Cryoturbated peat circles (that is, bare surface soil mixed by frost action; pH 3-4) in the Russian discontinuous permafrost tundra are nitrate-rich 'hotspots' of nitrous oxide (N 2 O) emissions in arctic ecosystems, whereas adjacent unturbated peat areas are not. N 2 O was produced and subsequently consumed at pH 4 in unsupplemented anoxic microcosms with cryoturbated but not in those with unturbated peat soil. Nitrate, nitrite and acetylene stimulated net N 2 O production of both soils in anoxic microcosms, indicating denitrification as the source of N 2 O. Up to 500 and 10 lM nitrate stimulated denitrification in cryoturbated and unturbated peat soils, respectively. Apparent maximal reaction velocities of nitrite-dependent denitrification were 28 and 18 nmol N 2 O g DW À1 h À1 , for cryoturbated and unturbated peat soils, respectively. Barcoded amplicon pyrosequencing of narG, nirK/nirS and nosZ (encoding nitrate, nitrite and N 2 O reductases, respectively) yielded E49 000 quality-filtered sequences with an average sequence length of 444 bp. Up to 19 species-level operational taxonomic units were detected per soil and gene, many of which were distantly related to cultured denitrifiers or environmental sequences. Denitrification-associated gene diversity in cryoturbated and in unturbated peat soils differed. Quantitative PCR (inhibition-corrected per DNA extract) revealed higher copy numbers of narG in cryoturbated than in unturbated peat soil. Copy numbers of nirS were up to 1000 Â higher than those of nirK in both soils, and nirS nirK À1 copy number ratios in cryoturbated and unturbated peat soils differed. The collective data indicate that the contrasting N 2 O emission patterns of cryoturbated and unturbated peat soils are associated with contrasting denitrifier communities.

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Wetlands are sources of denitrification-derived nitrous oxide (N 2 O). Thus, the denitrifier community of an N 2 O-emitting fen (pH 4.7 to 5.2) was investigated. N 2 O was produced and consumed to subatmospheric concentrations in unsupplemented anoxic soil microcosms. Total cell counts and most probable numbers of denitrifiers approximated 10 11 cells ⅐ g DW ؊1 (where DW is dry weight) and 10 8 cells ⅐ g DW ؊1 , respectively, in both 0-to 10-cm and 30-to 40-cm depths. Despite this uniformity, depth-related maximum reaction rate (v max ) values for denitrification in anoxic microcosms ranged from 1 to 24 and ؊19 to ؊105 nmol N 2 O h ؊1 ⅐ g DW ؊1 , with maximal values occurring in the upper soil layers. Denitrification was enhanced by substrates that might be formed via fermentation in anoxic microzones of soil. N 2 O approximated 40% of total nitrogenous gases produced at in situ pH, which was likewise the optimal pH for denitrification. Gene libraries of narG and nosZ (encoding nitrate reductase and nitrous oxide reductase, respectively) from fen soil DNA yielded 15 and 18 species-level operational taxonomic units, respectively, many of which displayed phylogenetic novelty and were not closely related to cultured organisms. Although statistical analyses of narG and nosZ sequences indicated that the upper 20 cm of soil contained the highest denitrifier diversity and species richness, terminal restriction fragment length polymorphism analyses of narG and nosZ revealed only minor differences in denitrifier community composition from a soil depth of 0 to 40 cm. The collective data indicate that the regional fen harbors novel, highly diverse, acid-tolerant denitrifier communities capable of complete denitrification and consumption of atmospheric N 2 O at in situ pH.

