Inhibition profile of three biological nitrification inhibitors and their response to soil pH modification in two contrasting soils.

Up to 70% of the nitrogen (N) fertilizer applied to agricultural soils is lost through microbially mediated processes, such as nitrification. This can be counteracted by synthetic and biological compounds that inhibit nitrification. However, for many biological nitrification inhibitors (BNIs), the interaction with soil properties, nitrifier specificity, and effective concentrations are unclear. Here, we investigated three synthetic nitrification inhibitors (SNIs) (DCD, DMPP, and nitrapyrin) and three BNIs [methyl 3(4-hydroxyphenyl) propionate (MHPP), methyl 3(4-hydroxyphenyl) acrylate (MHPA), and limonene] in two agricultural soils differing in pH and nitrifier communities. The efficacies of SNIs and BNIs were resilient to short-term pH changes in the neutral pH soil, whereas the efficacy of some BNIs increased by neutralizing the alkaline soil. Among the BNIs, MHPA showed the highest inhibition and was, together with MHPP, identified as a putative AOB/comammox-selective inhibitor. Additionally, MHPA and limonene effectively inhibited nitrification at concentrations comparable to those used for DCD. Moreover, we identified the effective concentrations at which 50% and 80% of inhibition is observed (EC50 and EC80) for the BNIs, and similar EC80 values were observed in both soils. Overall, our results show that these BNIs could potentially serve as effective alternatives to SNIs currently used.

Challenges in measuring nitrogen isotope signatures in inorganic nitrogen forms: An interlaboratory comparison of three common measurement approaches.

RATIONALE: Stable isotope approaches are increasingly applied to better understand the cycling of inorganic nitrogen (Ni ) forms, key limiting nutrients in terrestrial and aquatic ecosystems. A systematic comparison of the accuracy and precision of the most commonly used methods to analyze δ15 N in NO3 - and NH4 + and interlaboratory comparison tests to evaluate the comparability of isotope results between laboratories are, however, still lacking. METHODS: Here, we conducted an interlaboratory comparison involving 10 European laboratories to compare different methods and laboratory performance to measure δ15 N in NO3 - and NH4 + . The approaches tested were (a) microdiffusion (MD), (b) chemical conversion (CM), which transforms Ni to either N2 O (CM-N2 O) or N2 (CM-N2 ), and (c) the denitrifier (DN) methods. RESULTS: The study showed that standards in their single forms were reasonably replicated by the different methods and laboratories, with laboratories applying CM-N2 O performing superior for both NO3 - and NH4 + , followed by DN. Laboratories using MD significantly underestimated the "true" values due to incomplete recovery and also those using CM-N2 showed issues with isotope fractionation. Most methods and laboratories underestimated the at%15 N of Ni of labeled standards in their single forms, but relative errors were within maximal 6% deviation from the real value and therefore acceptable. The results showed further that MD is strongly biased by nonspecificity. The results of the environmental samples were generally highly variable, with standard deviations (SD) of up to ± 8.4‰ for NO3 - and ± 32.9‰ for NH4 + ; SDs within laboratories were found to be considerably lower (on average 3.1‰). The variability could not be connected to any single factor but next to errors due to blank contamination, isotope normalization, and fractionation, and also matrix effects and analytical errors have to be considered. CONCLUSIONS: The inconsistency among all methods and laboratories raises concern about reported δ15 N values particularly from environmental samples.

Down-regulation of the bacterial protein biosynthesis machinery in response to weeks, years, and decades of soil warming.

How soil microorganisms respond to global warming is key to infer future soil-climate feedbacks, yet poorly understood. Here, we applied metatranscriptomics to investigate microbial physiological responses to medium-term (8 years) and long-term (>50 years) subarctic grassland soil warming of +6°C. Besides indications for a community-wide up-regulation of centralmetabolic pathways and cell replication, we observed a down-regulation of the bacterial protein biosynthesis machinery in the warmed soils, coinciding with a lower microbial biomass, RNA, and soil substrate content. We conclude that permanently accelerated reaction rates at higher temperatures and reduced substrate concentrations result in cellular reduction of ribosomes, the macromolecular complexes carrying out protein biosynthesis. Later efforts to test this, including a short-term warming experiment (6 weeks, +6°C), further supported our conclusion. Down-regulating the protein biosynthesis machinery liberates energy and matter, allowing soil bacteria to maintain high metabolic activities and cell division rates even after decades of warming.

