Genetic and Environmental Controls on Nitrous Oxide Accumulation in Lakes

This is an electronic reprint of the original article. This reprint may differ from the original in pagination and typographic detail. Author(s): Title: Year: Version: Please cite the original version: All material supplied via JYX is protected by copyright and other intellectual property rights, and duplication or sale of all or part of any of the repository collections is not permitted, except that material may be duplicated by you for your research use or educational purposes in electronic or print form. You must obtain permission for any other use. Electronic or print copies may not be offered, whether for sale or otherwise to anyone who is not an authorised user. Genetic and Environmental Controls on Nitrous Oxide Accumulation in Lakes Saarenheimo, Jatta; Rissanen, Antti; Arvola, Lauri; Nykänen, Hannu; Lehmann, Moritz F.; Tiirola, Marja Saarenheimo, J., Rissanen, A., Arvola, L., Nykänen, H., Lehmann, M. F., & Tiirola, M. (2015). Genetic and Environmental Controls on Nitrous Oxide Accumulation in Lakes. PLoS ONE, 10(3), Article e0121201. https://doi.org/10.1371/journal.pone.0121201 2015 RESEARCH ARTICLE Genetic and Environmental Controls on Nitrous Oxide Accumulation in Lakes Jatta Saarenheimo 1 *, Antti J. Rissanen 1 , Lauri Arvola 2 , Hannu Nykänen 1 , Moritz F. Lehmann 3 , Marja Tiirola 1 1Department of Biological and Environmental Science, University of Jyväskylä, 40014, Jyväskylä, Finland, 2Lammi Biological Station, University of Helsinki, 16900, Lammi, Finland, 3Department for Environmental Science, University of Basel, CH-4058, Basel, Switzerland *[email protected] Abstract We studied potential links between environmental factors, nitrous oxide (N 2 O) accumulation, and genetic indicators of nitrite and N 2 O reducing bacteria in 12 boreal lakes. Denitrifying bacteria were investigated by quantifying genes encoding nitrite and N 2 O reductases (nirS/nirK and nosZ, respectively, including the two phylogenetically distinct clades nosZ I and nosZ II ) in lake sediments. Summertime N 2 O accumulation and hypolimnetic nitrate concentrations were positively correlated both at the inter-lake scale and within a depth transect of an individual lake (Lake Vanajavesi). The variability in the individual nirS,nirK,nosZ I , and nosZ II gene abundances was high (up to tenfold) among the lakes, which allowed us to study the expected links between the ecosystem’snir-vs-nos gene inventories and N 2 O accumulation. Inter-lake variation in N 2 O accumulation was indeed connected to the relative abundance of nitrite versus N 2 O reductase genes, i.e. the (nirS+nirK)/nosZ I gene ratio. In addition, the ratios of (nirS+nirK)/nosZ I at the inter-lake scale and (nirS+nirK)/nosZ I+II within Lake Vanajavesi correlated positively with nitrate availability. The results suggest that ambient nitrate concentration can be an important modulator of the N 2 O accumulation in lake ecosystems, either directly by increasing the overall rate of denitrification or indirectly by controlling the balance of nitrite versus N 2 O reductase carrying organisms. Introduction Nitrous oxide (N 2 O) is an important greenhouse gas and the single most important ozone destroying chemical [1]. N 2 O in the biosphere is produced as an intermediate molecule in denitrification or nitrifier-denitrification, or as a by-product during nitrification or dissimilatory nitrate reduction to ammonium (DNRA) [2,3]. The denitrification pathway includes four enzymatically catalyzed reductive steps: nitrate reduction (nar), nitrite reduction (nir), nitric oxide reduction (nor), and nitrous oxide reduction (nos)[4]. Reduction of nitrite, where the first gaseous form of fixed nitrogen (N) (i.e. NO) is produced, is catalyzed by two analogous genes: nirK and nirS genes encoding a copper nitrite reductase and a cytochrome cd1-nitrite