P ermafrost systems in the Northern Hemisphere cover about 16% of the global soil surface area, store substantial amounts of carbon and nitrogen, and are therefore important players in the global carbon and nitrogen cycles (54, 67). Palsas are elevations of peat soil above the ground level due to uplifting of peat layers by a frozen ice lens and are mainly encountered in the discontinuous permafrost zone (63). Palsa peatlands are widely distributed in the Arctic, including Canada, Norway, Sweden, Iceland, Russia (Siberia), the United States (Alaska), and Finland (62, 75). Palsa development is affected by various environmental factors, such as wind erosion, vegetation cover, snow cover, and ground water table depth (63). High-latitude peatlands have been intensively studied with respect to their capacity to emit methane due to the large amount of stored carbon in peat soils, but nitrous oxide (N 2 O) emissions from permafrost regions were generally considered to be insignificant (12,58,66). However, recent studies document significant but variable N 2 O emissions from permafrost systems including palsas (17,45,58). N 2 O is the major ozone-depleting substance in the atmosphere (57), and such N 2 O emissions might well impact climate change since the global warming potential of N 2 O is approximately 300 times that of CO 2 (20). Palsa peats are predicted to be strongly affected by global warming (2, 21, 63). Increasing temperatures are generally anticipated to reduce the water table in northern peatlands and to increase the amount of CO 2 , CH 4 , and N 2 O released from peatland soils (2,44,45). N 2 O is produced during nitrification, denitrification, or chemical processes in soils (8,13). N 2 O is an intermediate during denitrification, and denitrification is considered to be the main source of and hypothesized to represent a sink for N 2 O in water-saturated soils including peatlands and is an essential part of the nitrogen cycle (11,13,48). Nitrate or nitrite is sequentially reduced via nitric oxide (NO) and N 2 O to dinitrogen (N 2 ) during denitrification (76). Such reductions are catalyzed by a set of oxidoreductases, namely, nitrate reductases (encoded by narG and napA), nitrite reductases (encoded by nirK and nirS), NO reductases (encoded by norBC or norZ), and N 2 O reductases (encoded by nosZ) (76,77). Nitrate reductases likewise catalyze nitrate reduction by nondenitrifying dissimilatory nitrate reducers (52). N 2 O and N 2 can be released into the atmosphere, and the ratio of N 2 O to N 2 is determined by in situ parameters such as pH, temperature, and oxygen content, as well as nitrate/nitrite and electron donor availability (69). pH is one of the main factors impacting denitrification; low pH decreases overall denitrification rates and increases the product ratio of N 2 O to N 2 (64). Although pristine northern peatlands including palsas are characterized by low pH and although evidence is accumulating that northern peatlands emit N 2 O, palsas might represent temporary sinks for N 2 O; however, mic...

Ninety percent of cultured bacterial nitrate reducers with a 16S rRNA gene similarity of >97% had a narG or nosZ similarity of >67% or >80%, respectively, suggesting that 67% and 80% could be used as standardized, conservative threshold similarity values for narG and nosZ, respectively (i.e., any two sequences that are less similar than the threshold similarity value have a very high probability of belonging to different species), for estimating species-level operational taxonomic units. Genus-level tree topologies of narG and nosZ were generally similar to those of the corresponding 16S rRNA genes. Although some genomes contained multiple copies of narG, recent horizontal gene transfer of narG was not apparent.Nitrate reducers (i.e., both dissimilatory nitrate reducers and denitrifiers) reduce nitrate to nitrite, which can then be reduced to ammonium by dissimilatory nitrate reducers or sequentially reduced to nitric oxide, nitrous oxide, and dinitrogen by denitrifiers (29). narG codes for the alpha subunit of the dissimilatory nitrate reductase, which reduces nitrate to nitrite and is thus common to both dissimilatory nitrate reducers and denitrifiers (29). nosZ codes for nitrous oxide reductase, which reduces nitrous oxide to dinitrogen and is common to denitrifiers but not dissimilatory nitrate reducers (29). Both narG and nosZ are commonly used as gene markers for community level analysis of nitrate reducers (2,8,9,16,18,19,20,25). However, standardized criteria for assigning environmental narG and nosZ sequences to operational taxonomic units (OTUs) are required so that diverse data sets on nitrate-reducing communities can be normalized. The widespread ability of bacteria and archaea to denitrify (29) complicates the development of such criteria for genes involved in denitrification. Some closely related narG and closely related nosZ genes occur in distantly related taxa, and narG or nosZ phylogenies do not always reflect 16S rRNA phylogenies (17). However, nosZ-based phylogenies in general have a high degree of congruency with 16S rRNA gene-based phylogenies (3,10,30), and recent horizontal gene transfer of nosZ seems unlikely (10), indicating that denitrifier structural genes might be used for estimating the species-level novelty, as well as species-level diversity, of denitrifiers in environmental samples. The limited amount of data on horizontal gene transfer of narG (4, 24) identifies a need to extend such an approach to this gene. The limited number of studies that have compared 16S rRNA with narG or nosZ phylogenies accentuates the need for a more thorough analysis of the phylogenetic relatedness of these three genes (3, 4, 7). Thus, the main objectives of this study were to (i) resolve criteria for standardizing OTU assignment of environmental narG and nosZ sequences, (ii) determine whether those criteria can be used as indicators of novel species, and (iii) investigate the impact of horizontal gene transfer on narG.