Contrasting drivers of belowground nitrogen cycling in a montane grassland exposed to a multifactorial global change experiment with elevated CO2 , warming, and drought.

Depolymerization of high-molecular weight organic nitrogen (N) represents the major bottleneck of soil N cycling and yet is poorly understood compared to the subsequent inorganic N processes. Given the importance of organic N cycling and the rise of global change, we investigated the responses of soil protein depolymerization and microbial amino acid consumption to increased temperature, elevated atmospheric CO2 , and drought. The study was conducted in a global change facility in a managed montane grassland in Austria, where elevated CO2 (eCO2 ) and elevated temperature (eT) were stimulated for 4 years, and were combined with a drought event. Gross protein depolymerization and microbial amino acid consumption rates (alongside with gross organic N mineralization and nitrification) were measured using 15 N isotope pool dilution techniques. Whereas eCO2  showed no individual effect, eT had distinct effects which were modulated by season, with a negative effect of eT on soil organic N process rates in spring, neutral effects in summer, and positive effects in fall. We attribute this to a combination of changes in substrate availability and seasonal temperature changes. Drought led to a doubling of organic N process rates, which returned to rates found under ambient conditions within 3 months after rewetting. Notably, we observed a shift in the control of soil protein depolymerization, from plant substrate controls under continuous environmental change drivers (eT and eCO2 ) to controls via microbial turnover and soil organic N availability under the pulse disturbance (drought). To the best of our knowledge, this is the first study which analyzed the individual versus combined effects of multiple global change factors and of seasonality on soil organic N processes and thereby strongly contributes to our understanding of terrestrial N cycling in a future world.

Increased microbial expression of organic nitrogen cycling genes in long-term warmed grassland soils.

Global warming increases soil temperatures and promotes faster growth and turnover of soil microbial communities. As microbial cell walls contain a high proportion of organic nitrogen, a higher turnover rate of microbes should also be reflected in an accelerated organic nitrogen cycling in soil. We used a metatranscriptomics and metagenomics approach to demonstrate that the relative transcription level of genes encoding enzymes involved in the extracellular depolymerization of high-molecular-weight organic nitrogen was higher in medium-term (8 years) and long-term (>50 years) warmed soils than in ambient soils. This was mainly driven by increased levels of transcripts coding for enzymes involved in the degradation of microbial cell walls and proteins. Additionally, higher transcription levels for chitin, nucleic acid, and peptidoglycan degrading enzymes were found in long-term warmed soils. We conclude that an acceleration in microbial turnover under warming is coupled to higher investments in N acquisition enzymes, particularly those involved in the breakdown and recycling of microbial residues, in comparison with ambient conditions.

Composition and activity of nitrifier communities in soil are unresponsive to elevated temperature and CO2, but strongly affected by drought.

Nitrification is a fundamental process in terrestrial nitrogen cycling. However, detailed information on how climate change affects the structure of nitrifier communities is lacking, specifically from experiments in which multiple climate change factors are manipulated simultaneously. Consequently, our ability to predict how soil nitrogen (N) cycling will change in a future climate is limited. We conducted a field experiment in a managed grassland and simultaneously tested the effects of elevated atmospheric CO2, temperature, and drought on the abundance of active ammonia-oxidizing bacteria (AOB) and archaea (AOA), comammox (CMX) Nitrospira, and nitrite-oxidizing bacteria (NOB), and on gross mineralization and nitrification rates. We found that N transformation processes, as well as gene and transcript abundances, and nitrifier community composition were remarkably resistant to individual and interactive effects of elevated CO2 and temperature. During drought however, process rates were increased or at least maintained. At the same time, the abundance of active AOB increased probably due to higher NH4+ availability. Both, AOA and comammox Nitrospira decreased in response to drought and the active community composition of AOA and NOB was also significantly affected. In summary, our findings suggest that warming and elevated CO2 have only minor effects on nitrifier communities and soil biogeochemical variables in managed grasslands, whereas drought favors AOB and increases nitrification rates. This highlights the overriding importance of drought as a global change driver impacting on soil microbial community structure and its consequences for N cycling.

