class. The percentage of replicate trees in the bootstrap analysis (10 000 replicates), in which the associated taxa clustered together, are shown next to the branches (values below 50% have been omitted). nosZ of Haloarcula marismortui ATCC 43049 was used as outgroup. 71 3 Greenhouse gas production in pristine peatlands 3.3.3 Environmental factors shaping denitrifier communities in peatlands Statistical analyses based on the community composition of sequences received from pyrosequencing of narG,nirK,nirS, and nosZ from different peatland soils were conducted to assess the effect of different environmental parameters on community composition of nitrate reducers and denitrifiers. Environmental parameters were classified as primary environmental parameters (i.e., pH, temperature, precipitation, nitrate and ammonium content, total carbon, total nitrogen, C/N ratio) or derived parameters (i.e., N2O emission, ratio of N2O to total N-gases, relative abundance of narG or nosZ). Canonical correspondence analyses (CCA) were conducted for each gene marker on the basis of rarified species-level OTU tables to cluster the communities according to their habitat and to elucidate the primary or derived environmental parameters that influence community composition. CCA of narG revealed a clear separation of Puukkosuo and Schl¨oppnerbrunnen fen soil communities from each other and from the permafrost affected communities (Figure 23 A), as was already observed for direct comparison of OTU relative abundances (Figure 18 A). Community composition was affected by pH, total soil carbon and mean annual precipitation (Figure 23 A). The relative abundance of OTU 1 was negatively correlated with pH and soil water content (Spearman rank correlations with R=−0.8, P= 0.08 and R≈ −1.0, P= 0.02, respectively), and positively correlated with total soil carbon (R= 0.9, P= 0.08), while OTU 2 was positively and negatively correlated with soil water content and total soil carbon, respectively (R≈1.0, P < 0.001 and R=−0.9, P= 0.08, respectively). CCA of nirK showed a clear separation of all studied peatland communities (Figure 23 B). The nirK community of Puukkosuo fen was most dissimilar to the communities in the other peatland soil, as it was also indicated earlier (Figure 18 B, Table 9). pH and total soil carbon determined the community composition of detected 72 3.3 Processes involved in turnover of N2O Figure 23: Canonical correspondence analyses (CCA) based on relative species-level OTU abundances of narG (A), nirK (B), nirS (C), and nosZ (D). Blue circles represent the different sampling sites (Table 6). Primary and derived environmental parameters affecting the community composition of denitrification-associated genes are displayed in red. OTU abundances from rarified datasets(100 iterations at sampling depths of 500, 1000, 100, and 100 for narG,nirK,nirS, and nosZ, respectively) were used for the calculation of CCAs. nirK (Figure 23 B). Correlations between environmental parameters and individual OTUs were observed for OTU 1, which was predominant in frost-affected soils and was positively, negatively and negatively correlated with total soil carbon, soil water content and soil pH, respectively (Spearman rank correlations with R= 0.9, P= 0.08, R≈ −1.0, P= 0.02, and R=−0.8, P= 0.08, respectively). On the other hand, the relative abundance of OTU 5 which was abundant in Puukkosuo and 73 3 Greenhouse gas production in pristine peatlands Schl¨oppnerbrunnen fen soil was positively and negatively correlated with soil water content and total soil carbon, respectively (R= 0.98, P= 0.02 and R=−0.87, P= 0.08, respectively), indicating that total soil carbon does not only affect the nirK community composition in total but also more directly the relative abundance of individual OTUs. OTU 2 was the only OTU that showed a correlation with in situ N2O emission, the relative abundance of OTU 2 was negatively correlated with N2O emission (R=−0.9, P= 0.08), indicating that denitrifiers carrying this type of nirK might be important for N2O reduction in situ. CCA based on relative abundances of detected nirS revealed 3 distinct clusters of nirS communities, thus supporting the visual comparison (Figure 23 C, Figure 18 C). The nirS community from Puukkosuo fen was clearly distinct from the community in the other peatland soils, and Skalluvaara palsa peat and peatland tundra as well as Schl¨oppnerbrunnen fen and cryoturbated peat circle communities clustered together (Figure 23 C). Community composition of detected nirS was influenced by pH as a primary environmental factor and by the relative abundance of narG and the ratio of N2O to total N-gases (when 10 µM nitrite were supplied) as derived factors (Figure 23 C). The relative abundance of several OTUs was correlated to observed in situ N2O emissions: OTU 1 was positively correlated with in situ N2O emissions (Spearman rank correlation with R= 0.98, P= 0.02), while OTUs 2, 4 and 6 were negatively correlated with in situ N2O emissions (R=−0.98, P= 0.02, R=−0.87, P= 0.08, and R=−0.98, P= 0.02, respectively), indicating that denitrifiers harboring those types of nirS might be involved in production and consumption of N2O in peatland soils, respectively. CCA analysis based on relative abundances of nosZ in peatland soils likewise supported the observed grouping of peatland soils (Figure 23 D, Figure 18 D), as the nosZ community of Puukkosuo fen was clearly separated from the other soils, while 74 3.3 Processes involved in turnover of N2O nosZ communities of Schl¨oppnerbrunnen fen and Skalluvaara palsa peat as well as nosZ communities of permafrost tundra and cryoturbated peat circles grouped together (Figure 23 D). Community composition of nosZ was influenced by the primary environmental parameters pH and temperature as well as by the relative abundance of nosZ in peatland soils as a secondary parameter (Figure 23 D). Unlike OTUs of nirK and nirS, OTUs of nosZ were not correlated with observed in situ N2O emissions. However, OTUs 1 and 2 were negatively and positively correlated with the ratio of N2O to total N-gases in unsupplemented anoxic microcosms, respectively (Spearman rank correlations with R≈ −1.0, P= 0.02 and R= 0.9, P= 0.08, respectively), indicating that denitrifiers harboring nosZ of OTU 1 might be highly efficient in N2O consumption and cause the observed differences in N2O emissions. The collective data indicate that (i) nitrate reducer and denitrifier communities in different peatland soils are clearly distinct, (ii) the primary and derived environmental factors affecting community composition of denitrification associated genes differ between the genes, and (iii) several OTUs of nirK,nirS, and nosZ are likely determining N2O emissions from peatland soils. 75 3 Greenhouse gas production in pristine peatlands Figure 26: Effect of 6 month flooding on formate-dependent methanogenesis in anoxic microcosms with fen soil from 0 to 20 cm (A) and 20 to 40 cm depth (B). Microcosms were supplemented with 0 to 1 mM formate after 7 days of anoxic preincubation to reduce alternative electron acceptors. vmax are based on initial CH4production rates in microcosms. Black bars represent microcosms with soil from control plots, white bars represent microcosms with soil from flooded plots. Mean values of three replicate microcosms and standard errors are displayed. Post-flooding CH4production rates in microcosms with 0 to 20 cm soil from control plots did not follow apparent Michaelis-Menten kinetics (*). The insert in (B) represents a magnification of the y-axis to better visualize the low observed vmax. composition in 2008 and 2009 (Figure 28 A and B, respectively), with Methanocellaceae being to most abundant group in 2008 and Methanomicrobiales/-bacteriales being the most abundant group in 2009. This indicates that the composition of the methanogenic community is rather stable when subjected to short-term changes such as experimental drought or flooding but might be influenced by annual variations. 82 3.4 Effect of water table manipulations on anaerobic processes in a model peatland Figure 27: Effect of water table manipulations on the abundance of mcrA genes and transcripts in an acidic fen.mcrA gene copy numbers in relation to archaeal 16S rRNA genes were determined before and after a 42 day long experimental drought in 2006 with soil from 0 to 40 cm depth (A) as well as before and after 6 month of experimental flooding in 2009 with soil from 0 to 40 cm depth (B). mcrA transcript numbers were determined before and after a 42 day long experimental drought in 2006 with soil from 0 to 40 cm depth (C). Black bars represent abundances in control plots, white bars represent abundances in treatment plots. Mean values of three replicate plots (four sampling depths per plot) and standard errors are displayed. 83 3 Greenhouse gas production in pristine peatlands Figure 28: Effect of water table manipulations on the community composition of mcrA genes and transcripts in an acidic fen. Community composition was determined by TRFLP analysis before and after a 42 day long experimental drought in 2006 with soil from 0 to 40 cm depth on gene (A) and transcript (C) level as well as on gene level before and after 6 month of experimental flooding in 2009 with soil from 0 to 40 cm depth (B). All TRFs that could be assigned to a certain order/family of methanogens were grouped. Mean values of three replicate plots (four sampling depths per plot) are displayed. 84 3.4 Effect of water table manipulations on anaerobic processes in a model peatland 3.4.3 Effect of water table manipulations on denitrification in an acidic fen The effect of water table manipulations on the abundance of nitrate reducers and denitrifiers in acidic fen soil was assessed by qPCR of narG and nosZ in control and treatment plots before and after the experimental drought in 2008 as well as before and after experimental flooding in 2009 (qPCR assay described in [118]). narG and nosZ copy numbers ranged from 2% to 14% and from 0.02% to 0.2% of bacterial 16S rRNA gene copy numbers, respectively (Figure 29). After 42 days of experimental drought, relative abundances of narG and nosZ in drought plots were 2x and 1.5 as high as before the drought period, respectively, while they were slightly lower in control plots (0.75x and 0.6x as high as before the drought period, respectively) (Figure 29), indicating that growth of nitrate reducers and denitrifiers was stimulated by the experimental drought. Experimental drought leads to increased oxygen penetration into the soil and might thus promote the growth of facultatively aerobic denitrifiers. The observed effect of experimental flooding on narG and nosZ abundances was less pronounced than the effect of experimental drought. Relative abundances of narG were lower postthan pre-flooding in flooded and control plots, while relative abundances of nosZ were similar postthan pre-flooding in both plot types (Figure 29), indicating that increased water saturation does not influence the relative abundance of nitrate reducers or denitrifiers in fen soil. Moreover, the relative abundance of nitrate reducers undergoes likely stronger seasonal variations than the relative abundance of denitrifiers, as the relative abundance of narG was lower in the end of the experimental period in 2008 and 2009 (August and November, respectively), while the variation in nosZ relative abundance was less pronounced (Figure 29). The effect of water table manipulations on the community composition of nitrate reducers and denitrifiers was assessed by TRFLP analysis of narG and nosZ in con85 3 Greenhouse gas production in pristine peatlands Figure 29: Effect of water table manipulations on the abundance of narG (A,B) and nosZ (C,D) in an acidic fen.narG and nosZ gene copy numbers in relation to bacterial 16S rRNA genes were determined before and after a 42 day long experimental drought in 2006 with soil from 0 to 40 cm depth (A,C) as well as before and after 6 month of experimental flooding in 2009 with soil from 0 to 40 cm depth (B,D). Black bars represent abundances in control plots, white bars represent abundances in treatment plots. Mean values of three replicate plots (four sampling depths per plot) and standard errors are displayed. trol and treatment plots before and after the experimental drought in 2008 as well as before and after experimental flooding in 2009 (experimental procedure of