Successional changes in bacterial phyllosphere communities are plant-host species dependent.

Phyllosphere microbial communities are increasingly experiencing intense pulse disturbance events such as drought. It is currently unknown how phyllosphere communities respond to such disturbances and if they are able to recover. We explored the stability of phyllosphere communities over time, in response to drought stress, and under recovery from drought on temperate forage grasses. Compositional or functional changes were observed during the disturbance period and whether communities returned to non-stressed levels following recovery. Here, we found that phyllosphere community composition shifts as a result of simulated drought but does not fully recover after irrigation is resumed and that the degree of community response to drought is host species dependent. However, while community composition had changed, we found a high level of functional stability (resistance) over time and in the water deficit treatment. Ecological modeling enabled us to understand community assembly processes over a growing season and to determine if they were disrupted during a disturbance event. Phyllosphere community succession was characterized by a strong level of ecological drift, but drought disturbance resulted in variable selection, or, in other words, communities were diverging due to differences in selective pressures. This successional divergence of communities with drought was unique for each host species. Understanding phyllosphere responses to environmental stresses is important as climate change-induced stresses are expected to reduce crop productivity and phyllosphere functioning. IMPORTANCE: Leaf surface microbiomes have the potential to influence agricultural and ecosystem productivity. We assessed their stability by determining composition, functional resistance, and resilience. Resistance is the degree to which communities remain unchanged as a result of disturbance, and resilience is the ability of a community to recover to pre-disturbance conditions. By understanding the mechanisms of community assembly and how they relate to the resistance and resilience of microbial communities under common environmental stresses such as drought, we can better understand how communities will adapt to a changing environment and how we can promote healthy agricultural microbiomes. In this study, phyllosphere compositional stability was highly related to plant host species phylogeny and, to a lesser extent, known stress tolerances. Phyllosphere community assembly and stability are a result of complex interactions of ecological processes that are differentially imposed by host species.

Long-term soil warming changes the profile of primary metabolites in fine roots of Norway spruce in a temperate montane forest.

Climate warming poses major threats to temperate forests, but the response of tree root metabolism has largely remained unclear. We examined the impact of long-term soil warming (>14 years, +4°C) on the fine root metabolome across three seasons for 2 years in an old spruce forest, using a liquid chromatography-mass spectrometry platform for primary metabolite analysis. A total of 44 primary metabolites were identified in roots (19 amino acids, 12 organic acids and 13 sugars). Warming increased the concentration of total amino acids and of total sugars by 15% and 21%, respectively, but not organic acids. We found that soil warming and sampling date, along with their interaction, directly influenced the primary metabolite profiles. Specifically, in warming plots, concentrations of arginine, glycine, lysine, threonine, tryptophan, mannose, ribose, fructose, glucose and oxaloacetic acid increased by 51.4%, 19.9%, 21.5%, 19.3%, 22.1%, 23.0%, 38.0%, 40.7%, 19.8% and 16.7%, respectively. Rather than being driven by single compounds, changes in metabolite profiles reflected a general up- or downregulation of most metabolic pathway network. This emphasises the importance of metabolomics approaches in investigating root metabolic pathways and understanding the effects of climate change on tree root metabolism.

Does long-term soil warming affect microbial element limitation? A test by short-term assays of microbial growth responses to labile C, N and P additions.

Increasing global temperatures have been reported to accelerate soil carbon (C) cycling, but also to promote nitrogen (N) and phosphorus (P) dynamics in terrestrial ecosystems. However, warming can differentially affect ecosystem C, N and P dynamics, potentially intensifying elemental imbalances between soil resources, plants and soil microorganisms. Here, we investigated the effect of long-term soil warming on microbial resource limitation, based on measurements of microbial growth (18 O incorporation into DNA) and respiration after C, N and P amendments. Soil samples were taken from two soil depths (0-10, 10-20 cm) in control and warmed (>14 years warming, +4°C) plots in the Achenkirch soil warming experiment. Soils were amended with combinations of glucose-C, inorganic/organic N and inorganic/organic P in a full factorial design, followed by incubation at their respective mean field temperatures for 24 h. Soil microbes were generally C-limited, exhibiting 1.8-fold to 8.8-fold increases in microbial growth upon C addition. Warming consistently caused soil microorganisms to shift from being predominately C limited to become C-P co-limited. This P limitation possibly was due to increased abiotic P immobilization in warmed soils. Microbes further showed stronger growth stimulation under combined glucose and inorganic nutrient amendments compared to organic nutrient additions. This may be related to a prolonged lag phase in organic N (glucosamine) mineralization and utilization compared to glucose. Soil respiration strongly positively responded to all kinds of glucose-C amendments, while responses of microbial growth were less pronounced in many of these treatments. This highlights that respiration-though easy and cheap to measure-is not a good substitute of growth when assessing microbial element limitation. Overall, we demonstrate a significant shift in microbial element limitation in warmed soils, from C to C-P co-limitation, with strong repercussions on the linkage between soil C, N and P cycles under long-term warming.