reductase, respectively [4]. These two genes prevail in different organisms and their differential PLOS ONE | DOI:10.1371/journal.pone.0121201 March 10, 2015 1/14 OPEN ACCESS Citation: Saarenheimo J, Rissanen AJ, Arvola L, Nykänen H, Lehmann MF, Tiirola M (2015) Genetic and Environmental Controls on Nitrous Oxide Accumulation in Lakes. PLoS ONE 10(3): e0121201. doi:10.1371/journal.pone.0121201 Academic Editor: Yiguo Hong, CAS, CHINA Received: October 21, 2014 Accepted: January 28, 2015 Published: March 10, 2015 Copyright: © 2015 Saarenheimo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This study was supported by the Academy of Finland (MT project 260797 and HN project 136455), the VVY, the Maj and Tor Nessling Foundation, and EnSTE graduate school. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. distributions in nature seem to be modulated by the redoxconditions, with nirS being preferentially expressed under low dissolved oxygen conditions [5,6]. Recent studies have also revealed that nosZ genes encoding N 2 O reductase actually belong to two phylogenetically distinct clades [7,8], here referred to as nosZ I and nosZ II , which need to be analyzed by separate PCR primer sets. As with nir genes, the relative importance of nos genes seems to systematically differ between habitats and with environmental conditions [8], yet the exact controls that modulate their relative abundance in nature are uncertain. Some denitrifiers are lacking the nosZ gene completely and perform the truncated denitrification pathway, where N 2 O is produced as an end-product [9]. In fact, genome sequencing showed that one third of the cultivated denitrifying bacteria lack the nosZ gene [10]. Since denitrifier community structure is likely to have an effect on net N 2 O production and emission [11,12], denitrifier communities have been studied through the analysis of sequence variation and/or the abundance of nirS,nirK, and nosZ genes in many ecosystems [13,14,15, 16,17]. High availability of nitrate and nitrite has been shown to be conducive to N 2 O accumulation [18,19], fostering the increase the N 2 O/(N 2 O+N 2 ) ratio in the gaseous denitrification products [20,21]. Such correlations may simply indicate nitrate-induced enhancement of denitrification rates (and thus N2O accumulation), but they may also be the result of microbial community adaptation. Philippot et al. [22], for example, demonstrated that the relative abundance of the nosZ gene was a strong predictor of the N 2 O/(N 2 O+N 2 ) production ratio. In soils, microbially produced N 2 O is likely lost to the atmosphere by turbulent diffusive escape. In contrast, in aquatic environments, the diffusivity of gases is much slower (K z values on the order of 10 −5 to 10 −6 cm 2 s −1 ,[23]), reducing diffusive loss rates and improving the N 2 O availability for nosZ carrying bacteria. More complete denitrification and lower N 2 O/N 2 gas emission ratios should, therefore, be expected for the aquatic versus soil environments. Still, lake ecosystems have shown to be important sites of N 2 O emissions [19,24], and, as in soils, N 2 O production and accumulation in lakes appears to be dependent on the ambient nitrate and oxygen concentrations [25,26,24,27]. Although the importance of lacustrine N 2 O production is well recognized [19,26], and albeit the fact that benthic denitrifier community structure has been studied in some lakes [28,29], it is not known whether variations in the accumulation of N 2 O are mostly directly dependent on the environmental conditions, or whether they rather are indirectly constrained by the denitrifying community structure. With some recent exceptions [7,8] the role of the nosZ II clade remained mostly unconsidered in this context. Here, we evaluated genetic and environmental factors that likely modulate N 2 O production and accumulation in lake ecosystems, especially