Increased microbial growth, biomass, and turnover drive soil organic carbon accumulation at higher plant diversity.

Species-rich plant communities have been shown to be more productive and to exhibit increased long-term soil organic carbon (SOC) storage. Soil microorganisms are central to the conversion of plant organic matter into SOC, yet the relationship between plant diversity, soil microbial growth, turnover as well as carbon use efficiency (CUE) and SOC accumulation is unknown. As heterotrophic soil microbes are primarily carbon limited, it is important to understand how they respond to increased plant-derived carbon inputs at higher plant species richness (PSR). We used the long-term grassland biodiversity experiment in Jena, Germany, to examine how microbial physiology responds to changes in plant diversity and how this affects SOC content. The Jena Experiment considers different numbers of species (1-60), functional groups (1-4) as well as functional identity (small herbs, tall herbs, grasses, and legumes). We found that PSR accelerated microbial growth and turnover and increased microbial biomass and necromass. PSR also accelerated microbial respiration, but this effect was less strong than for microbial growth. In contrast, PSR did not affect microbial CUE or biomass-specific respiration. Structural equation models revealed that PSR had direct positive effects on root biomass, and thereby on microbial growth and microbial biomass carbon. Finally, PSR increased SOC content via its positive influence on microbial biomass carbon. We suggest that PSR favors faster rates of microbial growth and turnover, likely due to greater plant productivity, resulting in higher amounts of microbial biomass and necromass that translate into the observed increase in SOC. We thus identify the microbial mechanism linking species-rich plant communities to a carbon cycle process of importance to Earth's climate system.

A systemic overreaction to years versus decades of warming in a subarctic grassland ecosystem.

Temperature governs most biotic processes, yet we know little about how warming affects whole ecosystems. Here we examined the responses of 128 components of a subarctic grassland to either 5-8 or >50 years of soil warming. Warming of >50 years drove the ecosystem to a new steady state possessing a distinct biotic composition and reduced species richness, biomass and soil organic matter. However, the warmed state was preceded by an overreaction to warming, which was related to organism physiology and was evident after 5-8 years. Ignoring this overreaction yielded errors of >100% for 83 variables when predicting their responses to a realistic warming scenario of 1 °C over 50 years, although some, including soil carbon content, remained stable after 5-8 years. This study challenges long-term ecosystem predictions made from short-term observations, and provides a framework for characterization of ecosystem responses to sustained climate change.

Plant roots increase both decomposition and stable organic matter formation in boreal forest soil.

Boreal forests are ecosystems with low nitrogen (N) availability that store globally significant amounts of carbon (C), mainly in plant biomass and soil organic matter (SOM). Although crucial for future climate change predictions, the mechanisms controlling boreal C and N pools are not well understood. Here, using a three-year field experiment, we compare SOM decomposition and stabilization in the presence of roots, with exclusion of roots but presence of fungal hyphae and with exclusion of both roots and fungal hyphae. Roots accelerate SOM decomposition compared to the root exclusion treatments, but also promote a different soil N economy with higher concentrations of organic soil N compared to inorganic soil N accompanied with the build-up of stable SOM-N. In contrast, root exclusion leads to an inorganic soil N economy (i.e., high level of inorganic N) with reduced stable SOM-N build-up. Based on our findings, we provide a framework on how plant roots affect SOM decomposition and stabilization.