Uptake, Metabolism, and Accumulation of Tire Wear Particle-Derived Compounds in Lettuce.

Tire wear particle (TWP)-derived compounds may be of high concern to consumers when released in the root zone of edible plants. We exposed lettuce plants to the TWP-derived compounds diphenylguanidine (DPG), hexamethoxymethylmelamine (HMMM), benzothiazole (BTZ), N-phenyl-N'-(1,3-dimethylbutyl)-p-phenylenediamine (6PPD), and its quinone transformation product (6PPD-q) at concentrations of 1 mg L-1 in hydroponic solutions over 14 days to analyze if they are taken up and metabolized by the plants. Assuming that TWP may be a long-term source of TWP-derived compounds to plants, we further investigated the effect of leaching from TWP on the concentration of leachate compounds in lettuce leaves by adding constantly leaching TWP to the hydroponic solutions. Concentrations in leaves, roots, and nutrient solution were quantified by triple quadrupole mass spectrometry, and metabolites in the leaves were identified by Orbitrap high resolution mass spectrometry. This study demonstrates that TWP-derived compounds are readily taken up by lettuce with measured maximum leaf concentrations between ∼0.75 (6PPD) and 20 µg g-1 (HMMM). Although these compounds were metabolized in the plant, we identified several transformation products, most of which proved to be more stable in the lettuce leaves than the parent compounds. Furthermore, continuous leaching from TWP led to a resupply and replenishment of the metabolized compounds in the lettuce leaves. The stability of metabolized TWP-derived compounds with largely unknown toxicities is particularly concerning and is an important new aspect for the impact assessment of TWP in the environment.

Long-term soil warming alters fine root dynamics and morphology, and their ectomycorrhizal fungal community in a temperate forest soil.

Climate warming is predicted to affect temperate forests severely, but the response of fine roots, key to plant nutrition, water uptake, soil carbon, and nutrient cycling is unclear. Understanding how fine roots will respond to increasing temperature is a prerequisite for predicting the functioning of forests in a warmer climate. We studied the response of fine roots and their ectomycorrhizal (EcM) fungal and root-associated bacterial communities to soil warming by 4°C in a mixed spruce-beech forest in the Austrian Limestone Alps after 8 and 14 years of soil warming, respectively. Fine root biomass (FRB) and fine root production were 17% and 128% higher in the warmed plots, respectively, after 14 years. The increase in FRB (13%) was not significant after 8 years of treatment, whereas specific root length, specific root area, and root tip density were significantly higher in warmed plots at both sampling occasions. Soil warming did not affect EcM exploration types and diversity, but changed their community composition, with an increase in the relative abundance of Cenoccocum at 0-10 cm soil depth, a drought-stress-tolerant genus, and an increase in short- and long-distance exploration types like Sebacina and Boletus at 10-20 cm soil depth. Warming increased the root-associated bacterial diversity but did not affect their community composition. Soil warming did not affect nutrient concentrations of fine roots, though we found indications of limited soil phosphorus (P) and potassium (K) availability. Our findings suggest that, in the studied ecosystem, global warming could persistently increase soil carbon inputs due to accelerated fine root growth and turnover, and could simultaneously alter fine root morphology and EcM fungal community composition toward improved nutrient foraging.

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.

Isotopically characterised N2 O reference materials for use as community standards.

RATIONALE: Information on the isotopic composition of nitrous oxide (N2 O) at natural abundance supports the identification of its source and sink processes. In recent years, a number of mass spectrometric and laser spectroscopic techniques have been developed and are increasingly used by the research community. Advances in this active research area, however, critically depend on the availability of suitable N2 O isotope Reference Materials (RMs). METHODS: Within the project Metrology for Stable Isotope Reference Standards (SIRS), seven pure N2 O isotope RMs have been developed and their 15 N/14 N, 18 O/16 O, 17 O/16 O ratios and 15 N site preference (SP) have been analysed by specialised laboratories against isotope reference materials. A particular focus was on the 15 N site-specific isotopic composition, as this measurand is both highly diagnostic for source appointment and challenging to analyse and link to existing scales. RESULTS: The established N2 O isotope RMs offer a wide spread in delta (δ) values: δ15 N: 0 to +104‰, δ18 O: +39 to +155‰, and δ15 NSP : -4 to +20‰. Conversion and uncertainty propagation of δ15 N and δ18 O to the Air-N2 and VSMOW scales, respectively, provides robust estimates for δ15 N(N2 O) and δ18 O(N2 O), with overall uncertainties of about 0.05‰ and 0.15‰, respectively. For δ15 NSP , an offset of >1.5‰ compared with earlier calibration approaches was detected, which should be revisited in the future. CONCLUSIONS: A set of seven N2 O isotope RMs anchored to the international isotope-ratio scales was developed that will promote the implementation of the recommended two-point calibration approach. Particularly, the availability of δ17 O data for N2 O RMs is expected to improve data quality/correction algorithms with respect to δ15 NSP and δ15 N analysis by mass spectrometry. We anticipate that the N2 O isotope RMs will enhance compatibility between laboratories and accelerate research progress in this emerging field.