focusing on the benthic abundance of nirS,nirK,nosZ I , and nosZ II genes during the summertime N 2 O accumulation period. Anticipating close links between nitrate concentrations and the N 2 O accumulation, we hypothesized 1) that high hypolimnetic nitrate concentrations would decrease the relative abundance of the nosZ genes (i.e., increase the nir/nos ratio) within lacustrine sediments, and 2) that higher nir/nos ratios would lead to enhanced N 2 O accumulation. The linkage between benthic denitrification gene frequency and N 2 O accumulation was assessed in an inter-lake study of 12 boreal lakes in southern Finland, pooling the lakes into two groups based on their hypolimnetic nitrate concentrations (high-NO 3 − -lakes and low-NO 3 − -lakes). In addition, denitrification gene abundance and N 2 O accumulation was investigated along a littoral-to-pelagic transect in a large stratified lake (Vanajavesi) with relatively high hypolimnetic nitrate levels (24.0−44.9 μmol l −1 ). N 2 O Production in Boreal Lakes PLOS ONE | DOI:10.1371/journal.pone.0121201 March 10, 2015 2/14 Results Comparison of denitrification genes in highversus low-nitrate lakes Considerable inter-lake variation was observed with regards to the nitrate (0.4−79.1 μmol l −1 ), ammonium (0.6−61.4 μmol l −1 ), and oxygen (1.9−333.4 μmol l −1 ) concentrations (S1 Table). The lakes were classified into two groups based on their nitrate concentration, which generally reflected land use in the catchment area: high-NO 3 − -lakes comprised lakes mostly with extensive agricultural activity in their catchment area and one urban lake (Jyväsjärvi), while low- NO 3 − -lakes included lakes mostly with little agricultural land in their catchment area. Other environmental parameters did not differ significantly between the two groups (Table 1). Throughout the studied lakes, the abundances of nirS,nirK,nosZ I ,andnosZ II relative to 16S rRNA genes varied between 0.6−12.9% (Table 2), and the gene copy numbers ranged between 4.8 and 580 per ng of DNA (S2 Table). The ratio of nirS/nirK ranged between 0.5−2.0 (average 1.0), and the ratio of nosZ I /nosZ II varied between 0.5−5.7 (average 1.9). Neither environmental factors (oxygen, temperature, nitrate concentration) nor N 2 O accumulation showed any significant correlation with the gene abundance, gene copy numbers, or with nirS/nirKornosZ I /nosZ II gene ratios (Pearson correlations, p values >0.05). The relative proportion of the previously unaccounted nosZ II gene was of a similar magnitude as that of nosZ I , but showed a markedly higher inter-lake variability (Table 2). Although not statistically significant, nosZ I and nosZ II seemed slightly more abundant in the low-NO 3 − group of lakes, while nirS and nirK seemed less abundant, (Fig. 1A). The (nirS+nirK)/nosZ I ratio was higher in high-NO 3 − -lakes than in low-NO 3 − - lakes (Fig. 1B). In addition, the (nirS+nirK)/nosZ I gene ratio correlated positively with the estimated net N 2 O production, as well as with nitrate and phosphate concentrations (Table 3.). As for (nirS+nirK)/nosZ II and (nirS+nirK)/(nosZ I +nosZ II ), we also observed a tendency for higher ratios in the high-NO 3 − -lakes compared to low-NO 3 − lakes (Fig. 1B). However, correlation between nitrate and (nirS+nirK)/(nosZ I +nosZ II ) was only weakly significant (p = 0.06) (Table 3). N 2 O and N 2 accumulation in highversus low-nitrate lakes During the summer sampling (late July), most of the study lakes were oversaturated with respect to N 2 O (i.e. the depth-integrated mean N 2 O excess was >0). N 2 O excess in the water column varied between 0.9−37.1 nmol l −1 (11−337% oversaturation). The highest N 2 O excess Table 1. Environmental parameters (mean and SE) for high-NO 3 − -lakes (n = 6) and low-NO 3 − -lakes (n = 6), and results of a t-test or Mann- Whitney U-test*comparing the oxygen, nitrate, ammonium and phosphate