Microbial nitrogen transformations tracked by natural abundance isotope studies and microbiological methods: A review.

Nitrogen is an essential nutrient in the environment that exists in multiple oxidation states in nature. Numerous microbial processes are involved in its transformation. Knowledge about very complex N cycling has been growing rapidly in recent years, with new information about associated isotope effects and about the microbes involved in particular processes. Furthermore, molecular methods that are able to detect and quantify particular processes are being developed, applied and combined with other analytical approaches, which opens up new opportunities to enhance understanding of nitrogen transformation pathways. This review presents a summary of the microbial nitrogen transformation, including the respective isotope effects of nitrogen and oxygen on different nitrogen-bearing compounds (including nitrates, nitrites, ammonia and nitrous oxide), and the microbiological characteristics of these processes. It is supplemented by an overview of molecular methods applied for detecting and quantifying the activity of particular enzymes involved in N transformation pathways. This summary should help in the planning and interpretation of complex research studies applying isotope analyses of different N compounds and combining microbiological and isotopic methods in tracking complex N cycling, and in the integration of these results in modelling approaches.

Methanogenesis in biogas reactors under inhibitory ammonia concentration requires community-wide tolerance.

Ammonia (NH3) inhibition represents a major limitation to methane production during anaerobic digestion of organic material in biogas reactors. This process relies on co-operative metabolic interactions between diverse taxa at the community-scale. Despite this, most investigations have focused singularly on how methanogenic Archaea respond to NH3 stress. With a high-NH3 pre-adapted and un-adapted community, this study investigated responses to NH3 inhibition both at the community-scale and down to individual taxa. The pre-adapted community performed methanogenesis under inhibitory NH3 concentrations better than the un-adapted. While many functionally important phyla were shared between the two communities, only taxa from the pre-adapted community were robust to NH3. Functionally important phyla were mostly comprised of sensitive taxa (≥ 50%), yet all groups, including methanogens, also possessed tolerant individuals (10-50%) suggesting that potential mechanisms for tolerance are non-specific and widespread. Hidden Markov Model-based phylogenetic analysis of methanogens confirmed that NH3 tolerance was not restricted to specific taxonomic groups, even at the genus level. By reconstructing covarying growth patterns via network analyses, methanogenesis by the pre-adapted community was best explained by continued metabolic interactions (edges) between tolerant methanogens and other tolerant taxa (nodes). However, under non-inhibitory conditions, sensitive taxa re-emerged to dominate the pre-adapted community, suggesting that mechanisms of NH3 tolerance can be disadvantageous to fitness without selection pressure. This study demonstrates that methanogenesis under NH3 inhibition depends on broad-scale tolerance throughout the prokaryotic community. Mechanisms for tolerance seem widespread and non-specific, which has practical significance for the development of robust methanogenic biogas communities. KEY POINTS: • Ammonia pre-adaptation allows for better methanogenesis under inhibitory conditions. • All functionally important prokaryote phyla have some ammonia tolerant individuals. • Methanogenesis was likely dependent on interactions between tolerant individuals.

Estuarine plastisphere as an overlooked source of N2O production.

"Plastisphere", microbial communities colonizing plastic debris, has sparked global concern for marine ecosystems. Microbiome inhabiting this novel human-made niche has been increasingly characterized; however, whether the plastisphere holds crucial roles in biogeochemical cycling remains largely unknown. Here we evaluate the potential of plastisphere in biotic and abiotic denitrification and nitrous oxide (N2O) production in estuaries. Biofilm formation provides anoxic conditions favoring denitrifiers. Comparing with surrounding bulk water, plastisphere exhibits a higher denitrifying activity and N2O production, suggesting an overlooked N2O source. Regardless of plastisphere and bulk water, bacterial and fungal denitrifications are the main regulators for N2O production instead of chemodenitrification. However, the contributions of bacteria and fungi in the plastisphere are different from those in bulk water, indicating a distinct N2O production pattern in the plastisphere. These findings pinpoint plastisphere as a N2O source, and provide insights into roles of the new biotope in biogeochemical cycling in the Anthropocene.

Indications for enzymatic denitrification to N2O at low pH in an ammonia-oxidizing archaeon.