TRFLP described in [118]). Up to 8 and 9 TRFs were observed for narG and nosZ, respectively (Figure 30, Figure 31). Community composition of narG and nosZ was similar in control and treatment plots at all sampling timepoints (Figure 30,Figure 31), indicating that the composition of the nitrate reducer and denitrifier community is likely unaffected by short-term water table manipulations or by seasonal variability. Stability of denitrifier communities to water table fluctuations is observed in many wetland systems. Denitrifier communities in a constructed wetland vary between individual sites but are rather unaffected by hydrological pulsing (i.e., short-term drought and subsequent flooding) [155]. Moreover, the community composition of nosZ in fen grassland soil does not change significantly in response to variations in 86 3.4 Effect of water table manipulations on anaerobic processes in a model peatland water content [159]. Figure 30: Effect of water table manipulations on the community composition of narG genes in an acidic fen. Community composition was determined by TRFLP analysis before and after a 42 day long experimental drought in 2006 with soil from 0 to 40 cm depth (A) as well as before and after 6 month of experimental flooding in 2009 with soil from 0 to 40 cm depth (B). Mean values of three replicate plots (four sampling depths per plot) are displayed. The effect of prolonged flooding of the fen soil on nitrate-dependent denitrification was assessed in anoxic microcosms with fen soil taken before and after a 6 months flooding period in 2009 from 0 to 20 cm and 20 to 40 cm depth from control and treatment plots. Apparent Michaelis-Menten kinetics were determined based on initial N2O production rates in acetylene-amended nitrate-supplemented microcosms. vmax were higher in 0 to 20 cm than in 20 to 40 cm depth soil, thus denitrification potentials like fermentative and methanogenic potentials are mainly located in the upper soil layers (Figure 32). Prolonged flooding increased vmax in 87 3 Greenhouse gas production in pristine peatlands Figure 31: Effect of water table manipulations on the community composition of nosZ genes in an acidic fen. Community composition was determined by TRFLP analysis before and after a 42 day long experimental drought in 2006 with soil from 0 to 40 cm depth (A) as well as before and after 6 month of experimental flooding in 2009 with soil from 0 to 40 cm depth (B). Mean values of three replicate plots (four sampling depths per plot) are displayed. both depths in treatment plots, while vmax was similar in control plots preand post-flooding (Figure 32 A, B). The ratio of N2O to total N-gases was lower postthan pre-flooding in treatment plots, while it was similar in control plots at both time points (Figure 32 C, D). Thus, the higher water-saturation of fen soil as induced by prolonged flooding likely leads to increased denitrification and higher N2O consumption potentials. Higher in situ denitrification rates are detected in a constructed wetland when soil is flooded after an initial drought period [155]. N2O consumption is strongly affected by the amount of available oxygen in the system, and under water-saturated, mostly anoxic conditions the end-product of denitrifi88 3.4 Effect of water table manipulations on anaerobic processes in a model peatland cation is mainly N2[3, 26]. Water-saturated peat soil can be a sink for N2O rather than a source, while peat soil with a lowered water table water table is a source of N2O [42, 93, 136, 137, 141]. Moreover, drying of nutrient-rich peat soil increases the amount of emitted N2O [99, 136, 137]. Thus, a raised water table due to increased precipitation might enhance the N2O sink strength in peatland ecosystems. Figure 32: Effect of 6 month flooding on nitrate-dependent N2O production and the ratio of N2O to total N-gases in anoxic microcosms with fen soil from 0 to 20 cm (A, C) and 20 to 40 cm depth (B, D). Microcosms were supplemented with 0 to 100 µM nitrate and incubated with or without acetylene (15% vol/vol) in the gasphase. vmax are based on initial N2O production rates in microcosms with acetylene. The ratio of N2O to total N-gases equals the ratio of N2O production in microcosms without and with acetylene. Black bars represent microcosms with soil from control plots, white bars represent microcosms with soil from flooded plots. Mean values of three replicate microcosms and standard errors are displayed. 3.4.4 Conclusions: water table manipulations The short-term water table manipulations in the model fen Schl¨oppnerbrunnen affected methanogenic and denitrifying potentials in microcosms but had no obvious effect on fermentation potentials, thus future changes in water table height are likely to affect the turnover and release of greenhouse gases from the fen. Observed changes 89 3 Greenhouse gas production in pristine peatlands in physiological potentials were concomitant with small but detectable changes in relative abundances of the involved microbial groups, thus highlighting the capacity of fen microorganism to respond to changing environmental conditions. The community composition of methanogens and denitrifiers was not affected by the manipulation experiments, thus indicating a rather stable microbial community in fen soil. The collective data thus indicate that the acidic fen harbors a rather stable microbial community that is capable of adapting its activity to changing hydrological conditions quite rapidly. 90 3.5 General conclusions 3.5 General conclusions CH4is produced from polymers and sugars under anoxic conditions in wetlands, and different groups of organisms are involved in the conversion of sugars to CH4 (Figure 4; [30]). In Puukkosuo fen soil, the model sugar NAG is first converted to H2/CO2, acetate, and formate, which can then be used directly or indirectly by methanogens, leaving CH4as the end product (Figure 33). Methanogens in fen soil are thus trophically linked to fen soil fermenters, which supports the postulated Hypothesis 1 (3.1). Figure 33: Schematic model of processes contributing to methanogenesis in pH-neutral fen soil. Sugars like NAG are fermented to a variety of fermentation products, mostly H2/CO2and acetate, which in turn are used by methanogens. The relative contribution of each process is indicated by its arrow size. Dotted arrows indicate that more than one step/organism might be involved in the conversion of the compound. Methanol was not measured in microcosm experiments, and possible sources of methanol in fen soil were not investigated. 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The different threshold similarities of nosZ and narG suggest that nosZ is more conserved than narG, indicating that species-level OTU assignment of nosZ might be more reliable than that of narG. Conclusions. The above considerations indicate that narG or nosZ analyses can be used to estimate species-level diversity of the associated bacteria on the basis of sequence similarities. Such analysis is considered to yield a minimum number of species in a sample (i.e., the true species-level diversity might be much higher). Analyses of gene markers of different functional groups revealed similar threshold similarities indicating species-level OTUs (e.g., for dsrAB and amoA gene fragments, they were 80 to 90%) (11, 12). Since narG or nosZ sequences from organisms of the same genus generally form coherent clusters in phylogenetic trees (Fig. 2 and 3), distinct clusters of environmental sequences could provide evidence for new genus-level diversity. 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Ko¨rner. 2007. Nitrous oxide reductases, p. 67–81. In H. Bothe, S. J. Ferguson, and W. E. Newton (ed.), Biology of the nitrogen cycle, 1st ed. Elsevier Academic Press, Inc., Amsterdam, The Netherlands. 5174 PALMER ET AL. APPL.ENVIRON.MICROBIOL. 4 Manuscripts 130 Manuscript in preparation for ISME Journal Version 2 04.10.11 1 2 3 Contrasting denitrifier communities relate to contrasting N2O 4 emission patterns from acidic peat soils in arctic tundra 5 6 Katharina Palmer1, Christina Biasi2, and Marcus A. Horn1* 7 8 9 1 Department of Ecological Microbiology, University of Bayreuth, Dr.-Hans-Frisch-Straße 1-3, 10 95440 Bayreuth, Germany 11 2 Department of Environmental Science, University of Kuopio, Yliopistonranta 1 E, FIN-70210 12 Kuopio, Finland 13 14 ---------- 15 16 Key words: Permafrost affected soil, global change, wetland, barcoded amplicon 17 pyrosequencing, quantitative PCR 18 19 Running title: Denitrifiers in cryoand unturbated peat 20 21 ---------- 22 23 * Corresponding author. Mailing address: Department of Ecological Microbiology, 24 Dr.-Hans-Frisch-Str. 1-3, University of Bayreuth, 95440 Bayreuth, Germany. Tel: (+49) 25 (0)921-555620. Fax: (+49) (0)921-555799. E-Mail: [email protected] 26 27 4.1 Published articles in peer-reviewed journals 131 2 Cryoturbated peat circles (i.e., bare surface soil mixed by frost action; pH 3-4) in the 1 Russian discontinuous permafrost tundra are nitrate-rich ‘hotspots’ of N2O-emissions in arctic 2 ecosystems, while adjacent unturbated peat areas are not. N2O was produced and 3 subsequently consumed at pH 4 in unsupplemented anoxic microcosms with cryoturbated 4 but not in those with unturbated peat soil. Nitrate, nitrite, and acetylene stimulated net N2O 5 production of both soils in anoxic microcosms, indicating denitrification as the source of N2O. 6 Up to 500 and 10 µM nitrate stimulated denitrification in cryoturbated and unturbated peat 7 soil, respectively. Apparent maximal reaction velocities of nitrite-dependent denitrification 8 were 28 and 18 nmol N2O gDW-1h-1, for cryoturbated and unturbated peat soil, respectively. 9 Barcoded amplicon pyrosequencing of narG, nirK/nirS, and nosZ (encoding nitrate, nitrite, 10 and N2O reductases, respectively) yielded ≈49 000 quality filtered sequences with an 11 average sequence length of 444 bp. Up to 19 species-level operational taxonomic units were 12 detected per soil and gene, many of which were distantly related to cultured denitrifiers or 13 environmental sequences. Denitrification associated gene diversity in cryoturbated and in 14 unturbated peat soil differed. Quantitative PCR (inhibition-corrected per DNA extract) 15 revealed higher abundances of narG in cryoturbated than in unturbated peat soil. Copy 16 numbers of nirS were up to 1000x higher than those of nirK in both soils, and nirS nirK-1 copy 17 number ratios in cryoand unturbated peat soil differed. The collective data indicate that the 18 contrasting N2O emission patterns of cryoturbated and unturbated peat soils are associated 19 with contrasting denitrifier communities. 20 21 4 Manuscripts 132 3 Introduction 1 Nitrous oxide (N2O) is a major ozone depleting substance in the atmosphere and the 2 third most important greenhouse gas on earth (Forster et al., 2007; Ravishankara et al., 3 2009). The global warming potential of N2O is 300-fold higher than that of CO2 on a 100 year 4 basis, and the atmospheric concentration of N2O increased from 270 ppb to 319 ppb from 5 1750 to 2005 (Forster et al., 2007). Agricultural and pristine tropical soils are well recognized 6 major sources of N2O, while the importance of arctic peatlands as sources of N2O is just 7 emerging (Denman et al., 2007; Repo et al., 2009; Marushchak et al., 2011). 8 Areas of bare surface soil mixed by frost action in acidic tundra (pH 3 to 4) are termed 9 cryoturbated peat circles, and emit N2O at rates documented for tropical and agricultural soils 10 (Werner et al., 2007; Maljanen et al., 2007; Repo et al., 2009). The estimated global N2O 11 emission from cryoturbated peat circles is about 0.1 Tg N2O y-1, which is equivalent to 4% of 12 the global warming potential of the arctic methane emissions and to 0.6% of the total global 13 annual N2O emission (Christensen, 1993; Denman et al., 2007; Repo et al., 2009). 14 Vegetation is absent from about 12% of the area in the arctic, including cryoturbated peat 15 circles (Walker et al., 2005). Nitrate concentrations approximate 2 mM in the pore water of 16 such unvegetated cryoturbated peat soil, and are approximately 1000 x higher than in 17 adjacent vegetated unturbated peat areas where N2O emissions are negligible (Repo et al., 18 2009). Repeated freezing and thawing of the cryoturbated soil leads to breakdown of soil 19 aggregates, renders decomposable organic carbon more easily accessible to microbes, and 20 may thereby activate the microbial community including N2O-producers (Mørkved et al., 21 2006; Sharma et al., 2006). Thus, cryoturbated peat circles represent acidic ‘hotspots’ of 22 microbial N2O emission in tundra (Repo et al., 2009; Marushchak et al., 2011). 