concentrations, temperature, catchment field area (ha), averaged N 2 O excess concentrations, and maximum observed N 2excess concentrations between the two lake groups. O 2 (μmol l −1 ) NO 3 (μmol l −1 ) NH 4+ (μmol l −1 ) PO 4 − (μmol l −1 ) T (C°) Field area (ha) N 2 O excess (μmol m −3 ) N 2excess (μmol l −1 ) High-nitrate lakes Mean 101.15 39.30 15.48 0.15 11.41 37990 18.14 5.55 (±SE) (±51.30) (±8.76) (±8.55) (±0.02) (±1.49) (±35870) (±4.97) (±0.79) Low-nitrate lakes Mean 77.72 0.64 34.67 0.08 15.78 500 1.36 1.12 (±SE) (±35.77) (±0.11) (±17.68) (±0.03) (±2.42) (±245) (±1.50) (±0.47) Pairwise test results High vs. low nitrate high = low high >low high = low high = low high = low high >low high >low high >low Pns 0.007 Ns ns ns 0.012*0.001 0.001 doi:10.1371/journal.pone.0121201.t001 N 2 O Production in Boreal Lakes PLOS ONE | DOI:10.1371/journal.pone.0121201 March 10, 2015 3/14 concentrations were observed either in near-bottom waters of the lakes or, in the case of stratified lakes (five lakes were stratified with regards to oxygen and displayed an anoxic hypolimnion), at the oxic-anoxic interface within the water column (S1 Fig.). Maximum N 2excess concentrations measured using membrane inlet mass spectrometry (MIMS) were generally Table 2. Copy numbers (mean ±SE) of nirS,nirK,nosZ I , and nosZ II gene amplicons as percentages of 16S rRNA gene copy numbers (nd, no data). Inter-lake comparison Denitrification gene (% of 16S rRNA gene) High-nitrate/ Low-nitrate nirS nirK nosZ I nosZ II Pääjärvi 5.5 7.9 4.7 2.1 High (±SE) (±0.25) (±0.26) (±0.34) (±0.07) Mommilanjärvi 4.5 2.3 2.5 2.4 High (±SE) (±0.28) (±0.26) (±0.24) (±0.18) Ormajärvi 3.6 3.9 2.4 2.1 High (±SE) (±0.09) (±0.34) (±0.08) (±0.02) Vanajavesi 2.9 3.2 2.4 1.3 High (±SE) (±0.31) (±0.28) (±0.32) (±0.14) Jyväsjärvi 3.0 3.4 2.5 5.2 High (±SE) (±0.08) (±0.24) (±0.15) (±0.22) Suolijärvi 0.9 1.3 0.9 1.5 High (±SE) (±0.38) (±0.21) (±0.19) (±0.10) Ekojärvi 1.2 2.5 2.1 2.9 Low (±SE) (±0.07) (±0.31) (±0.23) (±0.19) Kataloistenjärvi 4.4 5.2 3.9 1.2 Low (±SE) (±0.20) (±0.4) (±0.32) (±0.24) Teuronjärvi 2.5 3.0 2.8 1.3 Low (±SE) (±0.28) (±0.28) (±0.29) (±0.04) Kyynäröjärvi 2.5 3.5 2.9 2.5 Low (±SE) (±0.25) (±0.26) (±0.09) (±0.07) Kastanajärvi 1.9 1.4 6.1 12.9 Low (±SE) (±0.04) (±0.05) (±0.25) (±0.25) Lehee 2.0 3.8 2.3 4.1 Low (±SE) (±0.21) (±0.22) (±0.12) (±0.26) Intra-lake depth transect Vanajavesi2 4.9 2.4 2.1 1.8 (±SE) (±0.28) (±0.20) (±0.28) (±0.08) Vanajavesi3 6.2 3.5 3.4 2.4 (±SE) (±0.33) (±0.32) (±0.43) (±0.09) Vanajavesi4 4.8 3.6 3.4 1.7 (±SE) (±0.26) (±0.32) (±0.36) (±0.05) Vanajavesi5 3.3 4.2 2.9 1.4 (±SE) (±0.29) (±0.30) (±0.22) (±0.10) Vanajavesi6 2.4 4.2 2.4 0.6 (±SE) (±0.23) (±0.27) (±0.26) (±0.13) Vanajavesi7 2.9 3.2 2.4 1.3 (±SE) (±0.31) (±0.28) (±0.32) (±0.14) Vanajavesi8 3.1 2.2 1.1 nd (±SE) (±0.20) (±0.19) (±0.14) doi:10.1371/journal.pone.0121201.t002 N 2 O Production in Boreal Lakes PLOS ONE | DOI:10.1371/journal.pone.0121201 March 10, 2015 4/14 slightly higher than the equilibrium concentration at given temperatures (<2% oversaturation). N 2excess was significantly higher in high-NO 3 − lakes than in low-NO 3 − lakes (Table 1) and correlated with nitrate concentrations (Table 3). Moreover, the depth-integrated N 2 O excess concentrations (0−20.3 μmol m −3 ) and net N 2 O production rates (0−11.2 μmol N m −2 d −1 ) estimated from the N 2 O concentration profiles were significantly higher in high-NO 3 − -lakes than in low-NO 3 − - lakes (Table 1), and both correlated with NO 3 − concentration (Table 3). Maximum N 2excess concentrations were found to correlate with the depth-integrated N 2 O excess concentration (Table 3). Denitrification genes and N 2 O accumulation in Lake Vanajavesi In Lake Vanajavesi, hypolimnetic temperature and oxygen concentrations were tightly correlated, indicating the effect of thermal water column stratification on the vertical distribution of