Nitrous oxide (N2O) is a key climate change gas and nitrifying microbes living in terrestrial ecosystems contribute significantly to its formation. Many soils are acidic and global change will cause acidification of aquatic and terrestrial ecosystems, but the effect of decreasing pH on N2O formation by nitrifiers is poorly understood. Here, we used isotope-ratio mass spectrometry to investigate the effect of acidification on production of N2O by pure cultures of two ammonia-oxidizing archaea (AOA; Nitrosocosmicus oleophilus and Nitrosotenuis chungbukensis) and an ammonia-oxidizing bacterium (AOB; Nitrosomonas europaea). For all three strains acidification led to increased emission of N2O. However, changes of 15N site preference (SP) values within the N2O molecule (as indicators of pathways for N2O formation), caused by decreasing pH, were highly different between the tested AOA and AOB. While acidification decreased the SP value in the AOB strain, SP values increased to a maximum value of 29‰ in N. oleophilus. In addition, 15N-nitrite tracer experiments showed that acidification boosted nitrite transformation into N2O in all strains, but the incorporation rate was different for each ammonia oxidizer. Unexpectedly, for N. oleophilus more than 50% of the N2O produced at pH 5.5 had both nitrogen atoms from nitrite and we demonstrated that under these conditions expression of a putative cytochrome P450 NO reductase is strongly upregulated. Collectively, our results indicate that N. oleophilus might be able to enzymatically denitrify nitrite to N2O at low pH.

Use of oxygen isotopes to differentiate between nitrous oxide produced by fungi or bacteria during denitrification.

RATIONALE: Fungal denitrifiers can contribute substantially to N2 O emissions from arable soil and show a distinct site preference for N2 O (SP(N2 O)). This study sought to identify another process-specific isotopic tool to improve precise identification of N2 O of fungal origin by mass spectrometric analysis of the N2 O produced. METHODS: Three pure bacterial and three fungal species were incubated under denitrifying conditions in treatments with natural abundance and stable isotope labelling to analyse the N2 O produced. Combining different applications of isotope ratio mass spectrometry enabled us to estimate the oxygen (O) exchange accelerated by denitrifying enzymes and the ongoing microbial pathway in parallel. This experimental set-up allowed the determination of δ18 O(N2 O) values and isotopic fractionation of O, as well as SP(N2 O) values, as a perspective to differentiate between microbial denitrifiers. RESULTS: Oxygen exchange during N2 O production was lower for bacteria than for fungi, differed between species, and depended also on incubation time. Apparent O isotopic fractionation during denitrification was in a similar range for bacteria and fungi, but application of the fractionation model indicated that different enzymes in bacteria and fungi were responsible for O exchange. This difference was associated with different isotopic fractionation for bacteria and fungi. CONCLUSIONS: δ18 O(N2 O) values depend on isotopic fractionation and isotopic fractionation may differ between processes and organism groups. By comparing SP(N2 O) values, O exchange and the isotopic signature of precursors, we propose here a novel tool for differentiating between different sources of N2 O.

Dual isotope and isotopomer signatures of nitrous oxide from fungal denitrification--a pure culture study.

RATIONALE: The contribution of fungal denitrification to the emission of the greenhouse gas nitrous oxide (N2O) from soil has not yet been sufficiently investigated. The intramolecular (15)N site preference (SP) of N2O could provide a tool to distinguish between N2O produced by bacteria or fungi, since in previous studies fungi exhibited much higher SP values than bacteria. METHODS: To further constrain isotopic evidence of fungal denitrification, we incubated six soil fungal strains under denitrifying conditions, with either NO3(-) or NO2(-) as the electron acceptor, and measured the isotopic signature (δ(18)O, δ(15)Nbulk and SP values) of the N2O produced. The nitrogen isotopic fractionation was calculated and the oxygen isotope exchange associated with particular fungal enzymes was estimated. RESULTS: Five fungi of the order Hypocreales produced N2O with a SP of 35.1 ± 1.7 ‰ after 7 days of anaerobic incubation independent of the electron acceptor, whereas one Sordariales species produced N2O from NO2(-) only, with a SP value of 21.9 ± 1.4 ‰. Smaller isotope effects of (15)Nbulk were associated with larger N2O production. The δ(18)O values were influenced by oxygen exchange between water and denitrification intermediates, which occurred primarily at the nitrite reduction step. CONCLUSIONS: Our results confirm that SP of N2O is a promising tool to differentiate between fungal and bacterial N2O from denitrification. Modelling of oxygen isotope fractionation processes indicated that the contribution of the NO2(-) and NO reduction steps to the total oxygen exchange differed among the various fungal species studied. However, more information is needed about different biological orders of fungi as they may differ in denitrification enzymes and consequently in the SP and δ(18)O values of the N2O produced.

Fungal oxygen exchange between denitrification intermediates and water.