23 The main source of N2O in water-logged anoxic soils including peatlands is 24 denitrification (Conrad, 1996; Pihlatie et al., 2004; Palmer et al., 2010). Complete 25 denitrification is the sequential reduction of nitrate or nitrite to dinitrogen (N2) via nitric oxide 26 (NO) and N2O; nitrite is likewise an intermediate when nitrate is utilized (Zumft, 1997). The 27 oxidoreductases involved in denitrification include dissimilatory nitrate reductases encoded 28 4.1 Published articles in peer-reviewed journals 133 4 by narG or napA, copperand cytochrome cd1-containing nitrite reductases (encoded by nirK 1 and nirS, respectively), NO reductases encoded by norBC, and N2O reductases encoded by 2 nosZ (Zumft, 1997). Nitrate reductases likewise occur in dissimilatory nitrate reducers (Stolz 3 & Basu, 2002). NirK and NirS are structurally different but functionally equivalent (Jones et 4 al., 2008). Organisms hosting both types of nitrite reductase are unknown to date (Heylen et 5 al., 2006). The genes coding for the above named oxidoreductases are commonly used as 6 structural gene markers for the analysis of nitrate reducer and denitrifier communities (e.g., 7 Bru et al., 2011; Enwall et al., 2010; Jones & Hallin, 2010; Palmer et al., 2010; Horn et al., 8 2006; Rich et al., 2003; Philippot et al., 2002; Prieme et al., 2002; Braker et al., 2000). Main 9 products of denitrification that are released into the atmosphere are N2 or N2O. Denitrifiers 10 might lack nitrate reductases and/or N2O reductases, and occupy diverse ecological niches 11 (Tiedje, 1988; Zumft, 1997; Shapleigh, 2006). Denitrification rates and the product ratio of 12 N2O to N2 are regulated by the denitrifying community and in situ conditions (e.g., pH, 13 temperature, C-to-N-ratio, as well as the availability of substrates and electron acceptors; 14 van Cleemput, 1998). Acidic pH < 5 impairs denitrification and increases the product ratio of 15 N2O to N2 (Simek & Cooper, 2002; Cuhel et al., 2010). The increased product ratio of N2O to 16 N2 is likely caused by post-transcriptional effects of low pH on N2O reductase formation (Liu 17 et al., 2010). However, information on denitrifier communities that thrive at pH < 5 in 18 peatlands is scarce (Palmer et al., 2010). 19 Denitrifier communities in permafrost-affected acidic tundra soils are unresolved to 20 date, despite the fact that such soils are prone to react sensitively to global warming, which 21 might accelerate cryoturbation and in turn increase N2O emissions (Bockheim, 2007; Repo et 22 al., 2009). It is hypothesized that the observed contrasting N2O emission patterns of 23 cryoturbated and unturbated acidic peat soil are associated with contrasting denitrifier 24 communities. The main objectives of the present study were (i) to compare ecophysiological 25 traits (i.e., capacities) of acid-tolerant denitrifier communities in cryoturbated and unturbated 26 peat soil, (ii) to develop pyrosequencing-based strategies for the in-depth analysis of 27 denitrifier communities by parallel analysis of multiple denitrification associated genes, (iii) to 28 4 Manuscripts 134 5 determine if contrasting and new denitrifier communities occur in cryoturbated and 1 unturbated peat soil by such pyrosequencing-based strategies and quantitative PCR, and (iv) 2 thus to identify potential microbial catalysts of the exceptionally high N2O emissions from 3 cryoturbated peat soil. 4 5 4.1 Published articles in peer-reviewed journals 135 6 Material and Methods 1 Site description and soil sampling 2 The sampling area is located in the Russian discontinuous permafrost zone (62°57'E, 3 67°03'N) and was described previously (Repo et al., 2009; Supplemental Material and 4 Methods). Cumulative N2O emissions in the field from the cryoturbated soil are 1.2 ± 0.3 g 5 N2O m-2, while those of the unturbated soil are negligible (<0.006 g N2O m-2). Topsoil was 6 identified as the site of highest N2O production in the peat profile (data not shown), and the 7 upper 5 cm were sampled from three cryoturbated peat circles and three adjacent, 8 unturbated areas in September 2010. Roots were removed from unturbated soil, and soil for 9 microcosm studies was stored at 4°C until further processing. Soil for DNA extraction was 10 suspended in RNAlater (Qiagen, Hilden, Germany) immediately after sampling to avoid 11 decomposition of nucleic acids, and stored at -20 °C upon arrival at the laboratory. 12 Experiments were conducted within 2 months after sampling. Moisture content was 13 determined by weighing the soil before and after drying at 60°C for 3 days and was 71% and 14 81% in cryoturbated and unturbated peat soil, respectively. 15 16 Assessment of denitrification in soil microcosms 17 Soil of the three replicate sampling sites was homogenized and pooled prior to 18 microcosm experiments. Soil slurries at in situ pHH2O of approximately 4 were prepared with 19 4-5 g of soil and 3 volumes of deionized water in 125-ml infusion flasks, and sealed with gas-20 tight rubber stoppers. The gasphase was 100% argon. Microcosms were incubated at 20 °C 21 in the dark and performed in triplicate unless stated otherwise. 22 Acetylene blocks nitrous oxide reductases and thus the reduction of N2O to N2 23 (Yoshinari & Knowles, 1976). Parallel microcosms with and without acetylene (15% [vol/vol] 24 in headspace) were used to differentiate between total denitrification and N2O-production 25 potentials. Total denitrification stopped after approximately 4 days (90 h) in unsupplemented 26 microcosms with cryoturbated peat soil and acetylene, indicating that internal nitrate and 27 nitrite were depleted (Figure 1 A). 28 4 Manuscripts 136 7 For apparent Michaelis-Menten kinetics, soil was pre-incubated for 7 days under 1 anoxic conditions to deplete internal nitrate and nitrite. Such soil was supplemented with 0 to 2 500 µM of NaNO3 or NaNO2. N2O did not accumulate in anoxic microcosms containing 1 mM 3 nitrite in sterile water at pH 4 within 2 days (data not shown). Apparent Michaelis-Menten 4 kinetics were based on the production of N2O in the presence of acetylene as described 5 (Segel, 1993; Palmer et al., 2010; Supplemental Material and Methods). Soil that was pre-6 incubated under anoxic conditions for 9 days was utilized to study the effect of the electron 7 donors acetate, ethanol, formate, propionate, butyrate, and lactate on denitrification in 8 microcosms supplemented with 1 mM nitrite and 0.5 mM of electron donors in the presence 9 of acetylene. After another 47 days of anoxic incubation, 1 mM nitrite and 2 mM of electron 10 donors (0.5 mM for propionate only) were resupplied. N2O production, nitrite and electron 11 donors were determined regularly after the initial supplementation and after 12 resupplementation. N2O production rates were calculated from 3-4 data points determined 13 within 8-25 h after addition of substrates (nitrite and/or electron donors) when N2O production 14 was linear. R2-values of the linear regressions were always greater than 0.88. 15 Concentrations of electron donors were assessed by high-performance liquid 16 chromatography, and nitrate as well as nitrite by ion chromatography (Palmer et al., 2010; 17 Supplemental Material and Methods). 18 19 Extraction of nucleic acids, and amplification of narG, nirK, nirS and nosZ 20 Nucleic acids were extracted from triplicate cryoturbated and unturbated peat soil 21 samples to account for lateral heterogeneity in microbial communities. A bead-beating 22 protocol tailored for the efficient removal of PCR-inhibiting humic acids by aluminum sulfate 23 precipitation prior to cell lysis was applied (Peršoh et al., 2008; Supplemental Material and 24 Methods). 25 narG, nirK, nirS, and nosZ were amplified using the primer pairs narG1960f (TAY 26 GTS GGS CAR GAR AA)/narG2650r (TTY TCR TAC CAB GTB GC; Philippot et al., 2002), 27 F1aCu (ATC ATG GTS CTG CCG CG)/R3Cu (GCC TCG ATC AGR TTG TGG TT; Throbäck 28 4.1 Published articles in peer-reviewed journals 137 8 et al., 2004), cd3aF (GTS AAC GTS AAG GAR ACS GG)/R3cd (GAS TTC GGR TGS GTC 1 TTG A; Throbäck et al., 2004), and nosZF (CGC TGT TCI TCG ACA GYC AG)/nosZR (ATG 2 TGC AKI GCR TGG CAG AA; Rich et al., 2003), respectively. Each primer was preceeded 3 by a 6 basepair-long barcode (AGCGTC for unturbated, and ATATAC for cryoturbated soil 4 samples) to separate sequences after pyrosequencing. Eight replicate 25 µl PCR reactions 5 per target gene were performed at 8 different annealing temperatures from 54.7 to 63.6 °C to 6 maximize the likelihood of detecting a high diversity of target genes. All replicate PCR 7 reactions that yielded products (i.e., amplicons) of the correct size were pooled prior to 8 subsequent analyses. For detailed PCR protocols refer to Supplemental Material and 9 Methods. 10 11 Barcoded amplicon pyrosequencing of structural genes 12 Previously published amplicon pyrosequencing strategies (Huber et al., 2007; Iwai et 13 al., 2010; Will et al., 2010) were modified to maximize the likelyhood of specific amplification 14 of denitrification associated structural genes during amplicon generation. Pyrosequencing 15 requires amplicons fused with sequencing adaptors. Published strategies utilize target gene 16 specific primers fused with a barcode and an approximately 30 bp long sequencing adaptor, 17 resulting in primers with more than 50% of the sequence being not complementary to the 18 target genes, and thus allowing for unspecific amplifications. In this study, amplicons were 19 generated during PCR with target gene specific primers fused with the barcode only (see 20 above) rather than utilizing primers that contain barcode and sequencing adaptors. 21 Sequencing adaptors were ligated after PCR to gel purified amplicons. 22 Amplicons of similar lengths of both soil types were combined in equal amounts (i.e., 23 narG and nosZ amplicons were pooled, as well as nirK and nirS). Amplicon mixtures were 24 treated with PreCR Repair Mix (New England Biolabs, Frankfurt am Main, Germany) to 25 eliminate possible PCR-blocking DNA damage that might have occurred during gel 26 purification or storage of amplicons, and purified via isopropanol precipitation. Sequencing 27 from 5’ (forward) and 3’ (reverse) ends of amplicons was performed after ligation of A (CGT 28 4 Manuscripts 138 9 ATC GCC TCC CTC GCG CCA TCA G) and B (CTA TGC GCC TTG CCA GCC CGC TCA 1 G) sequencing adaptors at the Göttingen Genomics Laboratory employing the Roche GS-2 FLX 454 pyrosequencer and GS FLX Titanium series reagents (Roche, Mannheim, 3 Germany) according to the manufacturer’s instructions. 