dissolved oxygen (correlation r = -0.98 and p = 0.000). Sampling sites 1−3(waterdepths2−6m) Fig 1. Abundance of nirS, nirK, nosZ I , and nosZ II genes relative to the amount of 16S rRNA genes (A), and ratios of nir and nos genes in sediments of lakes with high and low nitrate concentrations (high- NO3 − -lakes and low-NO3 − -lakes) (B). * = significantly different between the two lake groups (Mann-Whitney U-test, p = 0.006). doi:10.1371/journal.pone.0121201.g001 N 2 O Production in Boreal Lakes PLOS ONE | DOI:10.1371/journal.pone.0121201 March 10, 2015 5/14 were fully aerated, sites 4−6 (water depths 8−12 m) displayed lower oxygen concentrations, and the two deepest sampling sites (water depths 14 and 16 m) were anoxic at the bottom of the hypolimnion (S3 Table). Nitrate concentrations (24.0−44.9 μmol l −1 ) were consistently high at all sampling sites, whereas ammonium (1.1−57.4 μmol l −1 ) and phosphate (0.03−0.7 μmol l −1 ) concentrations displayed strong variability between strongly oxygen-depleted and oxygen-replete conditions (S3 Table). The relative abundances of nirS, nirK, nosZ I ,andnosZ II genes in Lake Vanajavesi varied between 0.6 and 6.2% of the total 16S rRNA genes (Table 2), with nosZ I or nosZ II being the least abundant of the denitrifying genes at all sites. In contrast to observation at the inter-lake scale (where nitrate concentrations were generally lower and more variable), we observed a strong positive correlation between nitrate concentrations and the (nirS+nirK)/nosZ I+II ratio (r = 0.98 and p = 0.001) (Fig. 2). The correlation with either nosZ I or nosZ II only was not significant (p >0.05). Table 3. Correlations of functional gene ratios and accumulated N 2 O and N 2 gas concentrations with environmental parameters in the inter-lake dataset. Correlation coefficients with 0.01 <p<0.05 and p <0.01 are written in normal text and bold, respectively. Gene ratios Gas accumulation measurements (nirS+nirK)/ nosZ I (nirS+nirK)/ nosZ II (nirS+nirK)/ (nosZ I + II ) N 2 O excess (μmol m −3 ) N 2 O production (μmol N m −2 d −1 ) N 2excess (μmol l −1 ) O 2 (μmol l −1 )- - - - - - NO 3 (μmol l −1 )0.78 - (0.55)*0.66 0.74 0.58 NH 4+ (μmol l −1) -- - - - - PO 4 − (μmol l −1 ) 0.67 - - - - - T (C°) - - - - - - N 2 O excess (μmol m −3 ) 0.61 - - 1 0.96 0.80 *Marginally significant (p = 0.06) doi:10.1371/journal.pone.0121201.t003 Fig 2. Relationship between hypolimnetic nitrate concentration and the sedimentary (0–2cm) (nirS+nirK)/nosZ I+II gene ratio (r = 0.98 and p = 0.001), and depth-integrated N 2 O excess (r = 0.89 and p = 0.02) in Lake Vanajavesi. doi:10.1371/journal.pone.0121201.g002 N 2 O Production in Boreal Lakes PLOS ONE | DOI:10.1371/journal.pone.0121201 March 10, 2015 6/14 At all sampling sites, essentially the entire water column was oversaturated with respect to equilibrium N 2 O concentrations (S2 Fig.). The N 2 O profiles of Sites 1, 2, and 3 indicated a homogenized water column, with an equal degree of oversaturation throughout. At the deeper Sites 4, 5, and 6, a markedly higher N 2 O oversaturation was observed at the bottom of the lake. The degree of N 2 O oversaturation was even higher at the oxic-anoxic interface in the water column of Sites 7 and 8 (S2 Fig.). Depth-integrated N 2 O excess varied between 5.7−36.0 nmol l −1 (62−337% oversaturation) and correlated positively with the nitrate concentration in Lake Vanajavesi (r = 0.89 and p = 0.02) (Fig. 2). A negative correlation was observed with respect to the oxygen concentration (r = -0.90 and p = 0.002) and temperature (r = -0.95 and p <0.001). Discussion To our knowledge, this is the first study combining N 2 O measurements and molecular analyses of denitrification genes in lake ecosystems. This is also the first time that the abundance of nirS and nirK genes together with both clades of nosZ genes were investigated in freshwater sediments. The total nir/nos