Rapid Transfer of Plant Photosynthates to Soil Bacteria via Ectomycorrhizal Hyphae and Its Interaction With Nitrogen Availability.

Plant roots release recent photosynthates into the rhizosphere, accelerating decomposition of organic matter by saprotrophic soil microbes ("rhizosphere priming effect") which consequently increases nutrient availability for plants. However, about 90% of all higher plant species are mycorrhizal, transferring a significant fraction of their photosynthates directly to their fungal partners. Whether mycorrhizal fungi pass on plant-derived carbon (C) to bacteria in root-distant soil areas, i.e., incite a "hyphosphere priming effect," is not known. Experimental evidence for C transfer from mycorrhizal hyphae to soil bacteria is limited, especially for ectomycorrhizal systems. As ectomycorrhizal fungi possess enzymatic capabilities to degrade organic matter themselves, it remains unclear whether they cooperate with soil bacteria by providing photosynthates, or compete for available nutrients. To investigate a possible C transfer from ectomycorrhizal hyphae to soil bacteria, and its response to changing nutrient availability, we planted young beech trees (Fagus sylvatica) into "split-root" boxes, dividing their root systems into two disconnected soil compartments. Each of these compartments was separated from a litter compartment by a mesh penetrable for fungal hyphae, but not for roots. Plants were exposed to a 13C-CO2-labeled atmosphere, while 15N-labeled ammonium and amino acids were added to one side of the split-root system. We found a rapid transfer of recent photosynthates via ectomycorrhizal hyphae to bacteria in root-distant soil areas. Fungal and bacterial phospholipid fatty acid (PLFA) biomarkers were significantly enriched in hyphae-exclusive compartments 24 h after 13C-CO2-labeling. Isotope imaging with nanometer-scale secondary ion mass spectrometry (NanoSIMS) allowed for the first time in situ visualization of plant-derived C and N taken up by an extraradical fungal hypha, and in microbial cells thriving on hyphal surfaces. When N was added to the litter compartments, bacterial biomass, and the amount of incorporated 13C strongly declined. Interestingly, this effect was also observed in adjacent soil compartments where added N was only available for bacteria through hyphal transport, indicating that ectomycorrhizal fungi were acting on soil bacteria. Together, our results demonstrate that (i) ectomycorrhizal hyphae rapidly transfer plant-derived C to bacterial communities in root-distant areas, and (ii) this transfer promptly responds to changing soil nutrient conditions.

Full 15N tracer accounting to revisit major assumptions of 15N isotope pool dilution approaches for gross nitrogen mineralization.

The 15N isotope pool dilution (IPD) technique is the only available method for measuring gross ammonium (NH4 +) production and consumption rates. Rapid consumption of the added 15N-NH4 + tracer is commonly observed, but the processes responsible for this consumption are not well understood. The primary objectives of this study were to determine the relative roles of biotic and abiotic processes in 15N-NH4 + sconsumption and to investigate the validity of one of the main assumptions of IPD experiments, i.e., that no reflux of the consumed 15N tracer occurs during the course of the experiments. We added a 15N-NH4 + tracer to live and sterile (autoclaved) soil using mineral topsoil from a beech forest and a grassland in Austria that differed in NH4 + concentrations and NH4 + consumption kinetics. We quantified both biotic tracer consumption (i.e. changes in the concentrations and 15N enrichments of NH4 +, dissolved organic N (DON), NO3 - and the microbial N pool) and abiotic tracer consumption (i.e., fixation by clay and/or humic substances). We achieved full recovery of the 15N tracer in both soils over the course of the 48 h incubation. For the forest soil, we found no rapid consumption of the 15N tracer, and the majority of tracer (78%) remained unconsumed at the end of the incubation period. In contrast, the grassland soil showed rapid 15N-NH4 + consumption immediately after tracer addition, which was largely due to both abiotic fixation (24%) and biotic processes, largely uptake by soil microbes (10%) and nitrification (13%). We found no evidence for reflux of 15N-NH4 + over the 48 h incubation period in either soil. Our study therefore shows that 15N tracer reflux during IPD experiments is negligible for incubation times of up to 48 h, even when rapid NH4 + consumption occurs. Such experiments are thus robust to the assumption that immobilized labeled N is not re-mobilized during the experimental period and does not impact calculations of gross N mineralization.