4 5 Sequence filtering and analysis 6 Sequences with ambiguities, and those with incorrect primer or barcode sequences 7 were discarded. narG as well as nosZ sequences shorter than 350 bp, and nirK as well as 8 nirS sequences shorter than 300 bp were likewise excluded from further analyses. Amplicon 9 sequences were sorted according to their barcodes and primers, and combined subsets of 10 sequences for each structural gene (i.e., containing sequences from both cryoturbated and 11 unturbated peat soil) were clustered [i.e., assigned to operational taxonomic units (OTUs)] at 12 species-level threshold distances of 33% [narG (Palmer et al., 2009)], 17% [nirK, (Depkat-13 Jakob et al., unpublished)], 18% (nirS, Depkat-Jakob et al., unpublished), or 20% [nosZ 14 (Palmer et al., 2009)] based on DNA sequences using the JAguc2 pipeline 15 (http://wwwagak.informatik.uni-kl.de/research/JAguc/; Nebel et al. unpublished 16 (wwwagak.informatik.uni-kl.de/staff/nebel/www_jaguc/www_jaguc.pdf; Figure S1). In brief, 17 JAguc2 generates a pairwise sequence alignment prior to calculation of a distance matrix 18 and clustering with the average similarity method. This approach is more reliable [i.e., less 19 sensitive to PCRand pyrosequencing noise, and thus less sensitive to an artifical inflation of 20 diversity (Kunin et al., 2010)] than multiple alignments and/or clustering with complete 21 linkage algorithms (Sun et al., 2009; Quince et al., 2009; Huse et al. 2010). Amplicon 22 sequences obtained by pyrosequencing from defined template mixtures were essentially at 23 most 10% dissimilar to template sequences due to PCRand pyrosequencing noise (Quince 24 et al., 2011; Behnke et al., 2011). The threshold-distances utilized to call OTUs in this study 25 were 17-33%, which is substantially greater than the above reported maximal PCRand 26 pyrosequencing noise (Figure S1). Thus, our approach was rather unaffected by PCRand 27 pyrosequencing noise, although we did not apply flowgram-based sequence correction 28 4.1 Published articles in peer-reviewed journals 139 3.4 Effect of experimental drought on abundance of mcrA genes and transcripts in the acidic fen Before the experimental drought, detected mcrA gene copy numbers were about 1.7% and 4.1% of detected archaeal 16S rRNA gene copy numbers in control and treatment plots, respectively (Figure 5), indicating a higher relative abundance of methanogens in drought plots before the onset of experimental drought. At the end of the drought period, the relative abundance of mcrA genes was significantly lower in drought plots than before the onset of drought (1.5%), while it had remained rather similar in control plots (1.2%), suggesting that the relative abundance of methanogens was lowered by the experimental drought. After rewetting, relative abundances of mcrA remained in the same range in the control plots (1.4%), while the relative abundance of mcrA genes in the drought plots increased to approximately 3.1%, indicating that rewetting led to increased relative abundances of methanogens in fen soil. Figure 5: Effect of experimental drought on the abundance of mcrA genes and transcripts. Black bars represent control plots, white bars represent drought plots. Mean values of three plots and four soil layers per plot type and standard errors are shown. t0=before drought (June 09th 2008), t1=after drought (July 27th 2008), t2=after rewetting (August 11th 2008). 4 Manuscripts 242 20 to 30 cm soil from both plot types yielded the highest relative abundances of mcrA (Figure 5). Experimental drought decreased the relative abundance of mcrA in 20 to 30 cm depth-soil. After rewetting, relative abundance of mcrA amounted to 10.4% in the drought plots, in the control plots they were about 3.3%. In the other soil layers relative mcrA gene copy numbers were below 5%, and changes in the relative abundance of mcrA were less pronounced than in the soil layer from 20 to 30 cm, indicating that this soil layer was more sensitive to experimental drought than the other soil layers. Transcript to gene ratios of mcrA (i.e., ratios of mcrA copies obtained from cDNA to mcrA copies obtained from DNA) were highly variable between plots and timepoints, ranging from 0.5% to 500%. The average transcript to gene ratio of mcrA (i.e., the average of all 3 plots and all 4 soil depths) was about 50% in both control and drought plots before the onset of drying (Figure 5). At the end of the drought period, the transcript to gene ratio of mcrA was about 100% in both plot types, indicating increased activity of fen methanogens in both plot types. In the control plots, the transcript to gene ratio of mcrA stayed at about 100% at the time after rewetting, while in the drought plots the expression dropped again to 50%, indicating a reduced activity of methanogens in drought plots. Transcript to gene ratios of mcrA were highest in the soil layers from 0 to 20 cm in both plot types (14 to 260%), and lowest in 30 to 40 cm soil (3 to 7%) (Figure 5), indicating a higher acitivity of methanogens in the upper soil layers. 3.5 Effect of experimental drought on the community composition of methanogens The effect of the experimental drought on the community composition of fen methanogens was assessed by mcrA TRFLP analysis from all plots on gene and transcript level. On gene level, 8 to 10 TRFs were found in each sample (Figure 6). The same TRFs were found on transcript level with the exception of the 230 bp TRF, which was absent in the transcript level TRFLP profiles. The comparison of the average gene-level TRFLP profiles (i.e., the average of all sampled soil depths from all three plots of a plot type) in control and drought plots revealed slight differences in the community composition of fen methanogens at the different timepoints and between the two plot types (Figure 6 A). Differences in mcrA community composition before the onset of experimental drought and after the drought period were observed in the drought plots, while mcrA community composition at those two timepoints was more similar in the control plots. This indicated a slight effect of 4.2 Manuscripts in preparation 243 Figure 6: Effect of experimental drought on the community composition of mcrA genes (A) and transcripts (B). Comparative TRFLP analysis of mcrA amplified from DNA and cDNA of control and drought plots. PCR products were digested with Hinf I. Combined relative abundances of the major phylogenetic groups are shown in color (right bars), relative abundances of individual TRFs are in black/white (left bars). Mean values of three plots and four soil layers per plot type are shown. t0=before drought (June 09th 2008), t1=after drought (July 27th 2008), t2=after rewetting (August 11th 2008). experimental drought on the community structure of fen methanogens. Seven major TRFs (i.e, TRFs with a relative abundance >1%) were observed in both plot types before the experimental drought. The community was dominated by TRFs indicative of 4 Manuscripts 244 Methanocellaceae, i.e., TRFs of 200 bp length (indicative of OTU 1 and 6), 586 bp length (indicative of OTU 2), and 638 bp length (indicative of OTU 2 and 7), with a combined relative abundance of 57% and 47% in control and treatment plots, respectively. After the drought period, the combined relative abundance of those TRFs was 53% and 37% in control and treatment plots, respectively, indicating a drought induced shift in community composition. Moreover, the relative abundance of TRFs indicative for Methanomicrobiales/Methanobacteriales, i.e., the 305 bp and >760 bp TRFs (indicative of OTUs 4 and 5) increased in the treatment plots, and 2 additional TRFs were detected in the control plots (64bp and 230 bp length, indicative of OTU 3 and 4 and of OTU 8, respectively) and one in the drought plots (64bp length). After rewetting, TRFLP profiles of control and treatment plots were again similar to each other, with combined relative abundances of TRFs indicative of Methanocellaceae (i.e., TRFs of 200, 586 and 638 bp length) of 27% and 35% in control and treatment plots, respectively, and increased abundances of TRFs indicative for Methanomicrobiales/Methanobacteriales (i.e., TRFs of 305 and >760 bp length) of 40% and 39%, respectively. TRFs indicative of Methanosarcinaceae (i.e., TRFs of 230 and 466 bp lengths) had similar relative abundances in control and drought plots at all timepoints, equaling 14-15% before and after the drought period and 20-23% after rewetting. This indicates that (i) a shift in community composition occured in both control and drought plots throughout the experimental period, (ii) drought reduced the relative abundance of Methanocellaceae, (iii) rewetting might equalize differences in community composition that were observed after the drought period, and (iv) Methanosarcinaceae are least affected by differences between the two plot types. At the transcript level, 8 major TRFs (i.e, TRFs with a relative abundance >1%) were found in the TRFLP profiles of control and treatment plots (Figure 6 B). Community composition of mcrA transcripts differed between control and treatment plots before the onset of drought. The combined relative abundances of TRFs indicative of Methanocellaceae (i.e, the 200 bp, 586 bp, and 638 bp length TRFs) amounted to 36% and 50% in control and drought plots, respectively, and the relative abundance of TRFs indicative of Methanomicrobiales/Methanobacteriales (i.e., TRFs of 305 and >760 bp length) was twice as high in control as in drought plots (36% and 18%, respectively). After the drought period, combined relative abundances of TRFs indicative of Methanocellaceae were 39% and 49% in control and drought plots, respectively. The relative abundance of TRFs indicative of Methanomicrobiales/Methanobacteriales was 21% and 34% in control and drought plots, respectively, after the experimental drought. The relative 4.2 Manuscripts in preparation 245 abundance of TRFs indicative of Methanosarcinaceae increased in the control plots from 12% to 30%, while it decreased in the drought plots from 28% to 16%. This indicates that the expression of mcrA (i) of Methanocellaceae is rather stable under experimental drought conditions, (ii) of Methanomicrobiales/Methanobacteriales is upregulated as a result of drought conditions, and (iii) of Methanosarcinaceae is downregulated as a result of drought conditions. After rewetting, Methanosarcina-related TRFs had the highest relative abundance in both plot types (45% and 38% in control and drought plots, respectively). The relative abundance of Methanocellaceae-related TRFs was low in both plot types after rewetting (17% and 28% in control and drought plots, respectively), while the relative abundance of Methanomicrobiales/Methanobacteriales-related TRFs was around 30% in both plot types. The data indicates (i) that rewetting restored similar expression patterns in both plot types and (ii) a shift in the active methanogenic community from Methanocellaceae in early summer to Methanosarcinaceae in late summer. TRFLPs profiles on transcript level differed from TRFLP profiles on gene level (Figure 6). Methanosarcina-related TRFs generally had a higher relative abundance on transcript than on gene level, while Methanomicrobiales/Methanobacteriales-related TRFs were generally more abundant on gene than on transcript level. The ratios of relative abundances of TRFs indicative for certain phylogenetic groups on gene and transcript level indicated that Methanosarcinaceae were inhibited by experimental drought, while Methanomicrobiales/Methanobacteriales were stimulated. 4 Manuscripts 246 4 Discussion 4.1 Effect of drying and rewetting on the methanogenic community in the acidic fen In the course of climate change, a higher frequency of more extreme weather events like prolonged drought periods or heavy precipitation events are anticipated [61]. Prolonged drought reduces methanogenic activity in the fen Schl¨oppnerbrunnen [33]. This is attributed to regeneration of electron acceptors due to higher soil aeration [33]. Thus, when conditions become anoxic methanogenesis competes with other anaerobic redox processes for electrons [8]. Indeed, after experimental drought methanogenic potentials of 0 to 10 and 30 to 40 cm soil were lower and the initial lag phases were longer in drought than in control plots (Figure 1). In an acidic fen soil in Finland, reduced CH4 emissions are observed in drained sites as compared to the wetter sites along a drainage gradient near a groundwater extraction plant [67]. Reduced CH4emissions from drained wetlands are also caused by methanotrophy occuring when O2penetrates into deeper soil layers [67]. In mesocosm studies with Schl¨oppnerbrunnen fen soil, CH4emissions decrease due to methanotrophy in more aerated parts above the water table when the water table is experimentally lowered, while the methanogenic pathways are mainly unaffected [32]. Methanotrophic activity has also been detected in microcosm studies with soil from Schl¨oppnerbrunnen fen, and methanotrophic taxa are detected in the fen soil [64]. After rewetting of peatland soil, recovery of methanogenesis is ofter delayed as a result of increased availability of alternative electron acceptors [7, 32, 33]. Methanogenesis is controlled by in situ parameteres such as temperature and the level of the ground water table [5, 31, 32, 57]. A water table drawdown like the one achived during the experimental drought results in higher soil aeration and a rise concomitant rise in the redox potential [33]. Methyl-coenzyme M reductase is inactivated during periods of raised O2concentrations [28]. Decreased abundances of methanogens were observed in drought plots after the drought period (Figure 5), however, the composition of the methanogenic community was not significantly affected. Detected mcrA were mainly affiliated with mcrA of Methanocella paludicola (Methanocellaceae), representative of ’Rice Cluster I’ [55], moreover mcrA affiliated with mcrA of the