Research Summary: Genetic And Environmental Controls On Nitrous Oxide Accumulation In Lakes

by Jatta Saarenheimo, Antti J. Rissanen, Lauri Arvola, Hannu Nykänen, Moritz F. Lehmann and Marja Tiirola
— March 23, 2016

Abstract

We studied potential links between environmental factors, nitrous oxide (N₂O) accumulation, and genetic indicators of nitrite and N₂O reducing bacteria in 12 boreal lakes. Denitrifying bacteria were investigated by quantifying genes encoding nitrite and N₂O reductases (nirS/nirKand nosZ, respectively, including the two phylogenetically distinct clades nosZ_I and nosZ_II) in lake sediments. Summertime N₂O 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’s nir-vs-nos gene inventories and N₂O accumulation. Inter-lake variation in N₂O accumulation was indeed connected to the relative abundance of nitrite versus N₂O 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 N₂O accumulation in lake ecosystems, either directly by increasing the overall rate of denitrification or indirectly by controlling the balance of nitrite versus N₂O reductase carrying organisms.

Lake Vanajavesi. (Credit: Wikimedia Commons User Alessio Damato via Creative Commons 3.0)

Introduction

Nitrous oxide (N₂O) is an important greenhouse gas and the single most important ozone destroying chemical [1]. N₂O 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 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 N₂O 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 N₂O 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 N₂O 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 N₂O accumulation [18, 19], fostering the increase the N₂O/(N₂O+N₂) 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 N₂O/(N₂O+N₂) production ratio.

In soils, microbially produced N₂O 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 cm² s⁻¹, [23]), reducing diffusive loss rates and improving the N₂O availability for nosZ carrying bacteria. More complete denitrification and lower N₂O/N₂ gas emission ratios should, therefore, be expected for the aquatic versus soil environments. Still, lake ecosystems have shown to be important sites of N₂O emissions [19, 24], and, as in soils, N₂O 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 N₂O 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 N₂O 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 N₂O production and accumulation in lake ecosystems, especially focusing on the benthic abundance of nirS, nirK,nosZ_I, and nosZ_II genes during the summertime N₂O accumulation period. Anticipating close links between nitrate concentrations and the N₂O 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 N₂O accumulation. The linkage between benthic denitrification gene frequency and N₂O 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-NO₃⁻-lakes and low-NO₃⁻-lakes). In addition, denitrification gene abundance and N₂O 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).