Biochar decelerates soil organic nitrogen cycling but stimulates soil nitrification in a temperate arable field trial.

Biochar production and subsequent soil incorporation could provide carbon farming solutions to global climate change and escalating food demand. There is evidence that biochar amendment causes fundamental changes in soil nutrient cycles, often resulting in marked increases in crop production, particularly in acidic and in infertile soils with low soil organic matter contents, although comparable outcomes in temperate soils are variable. We offer insight into the mechanisms underlying these findings by focusing attention on the soil nitrogen (N) cycle, specifically on hitherto unmeasured processes of organic N cycling in arable soils. We here investigated the impacts of biochar addition on soil organic and inorganic N pools and on gross transformation rates of both pools in a biochar field trial on arable land (Chernozem) in Traismauer, Lower Austria. We found that biochar increased total soil organic carbon but decreased the extractable organic C pool and soil nitrate. While gross rates of organic N transformation processes were reduced by 50-80%, gross N mineralization of organic N was not affected. In contrast, biochar promoted soil ammonia-oxidizer populations (bacterial and archaeal nitrifiers) and accelerated gross nitrification rates more than two-fold. Our findings indicate a de-coupling of the soil organic and inorganic N cycles, with a build-up of organic N, and deceleration of inorganic N release from this pool. The results therefore suggest that addition of inorganic fertilizer-N in combination with biochar could compensate for the reduction in organic N mineralization, with plants and microbes drawing on fertilizer-N for growth, in turn fuelling the belowground build-up of organic N. We conclude that combined addition of biochar with fertilizer-N may increase soil organic N in turn enhancing soil carbon sequestration and thereby could play a fundamental role in future soil management strategies.

Endogenous nitric oxide generation in protoplast chloroplasts.

KEY MESSAGE : NO generation is studied in the protoplast chloroplasts. NO, ONOO ( - ) and ROS (O ( 2 ) ( - ) and H ( 2 ) O ( 2 ) ) are generated in chloroplasts. Nitric oxide synthase-like protein appears to be involved in NO generation. Nitric oxide stimulates chlorophyll biosynthesis and chloroplast differentiation. The present study was conducted to better understand the process of NO generation in the leaf chloroplasts and protoplasts. NO, peroxynitrite and superoxide anion were investigated in the protoplasts and isolated chloroplasts using specific dyes, confocal laser scanning and light microscopy. The level of NO was highest after protoplast isolation and subsequently decreased during culture. Suppression of NO signal in the presence of PTIO, suggests that diaminofluorescein-2 diacetate (DAF-2DA) detected NO. Detection of peroxynitrite, a reaction product of NO and superoxide anion, further suggests NO generation. Moreover, generation of NO and peroxynitrite in the chloroplasts of wild-type Arabidopsis and their absence or weak signals in the leaf-derived protoplasts of Atnoa1 mutants confirmed the reactivity of DAF-2DA and aminophenyl fluorescein to NO and peroxynitrite, respectively. Isolated chloroplasts also showed signal of NO. Suppression of NO signal in the presence of 100 µM nitric oxide synthase inhibitors [L-NNA, Nω-nitro-L-arginine and PBIT, S,S'-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea] revealed that nitric oxide synthase-like system is involved in NO synthesis. Suppression of NO signal in the protoplasts isolated in the presence of cycloheximide suggests de novo synthesis of NO generating protein during the process of protoplast isolation. Furthermore, the lack of inhibition of NO production by sodium tungstate (250 µM) and inhibition by L-NNA, and PBIT suggest involvement NOS-like protein, but not nitrate reductase, in NO generation in the leaf chloroplasts and protoplasts.