Methanomicrobiales, Methanobacteriaceae and Methanosarcinaceae were detected (Figure 3, Figure 4). Even though small changes in community composition were observed (Figure 4, Figure 6), the relative abundances of the detected groups were not significantly affected by experimental drought. This is in good agreement with studies from rice field soils, where 4.2 Manuscripts in preparation 247 drainage and O2exposure reduce the abundance of methanogens while the community composition of methanogens is not affected [38, 39, 68]. Thus, the observed changes in the methanogenic potential of fen soil after drying and rewetting might be caused by reduced abundances of methanogens. Transcript to gene ratios of mcrA ranged from 50 to 110% in fen soil (Figure 5) and were thus of a similar magnitude as ratios of mcrA in British peatlands [13]. However, this ratio might be underestimated due to dead or inactive methanogens [13]. mcrA expression was similar in drought and control plots after the drought period (Figure 5). Peat methanogens maintain high abundances of mcrA transcripts, even when their activity is low, and similar gene and transcript abundances have been detected in peat soil with methanogenic potential reduced due to long-term soil storage in the lab [13]. Thus, lower methanogenic potentials after prolonged experimental drought are likely caused by post-transcriptional effects on mcrA, and might also be affected by other parameters than O2. Moreover, methanogens as well as copies of mcrA were also found in the upper soil layers in the acidic fen Schl¨oppnerbrunnen, even though more oxic conditions are found there troughout the year [23]. Methanogens are also detected in other rather oxic systems such as desert soils [50], and minor CH4production and emission is known from oxic upland soils [27, 66]. Reduced viability of methanogens is attributed to the effect of desiccation rather than to the effect of O2itself [10]. The mechanism of the survival or even growth under oxic conditions remains unresolved, since there is no evidence for the existence of resting stages in methanogens [10]. However, enzymatic protection against oxidative stress by the enzymes superoxide dismutase and katalase has been reported in methanogenic species like Methanobacterium bryantii and Methanobacterium thermoautotrophicum [29, 60]. Some mcrA sequences obtained from the acidic fen were related to those species, and it seems thus likely that they also harbor the capacities to produce protective enzymes. Additionally, wet and anoxic microenviroments in drought plots might protect methanogens from O2during drought periods [32]. However, O2inhibited methanogenesis in fen soil microcosms even at very low O2concentrations (Figure 2), thus it is likely that fen soil methanogens are inactive during periods of drought stress. The composition of mcrA transcripts was more variable than that of mcrA genes (Figure 6). However, differences were more pronounced between the different sampling timepoints than between the two plot types, even though transcript to gene ratios of TRFs suggest inhibitory and stimulatory effects of experimental drought on Methanosarcinaceae and Methanomicrobiales/Methanobacteriales, respectively. O2exposure alters the composition of mcrA transcripts in rice field soil [69]. In August, a 4 Manuscripts 248 greater abundance of Methanosarcina-related mcrA transcripts than in June or July was detected, indicating higher activity of Methanosarcina-related methanogens in late summer. Methanogens of the orders Methanobacteriales,Methanomicrobiales, and Methanocellales use hydrogenotrophic methanogenesis for energy conservation [55, 63], while Methanosarcinales conserve energy via acetoclastic and hydrogenotrophic methanogenesis [43]. It is thus feasible that the contribution of acetoclastic methanogenesis is variable troughout the year and is of greater influence in the late summer. 4.2 Conclusions and limitations Future climate change will likely increase the frequency of drought events in peatlands [61]. Experimental drought in the acidic fen Schl¨oppnerbrunnen decreases methane emissions [33], and reduced methanogenic potentials were observed in microcosm studies with fen soil. The abundance of methanogens was reduced in the drought plots while the composition of the methanogenic community was rather stable throughout the drought experiment based on analyses of mcrA gene and transcript abundances and TRFLP profiles. Unfortunately, owing to the experimental design, the effect of experimental drought on methanogenic potentials in microcosm incubations and on the abundance and community structure of fen methanogens were studied in two different years. It is therefore not possible to directly link the observed effects of experimental drought on process level to the observed changes in the methanogenic community. Within these limitations the data collected in this study nonetheless indicate that experimental drought (i) impacts on methanogenic potentials in the acidic fen, and (ii) moreover influences the abundance but not the community composition of fen methanogens. Further investigation would be needed to establish the link between observed effects on methanogenic potential and observed effects on community composition of fen methanogens. 5 Acknowledgments Support for this study was provided by the Deutsche Forschungsgemeinschaft (DFG HO 4020/2-2). Work was also supported by the DFG Research group FOR 562. 4.2 Manuscripts in preparation 249 References [1] Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. Basic local alignment search tool. Journal of Molecular Biology 215, 403–410 (1990). [2] Bapteste, E., Brochier, C., and Boucher, Y. Higher-level classification of the Archaea: evolution of methanogenesis and methanogens. Archaea 1, 353–363 (2005). [3] Blodau, C., Basiliko, N., and Moore, T. Carbon turnover in peatland mesocosms exposed to different water table levels. Biogeochemistry 67, 331–351 (2004). [4] Clymo, R. S. The ecology of peatlands. Science Progress 71, 593–614 (1987). [5] Conrad, R. Soil microorganisms as controllers of atmospheric trace gases (H2, CO, CH4, OCS, N2O, and NO). Microbiological Reviews 60, 609–640 (1996). [6] Denman, K. L., Brasseur, G., Chidthaisong, A., Ciais, P., Cox, P. M., Dickinson, R. 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Biogeosciences 7, 3893–3900 (2010). [17] Gorham, E. Northern peatlands - role in the carbon-cycle and probable responses to climatic warming. Ecological Applications 1, 182–195 (1991). [18] Griffiths, R. I., Whiteley, A. S., O’Donnell, A. G., and Bailey, M. J. Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNAand rRNA-based microbial community composition. Applied and Environmental Microbiology 66, 5488–5491 (2000). 4.2 Manuscripts in preparation 251 6 Supplemental Figures Figure S1: Effect of difluoromethane on methanogenesis and methanotrophy in fen soil microcosms. Mean values and standard errors of three replicate microcosms are shown. Squares represent anoxic microcosms, circles represent oxic microcosms. Open symbols represent microcosms without difluoromethane, closed symbols represent microcosms with difluoromethane. 4 Manuscripts 258 Figure S2: Correlation of DNA sequence similarities of mcrA versus 16S rRNA gene similarity. Dotted lines represent the similarity values, below which two sequences always had less than 97% 16S rRNA gene sequence similarity. The dashed lines represent the 90% quantile of pairwise sequence comparisons with a 16S rRNA gene sequence similarity of 97% (i.e., threshold similarity). The solid lines mark the 97% 16S rRNA gene similarities. 4.2 Manuscripts in preparation 259 Denitrifier communities in an acidic fen are stable during experimental drought Manuscript in preparation Katharina Palmer, Harold L. Drake, Marcus A. Horn Department of Ecological Microbiology, University of Bayreuth, 95440 Bayreuth, Germany 4 Manuscripts 260 Wetlands can act as sources or sinks of the greenhouse gas nitrous oxide (N2O). The acidic fen Schl¨oppnerbrunnen emits denitrification derived N2O and is also capable of N2O consumption. Global warming is predicted to cause more extreme weather events in future years, including prolonged drought periods, which will influence the denitrifier community in the acidic fen. Thus, the abundance and community composition of denitrifiers and their reaction to enhanced drought were investigated. Pyrosequencing of the structural gene markers narG,nirK,nirS, and nosZ revealed a high denitrifier diversity and 6, 13, 18 and 6 species-level operational taxonomic units (OTUs). Detected species-level OTUs were mainly related to Actinobacteria, Alpha-,Beta-, and Gammaproteobacteria. An experiment to simulate prolonged drought was conducted in summer 2008 by experimentally lowering the water table in 3 drought plots for 8 weeks followed by subsequent rewetting of the drought plots. 3 undrained plots served as controls. Samples were taken before and after drought and after rewetting. narG and nosZ were used to see assess changes in the nitrate reducer and denitrifier community to experimental drought. Higher copy numbers of narG and nosZ (2x and 1.5x, respectively) were detected after the experimental drought in drought but not in control plots by quantitative PCR (qPCR), indicating that nitrate reducers and denitrifiers are positively influenced by the drought treatment. Rewetting restored the copy numbers of narG and nosZ to the original level. Terminal restriction fragment length polymorphism (TRFLP) patterns of narG and nosZ were similar before and after the drought period, indicating a high stability of nitrate reducers and denitrifiers in the fen. The collective data indicate that (i) a high denitrifier diversity exists in the acidic fen Schl¨oppnerbrunnen, (ii) experimental drought increases denitrifier abundances, and (iii) the community composition is unaffected by experimental drought. 1 Introduction Extreme weather events such as prolonged drought periods or heavy rainfalls are predicted to increase in frequency as well as in intensity in future years due to global warming [38]. This will affect the water table in wetland soils and thus many biological processes in those soils, including processes involved in the turnover of nitrous oxide 4.2 Manuscripts in preparation 261 (N2O) [12, 33, 39]. N2O is a major greenhouse gas with a global warming potential that is 300 times higher than that of CO2and the major ozone-depleting substance in the atmosphere [9, 32]. The atmospheric concentration of N2O increased from 270 ppb to 319 ppb in the years 1750 to 2005 [9]. Soils are major sources of N2O, which can be nitrificationor denitrification derived [6, 7, 30]. Moreover, such wetland soils can be sources or sinks (transient or permanent) of atmospheric N2O [11, 10, 27, 36]. In water-logged wetland soils including fens denitrification is the main source of N2O [6, 27, 30]. Denitrification is the sequential reduction of nitrate or nitrite to N2via nitric oxide (NO) and N2O [41]. The denitrification processes is catalyzed by the enzymes nitrate reductase Nar or Nap (encoded by narGHJI or napEDABC, respectively), copperor cytochrome-dependent nitrite reductases NirK or NirS (encoded by nirK or nirS, respectively), NO recutase Nor (encoded by norBC ), and N2O reductase Nos (encoded by nosZ) [41]. Nitrate reductases are moreover found in many non-denitrifying nitrate-reducing microorganisms [41]. narG,nirK,nirS, and nosZ are structural gene markers commonly used in the analysis of nitrate reducer and denitrifier community composition [4, 15, 24, 29]. narG and nosZ from the acidic fen Schl¨oppnerbrunnen were studied earlier by conventional cloning based sequencing, revealing novel nitrate reducers and denitrifiers in the acidic fen [27]. However, new sequencing methods including amplicon pyrosequencing allow more in-depth analysis of nitrate reducers and denitrifier communities. Thus, one aim of the present study was to reassess the diversity of narG and nosZ from Schl¨oppnerbrunnen fen soil along with the diversity of the nitrite reductase encoding nirK /nirS using amplicon pyrosequencing. In the context of global warming, more extreme drought periods are expected to occur, which will effect water tables in fen soils [38]. However, it is so far unresolved how fen nitrate reducer and denitrifier communities react to changing water tables. Thus, a further aim of the study was to assess the effect of simulated drought conditions on nitrate reducer and denitrifier abundance and diversity in acidic Schl¨oppnerbrunnen fen soil to gain a better understanding of possible feedback effects of global warming. 