RATIONALE: Fungi can contribute greatly to N2O production from denitrification. Therefore, it is important to quantify the isotopic signature of fungal N2O. The isotopic composition of N2O can be used to identify and analyze the processes of N2O production and N2O reduction. In contrast to bacteria, information about the oxygen exchange between denitrification intermediates and water during fungal denitrification is lacking, impeding the explanatory power of stable isotope methods. METHODS: Six fungal species were anaerobically incubated with the electron acceptors nitrate or nitrite and (18)O-labeled water to determine the oxygen exchange between denitrification intermediates and water. After seven days of incubation, gas samples were analyzed for N2O isotopologues by isotope ratio mass spectrometry. RESULTS: All the fungal species produced N2O. N2O production was greater when nitrite was the sole electron acceptor (129 to 6558 nmol N2O g dw(-1) h(-1)) than when nitrate was the electron acceptor (6 to 47 nmol N2O g dw(-1) h(-1)). Oxygen exchange was complete with nitrate as electron acceptor in one of five fungi and with nitrite in two of six fungi. Oxygen exchange of the other fungi varied (41 to 89% with nitrite and 11 to 61% with nitrate). CONCLUSIONS: This is the first report on oxygen exchange with water during fungal denitrification. The exchange appears to be within the range previously reported for bacterial denitrification. This adds to the difficulty of differentiating N2O producing processes based on the origin of N2O-O. However, the large oxygen exchange repeatedly observed for bacteria and now also fungi could lead to less variability in the δ(18)O values of N2O from soils, which could facilitate the assessment of the extent of N2O reduction.

An enhanced technique for automated determination of 15N signatures of N2, (N2 +N2O) and N2O in gas samples.

RATIONALE: An enhanced analytical approach for analyzing gaseous products from (15)N-enriched pools has been developed. This technique can be used to quantify nitrous oxide (N2O) and dinitrogen (N2) fluxes from denitrification. It can also help in distinguishing different N2- and N2O-forming processes, such as denitrification, nitrification, anaerobic ammonium oxidation or co-denitrification. METHODS: The measurement instrumentation was based on a commercially available automatic preparation system allowing collection and separation of gaseous samples. The sample transfer paths, valves, liquid nitrogen traps, gas chromatography column and open split of the original system were modified. A reduction oven (Cu) was added in order to eliminate oxygen and measure N2O-N as N2. Gases leaving the separation system entered an isotope ratio mass spectrometer where masses (28)N2, (29)N2 and (30)N2 were measured. RESULTS: The enhanced technique enabled rapid simultaneous measurement of stable isotope ratios (29)N2/(28)N2 and (30)N2/(28)N2 originating from dinitrogen alone (N2) and from the sum of the denitrification products (N2 +N2O) as well as the determination of (15)N enrichment in N2O. The (15)N fraction in the N pool undergoing N2 and N2O production ((15)X(N)) and the contribution of N2 and N2O originating from this pool (d) were determined with satisfactory accuracy of better than 3.3% and 2.9%, respectively. CONCLUSIONS: The precision and accuracy of this method were comparable with or better than previously reported for similar measurements. The proposed method allows for the analysis of all quantities within one run, thus reducing the measurement and sample preparation time as well as increasing the reliability of the results.

From population-level effects to individual response: modelling temperature dependence in Gammarus pulex.

Population-level effects of global warming result from concurrent direct and indirect processes. They are typically described by physiologically structured population models (PSPMs). Therefore, inverse modelling offers a tool to identify parameters of individual physiological processes through population-level data analysis, e.g. the temperature dependence of growth from size-frequency data of a field population. Here, we make use of experiments under laboratory conditions, in mesocosms and field monitoring to determine the temperature dependence of growth and mortality of Gammarus pulex. We found an optimum temperature for growth of approximately 17°C and a related temperature coefficient, Q(10), of 1.5°C(-1), irrespective of whether we classically fitted individual growth curves or applied inverse modelling based on PSPMs to laboratory data. From a comparison of underlying data sets we conclude that applying inverse modelling techniques to population-level data results in meaningful response parameters for physiological processes if additional temperature-driven effects, including within-population interaction, can be excluded or determined independently. If this is not the case, parameter estimates describe a cumulative response, e.g. comprising temperature-dependent resource dynamics. Finally, fluctuating temperatures in natural habitats increased the uncertainty in parameter values. Here, PSPM should be applied for virtual monitoring in order to determine a sampling scheme that comprises important dates to reduce parameter uncertainty.