ratio was above 1:1 in nearly all study lakes, indicating that the microbial community had a higher potential to produce N 2 O than to reduce it. This implies that the accumulation of N 2 O is linked to genetic factors. All the studied denitrification genes (nir and nos variants) were present in the lake sediments, although their abundance largely varied among the lakes and along the Vanajavesi transect. The qPCR results also revealed that nosZ II genes are as frequent as the canonical nosZ I genes in the freshwater sediments, which emphasizes the need to further study the ecology of nosZ II encoding organisms in future studies. The relatively high abundance of individual nirS, nirK,nosZ I , and nosZ II genes highlights the important biogeochemical role of denitrification in boreal lake sediments. For comparison, the abundance of individual denitrification genes nirS, nirK, and nosZ have previously been found to range between 0.5 and 6.8% of the 16S rRNA gene abundance in various soil and sediment samples [14,21,30,31]. Bioavailability of copper (Cu) and iron (Fe) can control the expression and activity of nitrite and nitrous oxide reductases. While nirK and nosZ are copper-containing reductases, nirS is an iron containing cd1- type reductase. Possible Cu limitation may lead to nirS dominance and, thus, to increased N 2 O accumulation. Unfortunately, data on Fe and Cu concentrations were not available, and we cannot fully exclude Cu versus Fe limitation as a controlling factor in N 2 O accumulation in the study lakes. Yet, the equal abundance of nirS and nirK genes does not suggest any adaptation of the microbial community to Cu limitation. Data on the nirS/nirK gene ratios in lakes are rare. The only study we know of in this context is by Martins et al. [31], who reported that nirS genes were more abundant than nirK genes in sediments of freshwater lakes on the Azores. In contrast, the average nirS/nirK gene ratio observed in this study was 1:1. Different from the subtropical lakes studied by Martins et al. [31], boreal lakes experience seasonal variations in redox and other physico-chemical conditions, which may increase the diversity of ecological niches and prevent certain microbial ecotypes from dominating an ecosystem. Since the distribution of nirS and nirK genes is phylogenetically scattered [10], the ratio of these two evolutionarily separate, but functionally equivalent, nitrite reductase gene types does not necessarily reflect the dominance of one taxonomical group over another as a function of environmental conditions. Instead, the relatively strong variability in the nirS and nirK gene ratio between the existing studies highlights the need to quantify both genes when studying the factors affecting N 2 O accumulation. Although the nir/ nos ratio at the DNA level does not necessarily correspond to the respective ratios at the level of mRNA transcripts or enzyme molecules on short-term time scales, it may indicate longerterm genetic adaptation, which was the focus of this study. N 2 O Production in Boreal Lakes PLOS ONE | DOI:10.1371/journal.pone.0121201 March 10, 2015 7/14 When comparing lakes at different spatial scales and between various geographical regions, denitrification rates have shown a clear positive correlation with nitrate availability [32]. This correlation was further corroborated by the observed co-variation of NO 3 − and N 2excess in the lakes studied here. Our study also showed the linkage between NO 3 − concentration and N 2 O accumulation, which is in agreement with previous work in boreal lakes [18]. Based on previously published N 2 production rates for five of the lakes in this study [29,32] (unpublished results), the N 2 O production rates reported here correspond to 0.2−1.7% of the total gaseous N production (N 2 O/(N 2 +N 2 O) ratio). These values fall within the range of previously reported estimates (0.1–4.1%) for freshwater systems [33]. Besides total denitrification rates, it is the balance between nitrite reduction and N 2 O reduction which controls the build-up of N 2 O. This balance has been shown to be sensitive to changes in redox conditions [34]; however, the role of longer-term nitrate availability in modulating this balance is uncertain. NO 3 − is generally the preferred electron acceptor for the denitrifying community when compared to N 2 O (except for some nosZ II carrying organisms, see the discussion below). Hence, when the competition for nitrate is tighter, reduction of N 2 O becomes a more feasible trait for the heterotrophic micro-organisms [35]. At the inter-lake scale, nir/nosZ I ratios correlated with the nitrate concentrations and N 2 O excess . These correlations suggest that the denitrifying communities were adapted to varying nitrate levels within the lake and that they control the ratio of N 2 O production versus reduction. Moreover, both at the inter-lake scale and within the Lake Vanajavesi transect the combined nir/nos ratio (i.e. [nirS+nirK]/nosZ I+II ) correlated with ambient nitrate. In contrast, the nir/nosZ I ratio did not display any statistically significant correlation with (the less variant) nitrate concentration in Lake Vanajavesi. This apparent difference with regards to the role of nosZ I and nosZ II may be related to the known genetics of nosZ II carrying organisms. The N 2 O reductase nosZ I has only been found for Alpha-, Beta-, and Gammaproteobacteria and some archaea, whereas nosZ II reductases may be common in a wider range of bacterial and archaeal phyla [7,8]. While most of the typical nosZ I -harboring microbes have the complete set of denitrification genes, less than half of the known nosZ II -carrying microorganisms possess genes of the “upstream”denitrification steps, and nosZ II -type reductase was thus named as “non-deni- trifier nitrous oxide reductase”[7]. As a consequence, many of the nosZ II -carrying microbes are incapable of using nitrate (or nitrite) as an electron acceptor, and are, therefore, less affected by ambient nitrate availability. The variable prevalence of denitrifying versus non-denitrifying nosZ II subsets may explain the above-described differences in the correlation analyses between the inter-lake and intra-lake studies (genetic relationships versus NO 3 − levels). Although it has been shown that denitrification is the major N 2 O source in lake ecosystems [19,27], it is likely that nitrifiers (i.e. ammonium oxidation and nitrifier-denitrification) also contribute to N 2 O production in these environments. In the lake transect, where sampling sites where characterized by different hypolimnetic oxygen regimes, N 2 O accumulation patterns were clearly linked to oxygen concentration. Concentration of N 2 O peaked near the oxic-anox- ic interface, which was located either in the sediment surface or in the water column. This could be due to O 2 availability just above the oxic-anoxic interface, which would increase N 2 O production via nitrification [36,37]. On the other hand, the presence of O 2 even at low levels likely inhibits N 2 O reduction compared to other reduction steps in denitrification [37]. Therefore, truncated denitrification would also lead to observed accumulation patterns of N 2 O, with concentration maxima in the vicinity of the redox transition zones. The lack of N 2 O accumulation in the anoxic water layers of the lakes further supports the notion that stable anoxic conditions are conducive to full denitrification to N 2 , while microaerophilic conditions would rather support truncated denitrification and/or slowed nitrous oxide reduction. In addition, dissimilatory nitrate reduction to ammonium (DNRA), in which N 2 O can also be formed as a by- N 2 O Production in Boreal Lakes PLOS ONE | DOI:10.1371/journal.pone.0121201 March 10, 2015 8/14