4 Manuscripts 262 2 Material and Methods 2.1 Study site and experimental setup The minerotrophic fen Schl¨oppnerbrunnen is located in the Lehstenbach catchment in the Fichtelgebirge, Bavaria, Germany (50◦07’ 53” N, 11◦52’ 51” E), at approximately 700 m above sea level. The sampling site is described in more detail in [14, 25, 27]. In 2008, the water table was artificially lowered in 3 treatment (i.e., drought) plots (size 7.2 m x 5 m). Roofs were erected to shield the plots from rain water, and additional drainage was accomplished by ditches. The roofs had large open side walls to minimize temperature and wind speed effects. Additionally, 3 untreated plots served as control sites. Roofs and drainage on the treatment plots were installed on June 10th 2008 and kept in place until August 7th. On average, experimental drought lowered the water table to 0.60 m below the surface. After the experimental drought period, roofs and drainage were removed to allow rewetting of the drought plots. For more detailed description of the experimental setup refer to [25]. Soil was sampled for molecular analyses before the start of the drought period (June 09th), at the end of the experimental drought period (July 27th), and 4 days after rewetting (August 11th). Soil samples were taken from each plot from soil depths 0 to 40 cm at 10-cm intervals with a soil corer. Soil samples were quick-frozen in liquid nitrogen and stored at -80◦C until further processing. 2.2 Extraction of nucleic acids Nucleic acids from all sampled timepoints, plots, and soil layers were extracted using a bead-beating protocol [13] followed by separation of DNA and RNA using the Qiagen RNA/DNA Mini Kit (QIAGEN GmbH, Hilden, Germany) to the manufacturer’s instructions. 2.3 Barcoded amplicon pyrosequencing of narG, nirK, nirS, and nosZ narG,nirK,nirS, and nosZ were amplified from pooled DNA extracts (i.e., from all timepoints, plots, and soil layers) using published primers tagged with barcodes, purified and pyrosequenced as previously described [26]. Quality filtering of sequences included denoising (i.e., removal of pyrosequencing and PCR-amplification errors by PyroNoise and SeqNoise algorithms [31]) as described [28]. Denoised sequence data sets 4.2 Manuscripts in preparation 263 were grouped into species-level operational taxonomic untis (OTUs) at threshold similarities of 67%, 83%, 82%, and 80% for narG,nirK,nirS, and nosZ, respectively. For narG and nosZ only sequences obtained from forward reads were used, as mean sequence lenghts of approximately 400 bp yielded to little overlap of forward and reverse reads, while forward and reverse reads of nirK and nirS were combined [26]. OTU representatives were aligned with sequences of cultured and uncultured references (determined by BLAST analysis [1]) using ClustalW and phylogenetic trees were constructed using the neighbor joining algorithm [34] based on pairwise sequence distances in MEGA 5.0 [22]. The stability of tree topologies was tested using the bootstrap method with 10,000 replications. 2.4 Quantitative PCR Quantitative PCR (qPCR) of narG and nosZ was modified from [26]. Reactions were set up in triplicate reactions per DNA extract. The forward primers were fluoresecently labeled to allow the use of the products from qPCR in downstream TRFLP analyses. This approach allows direct coupling of quantification and analysis of community composition of a specific gene marker. Inhibition of qPCR assays by contaminating humic substances was conducted as described in [26]. 2.5 Terminal restriction fragment length polymorphism analysis (TRFLP) Triplicate qPCR reactions of narG and nosZ were pooled and gel purified using the Montage Gel Extraction Kit (Millipore Corporation, Bedford, MA, USA) prior to TRFLP analysis. The purified PCR products were digested with Mung Bean Nuclease (New England Biolabs, Frankfurt am Main, Germany) to remove single stranded DNA and reduce the probability of pseudo-terminal restriction fragments [8]. The restricted DNA was purified using the Millipore Multiscreen 96-well Filtration System (Millipore Corporation, Bedford, MA, USA). PCR products of narG were digested with the restriction enzyme CfoI, nosZ PCR products were digested with Fnu4HI. Polyacrylamide gel electrophoresis was performed as described previously [27]. Terminal restriction fragments (TRFs) were assigned to narG and nosZ sequences via in silico TRF analysis with sequences obtained from the fen Schl¨oppnerbrunnen [27]. 4 Manuscripts 264 3 Results 3.1 Diversity of denitrification-associated genes in Schl¨oppnerbrunnen fen soil Diversity of Schl¨oppnerbrunnen fen denitrifiers was assessed by barcoded amplicon pyrosequencing of denitrification-associated genes. Amplicon pyrosequencing of narG forward reads yielded 1,325 quality filtered (i.e., denoised) sequences. Sequences were grouped into 6 species-level OTUs, 3 OTUs had relative abundances greater than 1%. Of those 3 OTUs, OTU 1 had a relative abundance of 71% and thus dominated detected narG in Schl¨oppnerbrunnen fen soil (Figure 1). Representative sequences of OTU 1 affiliated with the Actinobacteria and were distantly related to narG of Actinosynnema mirum and Streptomyces coelicolor (Figure 1). OTU 2 accounted for 22% of narG, affiliated with the Gammaproteobacteria, and was related to narG of Pseudomonas spp. (Figure 1). OTU 3 accounted for 6% of narG and was distantly related to the Deinococcal genera Thermus,Oceanithermus, and Marinithermus (Figure 1). 2,401 quality filtered sequences were obtained for nirK. Sequences grouped into 13 species-level OTUs, and 7 OTUs had a relative sequence abundance greater than 1% (Figure 2). Those OTUs occured with relative abundances of 55%, 21%, 11%, 7%, 2%, 2%, and 1% (OTUs 1, 2, 3, 4, 5, 6, and 7, respectively; Figure 2). Sequences affiliated with Proteobacterial nirK (Figure 2). OTU representatives of OTUs 1 and 6 were distantly related to nirK of Enterococcus sp., OTUs 3 and 5 were related to nirK of Bradyrhizobium sp., OTU 2 was related to Rhizobial nirK, while OTUs 4 and 7 did were not clearly related to published nirK (Figure 2). 334 quality filtered sequences were obtained for nirS which grouped into 16 specieslevel OTUs. 6 OTUs had relative sequence abundances greater than 1%. OTUs 1 and 2 dominated nirS in Schl¨oppnerbrunnen fen soil with relative abundances of 52% and 33%, respectively, while OTUs 3, 4, 5, and 6 were detected with relative abundances of 4%, 3%, 2%, and 2%, respectively (Figure 3). OTU representatives affiliated with Alpha-,Beta-, and Gammaproteobacteria. Representative sequences of OTUs 1 and 2 were related to nirK of Bradyrhizobium sp., while representative sequences of OTUs 3, 4, 5, and 6 were related to Pseudomonas spp., Ralstonia sp., Thiobacillus denitrificans, and T. denitrificans, respectively (Figure 3). 384 quality filtered forward read sequences were obtained for nosZ which grouped into 7 species-level OTUs, of which 3 had relative sequence abundances greater than 1%. OTU 1 dominated nosZ in Schl¨oppnerbrunnen fen soil with relative abundances 4.2 Manuscripts in preparation 265 Figure 1: Phylogenetic tree of narG OTU representatives. Sequences were retrieved from pooled DNA extracts. The tree is based on in silico tranlated amino acid sequences. OTUs were calculated at a species-level threshold similarity of 67% from a total of 1,325 forward read sequences. Only OTUs that contain ≥1% of sequences are displayed, relative abundances of OTUs are given in parentheses. Codes preceeding sequence names represent sequence accession numbers of reference sequences in public databases. Grey boxes indicate branches were the majority of the sequences belong to a given phylogenetic group (indicated on the right). The percentage of replicate trees that produced the same clustering in the bootstrap analysis (10,000 replications) are shown next to the branches, bootstrap support values below 50% are omitted. The tree was rooted using narG of Haloarcula marismortui ATCC 43049 as the outgroup (NC 006396). of 92%, while OTUs 2, and 3 were detected with relative abundances of 6%, and 1%, respectively (Figure 4). OTU representatives affiliated with Alphaproteobacterial nosZ. Representative sequences of OTUs 1, 2, and 3 were related to nosZ of Achromobacter sp., Mesorhizobium sp., and Azospirillum sp., respectively (Figure 4). 4 Manuscripts 266 Figure 2: Phylogenetic tree of nirK OTU representatives. Sequences were retrieved from pooled DNA extracts. The tree is based on in silico tranlated amino acid sequences. OTUs were calculated at a species-level threshold similarity of 83% from a total of 2,401 sequences. Only OTUs that contain ≥1% of sequences are displayed, relative abundances of OTUs are given in parentheses. Codes preceeding sequence names represent sequence accession numbers of reference sequences in public databases. Grey boxes indicate branches were the majority of the sequences belong to a given phylogenetic group (indicated on the right). The percentage of replicate trees that produced the same clustering in the bootstrap analysis (10,000 replications) are shown next to the branches, bootstrap support values below 50% are omitted. The tree was rooted using nirK of Alcaligenes sp.CJANPY1 as the outgroup (EF202175). 4.2 Manuscripts in preparation 267 4 Discussion 4.1 Detected diversity of nitrate reducers and denitrifiers in acidic Schl¨oppnerbrunnen fen soil Up to 16 species-level OTUs of denitrification-associated genes were detected in the acidic fen Schl¨oppnerbrunnen (Figure 1, Figure 2, Figure 3, Figure 4), indicating a high species-level diversity of fen denitrifiers. OTUs of detected narG affiliated with Actinobacteria,Gammaproteobacteria, and Deinococci (Figure 1). Earlier cloning-based studies of narG from Schl¨oppnerbrunnen fen soil also indicate the presence of the mentioned groups, however also Alphaand Betaproteobacterial narG are detected by cloning-based approaches, indicating that the detected diversity varies between the 2 approaches, likely because of the shorter fragment length obtained from amplicon pyrosequencing as well as by variability in DNA extraction. The noise-removal algorithms employed in the present study remove errors caused by pyrosequencing (PyroNoise) as well as by PCR-amplification (SeqNoise) [31]. As PCR-amplification errors affect conventional sequencing and pyrosequencing in a simialr magnitude, it is moreover feasible that earlier cloning based sequencing efforts overestimate the genetic diversity of narG because of those PCR-amplification errors. Sequences related to Actinobacterial narG dominated amplicon libraries (Figure 1). Actinobacterial predominance is observed in many other wetland soils including peatland soils [26, 28, 29]. Sequences of nirK and nirS affiliated with sequences of Alpha-,Betaand Gammaproteobacterial nitrite reductases (Figure 2,Figure 3). nirK was dominated by sequences distantly related to Enterococcus sp., indicating that denitrifiers harboring new NirKtype nitrite reductases occur in Schl¨oppnerbrunnen fen soil. nirS was dominated by sequences related to Bradyrhizobium sp., indicating the presence of this common soil bacterium in Schl¨oppnerbrunnen fen soil. Detected nosZ affiliated with Alphaproteobacterial nosZ, confirming the results of earlier cloning based sequencing efforts [27]. 4.2 Relative abundance of denitrifiers in the acidic fen The relative narG gene copy number ranged between 6 and 13% (Figure 5 A). The detected narG copy numbers are in the same range as narG copy numbers in permafrost affected peat peat soil in Russia, agricultural soils of the Burgundy region in France, freshwater habitats such as rivers and floodplains as well as glacier forelands [2, 3, 19, 26] In some agricultural soils lower narG copy numbers of about 1% or even 4 Manuscripts 274 lower are detected [3, 18]. This indicates a high abundance of nitrate reducers in the fen Schl¨oppnerbrunnen. Indeed, nitrate-dependent MPNs show similar numbers of nitrate reducers in the acidic fen [27]. On the other hand, relative nosZ copy numbers ranged from 0.05% to 3% (Figure 5 C), indicating that about 1% to 10% of nitrate reducers in Schl¨oppnerbrunnen fen soil possess the genetical potential for complete denitrification. Indeed, culture-dependent studies also reveal a much higher proportion of nitrate reducers than of denitrifiers in soils [5], and many culture-independent studies also show that nitrate reducers are about 100-fold than denitrifiers [2, 26]. Denitrifier numbers from MPN counts from the acidic fen approximated 0.02% of the total cell count [27]. The detectability of denitrifiers by quantitative PCR is therefore slightly higher than the detectability by MPN counts. In glacier foreland soils relative nosZ gene copy numbers ranged from about 0.02% to about 1% [19], the denitrifier abundance in the acidic fen is therefore in the same range as in other soils or even higher. 4.3 Effect of drying and rewetting on denitrifier communities in the acidic fen Climate change models predict a higher frequency of extreme weather events, including prolonged periods of drought [38]. Experimental drought increases fluxes of N2O from the fen, especially when a significant drawdown of the water table is obtained [10]. Subsequent wetting of the soil increases N2O emission from previously drained fen soil [10]. The increase in N2O production after rewetting may be attributed to regeneration of electron acceptors such as nitrate due to higher soil aeration during the preceeding drought [21]. N2O production is most strongly affected in the top soil of the fen [10]. Long-term water table drawdown does not lead to a strong increase in the yearly N2O emissions in nutrient-poor peatlands, while it increases N2O emissions from nutrient-rich peatlands [23], but short-term drainage of peatland mesocosms leads to increased N2O emission from the soil [33]. Short-term drainage mostly effects upper soil layers of fen soil and leads to a more similar microbial community composition in different soil layers [16]. Water table drawdown in wetlands does not affect the community composition of denitrifiers, even though pronounced impacts on the denitrification process are observed [20, 35]. Actinobacterial community composition in a mesotrophic pine fen is relatively drought-resistant, while drought increases the overall biomass and C-turnover [17]. Indeed, the community composition of narG did not change during the drought treatment (Figure 6 A), as the narG community in the fen Schl¨oppnerbrunnen is dominated by 4.2 Manuscripts in preparation 275 Actinobacterial narG [27]. The response of denitrifier abundance to drought differs between different wetland types, ranging from a decrase in abundance in bogs and fens to no changes in riparian wetlands and marshes [20, 35]. Indeed, the observede increase in nosZ abundance was smaller than the increase in narG abundance, suggesting that nitrate reducers are favoured more strongly by drought conditions than denitrifiers. Denitrifiers are facultative aerobes that generally prefer oxygen as a terminal electron acceptor [41]. Thus, the growth of denitrifiers is likely favoured when lowering of the water table allows the diffusion of oxygen into the soil. However, denitrification can also occur in the presence of oxygen, and some organisms simultaneously use nitrate or oxygen as terminal electron acceptor [37]. Warming experiments in arctic soil show that denitrifier community composition is more strongly influenced by site characteristics than by warming, even though warming did not lead to significant changes in soil water content [40]. Moreover, denitrifier community composition in salt marshes and riparian wetlands is not significantly affected by water table alterations [20, 35]. Long-term water table drawdown in boreal peatlands does not significantly affect Actinobacterial community composition [17]. Indeed, most detected narG from Schl¨oppnerbrunnen fen soil affiliated with Actinobacterial narG (Figure 1), indicating that Actinobacteria in Schl¨oppnerbrunnen fen might be one major component affecting the observed stability of the nitrate reducer community composition in Schl¨oppnerbrunnen fen soil. 4.4 Conclusions and limitations Prolonged periods of drought are anticipated in future years due to climate warming [38]. Experimental drought and subsequent increase in situ emissions of N2O from the acidic fen Schl¨oppnerbrunnen [10]. The abundance of nitrate reducers and denitrifiers was increased after the drought period and decreased after the rewetting, indicating that the more oxic conditions favour growth of denitrifiers. The composition of the denitrifier community remained largely unaltered. 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CH4and N2O emissions from a forest-alas ecosystem in the permafrost taiga forest region, eastern Siberia, Russia. Journal of Geophysical Research - Biogeosciences 113, G02002 (2008). [37] Thomas, K. L., Lloyd, D., and Boddy, L. Effects of oxygen, pH and nitrate concentration on denitrification by Pseudomonas species. FEMS Microbiology Letters 118, 181–186 (1994). [38] Trenberth, K., Jones, P., Ambenje, P., Bojariu, R., Easterling, D., Klein Tank, A., Parker, D., Rahimzadeh, F., Renwick, J., Rusticucci, M., Soden, B., and Zhai, P. Observations: Surface and atmospheric climate change. In: Climate Change 2007: The Physical Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change, edited by Solomon, S., Qin, D., Manning, M., Chen, Z., Marquis, M., Averyt, K. B., Tignor, M., and Miller, H., pp. 235–336. Cambridge University Press (2007). [39] Venterink, H. O., Davidsson, T. E., Kiehl, K., and Leonardson, L. Impact of drying and re-wetting on N, P and K dynamics in a wetland soil. Plant and Soil 243, 119–130 (2002). [40] Walker, J. K. M., Egger, K. N., and Henry, G. H. R. Long-term experimental warming alters nitrogen-cycling communities but site factors remain the 4.2 Manuscripts in preparation 281 primary drivers of community structure in high arctic tundra soils. ISME Journal 2, 982–995 (2008). [41] Zumft, W. G. Cell biology and molecular basis of denitrification. Microbiology and Molecular Biology Reviews 61, 533–615 (1997). 4 Manuscripts 282 4.3 Previous peer-reviewed publications not included in the dissertation 4.3 Previous peer-reviewed publications not included in the dissertation 283 part of the fen denitrifier community is similar to previously resolved genera. Communities of different soil layers were phylogenetically similar, indicating that fen denitrifiers were derived from the same pool of microorganisms. TRFLP analysis of nosZ revealed only minor differences between different soil layers. In contrast, narG-associated differences were more pronounced, suggesting that the detected dissimilatory nitrate reducers (which have narG but lack nosZ [60]) in the fen are more dissimilar between soil layers than are detected denitrifiers. The largest difference observed in the narG TRFLP profiles was between the profile of 10to 20-cm-depth soil and profiles of the other soil layers, indicating that 10to 20-cmdepth soil harbors a nitrate-reducing population that is not identical to the populations of the other soil layers. In situ consequences of denitrifier activity. Nitrate concentrations in the Schlo¨ppnerbrunnen fen are generally low and FIG. 5. Comparative TRFLP analyses of narG (A to C) and nosZ (D to F) amplified from different soil layers of the acidic fen. PCR products were digested with CfoI (A), HaeIII (B), XhoI (C), BtgI (D), NlaIV (E), and PvuI and SacI (F). Mean values of three replicates are shown. Detected TRFs could be assigned to sequences from OTUs 1, 3, and 4 for narG and OTUs 1, 3, and 11 for nosZ. Soil layers 1, 2, 3, and 4 refer to soil depths of 0 to 10 cm, 10 to 20 cm, 20 to 30 cm, and 30 to 40 cm, respectively. Lengths of detected TRFs (in base pairs) are given in boxes next to the diagrams. VOL. 76, 2010 NEW FEN DENITRIFIERS 1131 by on February 8, 2010 aem.asm.orgDownloaded from 4 Manuscripts 290 often below the detection limit but can be as high as 0.13 mM in the upper 20 cm of soil (42, 55). Unsupplemented fen soil produced minor amounts of N 2 O, reflecting the low in situ nitrate concentrations in the fen (30, 42, 55). Supplemental nitrate caused a rapid increase in the production of N 2 O without apparent delay, indicating that (i) fen denitrifiers are poised to respond rapidly to nitrate and have a high potential to denitrify, and (ii) in situ denitrification is likely limited by nitrate availability. Increased concentrations of nitrate caused an increase in the relative proportion of N 2 O in total N gases, a phenomenon that has been observed with other soils (3, 18). The fen Schlo¨ppnerbrunnen is a net source of N 2 O, and N 2 O concentrations of up to 100 ppm occur in the pore water (20). There is a large difference between nitrate input and detected nitrate concentrations in the fen soil as nearby oxic soils receive the same amount of nitrate input but have nearly 100fold higher nitrate concentrations (42). This difference is suggestive of a high turnover of nitrate that is due in part to denitrification. The K m values (⬍20 ␮M) (Table 1) for nitrate are well below the maximum nitrate concentrations found in situ, indicating that fen denitrifiers have a high affinity for nitrate and can cope with low nitrate concentrations. Higher nitrate concentrations increased the relative amount of N 2 O formed by 0to 20-cm-depth soils (see Fig. S1 in the supplemental material). Nitrate concentrations above 40 ␮M are rarely encountered in situ (30, 42, 55). Thus, the complete reduction of nitrate to N 2 might occur under most in situ conditions, and the relative emissions of N 2 O versus N 2 might increase when nitrate concentrations are periodically elevated. The rapid increase of N 2 O production in response to nitrate and the capacity of fen soil to consume supplemental N 2 O without apparent delay (Fig. 1) suggest that denitrifiers are active in situ. High denitrification potentials in upper soil layers (0 to 20 cm) are coincident with higher concentrations of nitrate in those layers (42, 55). Rain events likely contribute to the larger amounts of nitrate in surface soils. The percentage of N 2 Oin total N gases formed by fen soils increased with increasing soil depth, a trend that might be due to the limitation of readily available organic carbon in deeper layers in the Schlo¨ppnerbrunnen fen (72). Electron donor limitation can enhance the percentage of N 2 O in total N gases produced by pure cultures of denitrifiers (53). Nitrification versus denitrification as possible sources of N 2 O. Isotope signatures of the N 2 O indicate that denitrification is the main source of the N 2 O emitted from the Schlo¨ppnerbrunnen fen (20). Although denitrification tends to be the dominant source of N 2 O under water-saturated conditions (44), nitrification likely occurs in the acidic fen when oxic conditions are augmented during dryer periods. Nitrification during dryer and more oxic conditions would theoretically provide additional nitrate for denitrification in anoxic microzones or subsequent to a rain event. In this regard, N 2 O emissions from the fen increase after rewetting events following periods of drought (19). Denitrification as an N 2 O sink. Wetlands can consume N 2 O (4). The capacity of wetlands to consume N 2 O is influenced by environmental factors such as pH and temperature, as well as the composition of the microbial community (6). The capacity of Schlo¨ppnerbrunnen fen soil to consume N 2 O to subatmospheric levels under anoxic conditions and the periodic occurrence of N 2 O at subatmospheric concentrations in fen pore water (20) are indirect evidence that N 2 O consumption occurs in situ. Isotope signatures of N 2 O from the fen indicate that the upward diffusion of the N 2 O produced in lower soil layers is subject to reduction to N 2 in the upper soil layers (20). Indeed, N 2 O consumption rates were higher in upper soil layers than in lower soil layers. These collective findings suggest that the Schlo¨ppnerbrunnen fen functions as not only an N 2 O source but also an N 2 O sink. Ecophysiology of fen denitrifiers. K m values for denitrification ranged from 6 to 19 ␮M nitrate, indicating that fen denitrifiers had a high affinity for nitrate. K m values were in the same range or lower than those of other soil types (32, 37, 62) and in the range of those of pure cultures that display a high affinity for nitrate (e.g., species of Alcaligenes,Pseudomonas, and Flavobacterium) (2, 41, 64). Denitrification rates of different fen soil layers were optimal at 34 to 46°C, optimal temperatures that approximate those of many model soil denitrifiers (e.g., Pseudomonas denitrificans, which denitrifies optimally at 38°C) (69). In contrast, the highest denitrifier activity and highest numbers of cultured denitrifiers of different soils occur between 25 and 30°C (50, 51), indicating that denitrifiers in the Schlo¨ppnerbrunnen fen have a temperature optimum that is slightly higher than the optima of denitrifiers from other soils. Enhanced denitrification capacities at temperatures that exceed most in situ conditions are a common phenomenon, and higher rates of denitrification in soils in summer can be attributed to increased soil temperatures (10, 24, 33). The capacity of fen soil to consume N 2 O under anoxic conditions was blocked by acetylene (Fig. 1B) and is therefore assumed to be due to the reduction of N 2 OtoN 2 by N 2 O reductase (74). The consumption of N 2 O by different soil layers was highest between 6 and 30°C, temperatures lower than those for nitrate-dependent denitrification. These contrasting temperature optima suggest that different denitrifier subpopulations in the acidic fen have different temperature and electron acceptor (i.e., nitrate or N 2 O) preferences. The terminal reaction of the denitrification pathway appears to be more adapted to in situ temperatures than the preceding reactions. The degree to which N 2 O reductase of soil denitrifiers is inhibited by lower temperatures varies, with effects ranging from no inhibition to almost complete inhibition (12). Denitrification rates were highest at in situ pH, indicating that Schlo¨ppnerbrunnen fen denitrifiers are well adapted to the moderately acidic fen environment. Denitrification activities of acidic agricultural soils can be highest at in situ pH even though denitrification capacities might be higher in more pHneutral soils (40), suggesting that soils of different pH values harbor distinct denitrifier communities adapted to in situ pH. Many pure cultures of denitrifiers (e.g., Pseudomonas sp.) have nearly neutral to slightly alkaline pH optima (63). Denitrification also occurred at very low pH by all fen soil layers, whereas only the upper 10-cm soil layer was capable of denitrification under slightly alkaline conditions. Therefore, alkaline conditions appear to be more limiting for fen denitrifiers than acidic conditions, which is consistent with the in situ conditions fen denitrifiers are subjected to. Acidic pH increases the percentage of N 2 O in total N gases (57, 70). Denitrification by Para1132 PALMER ET AL. APPL.ENVIRON.MICROBIOL. by on February 8, 2010 aem.asm.orgDownloaded from 4.3 Previous peer-reviewed publications not included in the dissertation 291 coccus denitrificans yields nitrite and nitrous oxide as transient intermediates at pH 5.5, whereas the amounts of these intermediates are low or not detectable at pH 8.5 (64). Up to 5 ␮M N 2 O occurs in the fen pore water (20), indicating that those intermediates occur in situ. The relative percentage of N 2 Oin total N gases formed by Schlo¨ppnerbrunnen fen soil was highest at pHs of 2 to 3 but was similar at in situ pH to values obtained at pH 7, indicating that the N 2 O reductases of fen denitrifiers are not inhibited by the moderately acidic in situ conditions. Conclusions. Schlo¨ppnerbrunnen fen soil produces formate, ethanol, and acetate under anoxic conditions via fermentation (22, 72). Such substrates are utilized by pure cultures of denitrifiers such as Pseudomonas denitrificans,Pseudomonas stutzeri, and Paracoccus denitrificans (38, 61); formate and acetate are detectable in the fen pore water (22, 30, 72); and the augmentation of denitrification in fen soil microcosms by these substrates suggests that fen denitrifiers might form trophic links to fen fermenters. Denitrification optima by fen denitrifiers at moderately acidic pH are dissimilar to those of model denitrifiers (such as those listed above). That the temperature optima of fen denitrifiers are above temperatures usually occurring in situ indicates that the fen denitrifiers are prone to respond to global warming with increased activity. Thus, the source and sink functions of the fen for N 2 O might be enhanced. These physiological findings and the novel phylogeny of denitrifier community members indicate that the fen contains heretofore unknown denitrifiers that are adapted to in situ conditions and are integrated in the intermediary ecosystem metabolism (i.e., processes that link input and output) of the fen (13). ACKNOWLEDGMENTS Support for this study was provided by the Deutsche Forschungsgemeinschaft (DFG DR 310/3-3 and DFG HO 4020/2-2) and the University of Bayreuth. REFERENCES 1. Balch, W. E., G. E. Fox, L. J. Magrum, C. R. Woese, and R. S. Wolfe. 1979. Methanogens: reevaluation of a unique biological group. Microbiol. Rev. 43:260–296. 2. Betlach, M. R., and J. M. Tiedje. 1981. 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APPL.ENVIRON.MICROBIOL. by on February 8, 2010 aem.asm.orgDownloaded from 4.3 Previous peer-reviewed publications not included in the dissertation 293 4 Manuscripts Curriculum vitae 294 Katharina Palmer Dipl. Biol. Lehrstuhl für Ökologische Mikrobiologie, Universität Bayreuth Dr.-Hans-Frisch-Str. 1-3, 95440 Bayreuth H+49172-9314685 T+49921-555672 [email protected] Ausbildung 2008–2012 Promotion,Lehrstuhl für Ökologische Mikrobiologie (Betreuung: PD Dr. Marcus A. Horn), Universität Bayreuth. Thema: Greenhouse gas metabolizing prokaryotes in peatlands 2007–2008 Diplomarbeit,Lehrstuhl für Ökologische Mikrobiologie (Betreuung: PD Dr. Marcus A. Horn, Prof. Harold L. Drake, Universität Bayreuth. Thema: Phylogenetische und funktionelle Diversität N2O-produzierender und - verbrauchender Prokaryoten in einem sauren, N2O-emittierenden Niedermoor 2003–2008 Studium der Biologie, Universität Bayreuth. 2006–2007 Auslandssemester (ERASMUS), Oulun yliopisto, Oulu, Finnland. 2002–2003 Studium der Physik und Geowissenschaften, Ruhruniversität Bochum. Berufserfahrung Juli-August 2010 Forschungsaufenthalt im Ausland (Finnland),im Rahmen des EU-Projektes „Lapland Atmosphere-Biosphere Facility“ (LAPBIAT), Forschungsstation Kevo (University of Turku), Forschungsstation Oulanka (University of Oulu), Thema: Effect of high latitude on denitrification-dependent N2O-fluxes and denitrifier community structure in acidic mires. 2006–2007 Projektarbeit,Department of Biology, Plant physiology, Dr. Anna Maria Mattila, Oulun yliopisto, Oulu, Finnland. Themen: 1. ENDIS: Discovery and development of new antibacterial compounds from endophytes 2. Role of endophytic Methylobacterium in plant tissue 2004–2006 Studentische Hilfskraft,Lehrstuhl für Pflanzensystematik, Abteilung Mykologie, Prof. Dr. Gerhard Rambold, Universität Bayreuth. 1/4 4.3 Previous peer-reviewed publications not included in the dissertation 295 Erhaltene Förderungen Kurzstipendium für Doktoranden,Deutscher Akademischer Austauschdienst (DAAD), Juli-August 2010: Unterstützung des Forschungsaufenthaltes in Finnland (Aussetzung der DFGStelle) Stipendium zur Förderung von Kongressreisen, Deutscher Akademischer Austauschdienst (DAAD), April 2011: Kongress „Ecology of Soil Microorganisms“, Prag Reisekostenzuschüsse, Frauenförderung aus den Mitteln des Zentralansatzes zur Gleichstellung, Universität Bayreuth: Tagungsreisen: BAGECO-10 (2009), ISME-13 (2010), NordSIR-Meeting (2010), Gordon Research Conference on Applied and Environmental Microbiology (2011) Mitbetreute Abschlussarbeiten Schulz, K. 2009. Einfluss von Austrocknungsereignissen auf die Methanogenenpopulation im sauren Niedermoor. Bachelor-Arbeit, Universität Bayreuth. Mundinger, A. 2010. Einfluss von Sauerstoff auf die Methanogenese im sauren Niedermoor. Bachelor-Arbeit, Universität Bayreuth. Perras, S. 2011. Trophische Interaktionen im mikrobiellen Nahrungsnetz eines Methan-emittierenden pH-neutralen Niedermoores. Bachelor-Arbeit, Universität Bayreuth. Guttmann, T. 2011 (laufend). (Toxische) Effekte von Eisennanopartikeln auf Bodenmikroorganismen. Bachelor-Arbeit, Universität Bayreuth. 2/4 4 Manuscripts 296 Publikationen Palmer, K., Drake, H.L., Horn, M.A. 2009. Genome-derived criteria for assigning environmental narG and nosZ sequences to operational taxonomic units of nitrate reducers. Appl. Environ. Microbiol. 75:5170-5174. Palmer, K., Drake, H.L., Horn, M.A. 2010. Association of novel and highly diverse acid-tolerant denitrifiers with N2O fluxes of an acidic fen. Appl. Environ. Microbiol. 76:1125-1134. Palmer, K., Biasi, C., Horn, M.A. 2011. Contrasting denitrifier communities relate to contrasting N2O emission patterns from acidic peat soils in arctic tundra. ISME Journal 6: 1058-1077. Publizierte Abstracts auf Tagungen und Workshops Palmer, K., Horn, M.A. 2012. Unknown denitrifier diversity in a pH neutral fen soil in Finnish Lapland. Annual Meeting Vereinigung für Allgemeine und Angewandte Mikrobiologie, BioSpectrum. Abstract SMV003, S. 210 Palmer, K., Horn, M.A. 2012. Palsa peats represent hitherto underappreciated reservoirs of new denitrifier diversity associated with N2O fluxes. International Polar Year 2012 Conference. Abstract online. Palmer, K., Biasi, C., Drake, H.L., Horn, M.A. 2011. Cryoturbation affects denitrifier communities in N2O-emitting arctic permafrost peat soil. Annual Meeting Vereinigung für Allgemeine und Angewandte Mikrobiologie, BioSpectrum. Abstract EMP104, S. 116. Palmer, K., Biasi, C., Drake, H.L., Horn, M.A. 2011. Cryoturbation affects denitrifier communities in N2O-emitting arctic permafrost peat soil. Annual Meeting Vereinigung für Allgemeine und Angewandte Mikrobiologie, BioSpectrum. Abstract EMP104, S. 116. Palmer, K., Biasi, C., Drake, H.L., Horn, M.A. 2011. Impact of cryoturbation on denitrifier community structure and activity in N2O-emitting arctic permafrost peat soil. Ecology of Soil Microorganisms. Abstract 99 Palmer, K., Schulz, K., Horn, M.A., Drake, H.L. 2010. Stability of the methanogenic community in an acidic fen to experimental drought. Annual Meeting Vereinigung für Allgemeine und Angewandte Mikrobiologie, BioSpectrum. Abstract ECV02, S. 81. Palmer, K., Schulz, K., Horn, M.A., Drake, H.L. 2010. Effects of enhanced drought on the diversity of methanogens in an acidic fen. Bayreuth Center of Ecology and Environmental Research (BayCEER) Workshop 2010, Abstract O 1.5. Palmer, K., Schulz, K., Horn, M.A., Drake, H.L. 2010. Impact of artificial drought on diversity, abundance, and gene expression of methanogens in an acidic fen. 13th International Symposium on Microbial Ecology (ISME-13), Abstract on disk Palmer, K., Drake, H.L., Horn, M.A. 2009. Activity and diversity of phylogenetically novel acid-tolerant nitrate reducer and denitrifier communities in an N2O-emitting fen. Annual Meeting Vereinigung für Allgemeine und Angewandte Mikrobiologie, BioSpectrum. Abstract PN22, S. 127. 3/4 4.3 Previous peer-reviewed publications not included in the dissertation 297 Palmer, K., Drake, H.L., Horn, M.A. 2009. Activity and diversity of phylogenetically novel acid-tolerant nitrate reducer and denitrifier communities in an N2O-emitting fen. Bayreuth Center of Ecology and Environmental Research (BayCEER) Workshop 2009, Abstract O 1.7. Palmer, K., Drake, H.L., Horn, M.A. 2009. Novel and highly diverse acid-tolerant nitrate reducers are associated with N2O-fluxes of an acidic N2O-emitting fen. 10th International Symposium on Bacterial Genetics and Ecology (BAGECO-10), Abstract P197, S. 279. Sonstige Tagungsbeiträge Palmer, K., Biasi, C., Horn, M.A. 2011. Denitrifier communitiy composition impacts N2O emission patterns in acidic tundra permafrost soils. Nordic Network for Stable Isotope Research (NordSIR) Meeting Palmer, K., Drake, H.L., Horn, M.A. 2011. Effect of high latitude on denitrification-dependent N2O-fluxes and denitrifier community structure in peatlands. Gordon Research Conference on Applied and Environmental Microbiology Palmer, K., Drake, H.L., Horn, M.A. 2010. Novel and highly diverse acid-tolerant nitrate reducers are associated with N2O-fluxes of an acidic N2O-emitting fen. Nordic Network for Stable Isotope Research (NordSIR) Meeting Schmidt, O., Horn, M.A., Palmer, K., Drake, H.L. 2010. H2A hidden link between fermentation and methanogenesis in the intermediary ecosystem metabolism of a wetland soil. Gordon Research Conference on the Molecular Basis of Microbial One-Carbon Metabolism 4/4 4 Manuscripts 298