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.

Applying the 15N labelling technique to material derived from a landfill simulation experiment to understand nitrogen cycle processes under aerobic and anaerobic conditions.

Reactive nitrogen (N) species, such as ammonium (NH4+), nitrate (NO3) and gaseous nitrous oxide (N2O), are released into the environment during the degradation of municipal solid waste (MSW), causing persistent environmental problems. Landfill remediation measures, such as in-situ aeration, may accelerate the degradation of organic compounds and reduce the discharge of ammonium via leachate. Nonetheless, the actual amount of N in the waste material remains relatively constant and a coherent explanation for the decline in leachate ammonium concentrations is still lacking. Hence, the present study aimed to elucidate the dynamics of N and its transformation processes during waste degradation. To this end, the gross rates of organic N mineralization and nitrification were measured using 15N pool dilution in waste material derived from a landfill simulation reactor (LSR) experiment. The results revealed a high potential for N mineralization and nitrification, the latter of which declined with the diminishing amount of extractable ammonium (after aeration). The analysis of the concentration and isotopic composition of N2O formed confirmed incomplete denitrification as the main source for N2O. Moreover, the natural abundance of 15N was investigated in various waste N pools to verify the conclusions drawn from the 15N tracing experiment. δ15N values of total waste N increased during aeration, indicating that nitrification is the major driver for N losses from aerated waste. The application of stable isotopes thereby allowed unprecedented insights into the complex N dynamics in decomposing landfill waste, of their response to aeration and their effect on hydrological versus gaseous loss pathways.

Warming and elevated CO2 intensify drought and recovery responses of grassland carbon allocation to soil respiration.

Photosynthesis and soil respiration represent the two largest fluxes of CO2 in terrestrial ecosystems and are tightly linked through belowground carbon (C) allocation. Drought has been suggested to impact the allocation of recently assimilated C to soil respiration; however, it is largely unknown how drought effects are altered by a future warmer climate under elevated atmospheric CO2 (eT_eCO2 ). In a multifactor experiment on managed C3 grassland, we studied the individual and interactive effects of drought and eT_eCO2 (drought, eT_eCO2 , drought × eT_eCO2 ) on ecosystem C dynamics. We performed two in situ 13 CO2 pulse-labeling campaigns to trace the fate of recent C during peak drought and recovery. eT_eCO2 increased soil respiration and the fraction of recently assimilated C in soil respiration. During drought, plant C uptake was reduced by c. 50% in both ambient and eT_eCO2 conditions. Soil respiration and the amount and proportion of 13 C respired from soil were reduced (by 32%, 70% and 30%, respectively), the effect being more pronounced under eT_eCO2 (50%, 84%, 70%). Under drought, the diel coupling of photosynthesis and SR persisted only in the eT_eCO2 scenario, likely caused by dynamic shifts in the use of freshly assimilated C between storage and respiration. Drought did not affect the fraction of recent C remaining in plant biomass under ambient and eT_eCO2 , but reduced the small fraction remaining in soil under eT_eCO2 . After rewetting, C uptake and the proportion of recent C in soil respiration recovered more rapidly under eT_eCO2 compared to ambient conditions. Overall, our findings suggest that in a warmer climate under elevated CO2 drought effects on the fate of recent C will be amplified and the coupling of photosynthesis and soil respiration will be sustained. To predict the future dynamics of terrestrial C cycling, such interactive effects of multiple global change factors should be considered.

Quantifying microbial growth and carbon use efficiency in dry soil environments via 18 O water vapor equilibration.

Soil microbial physiology controls large fluxes of C to the atmosphere, thus, improving our ability to accurately quantify microbial physiology in soil is essential. However, current methods to determine microbial C metabolism require liquid water addition, which makes it practically impossible to measure microbial physiology in dry soil samples without stimulating microbial growth and respiration (namely, the "Birch effect"). We developed a new method based on in vivo 18 O-water vapor equilibration to minimize soil rewetting effects. This method allows the isotopic labeling of soil water without direct liquid water addition. This was compared to the main current method (direct 18 O-liquid water addition) in moist and air-dry soils. We determined the time kinetics and calculated the average 18 O enrichment of soil water over incubation time, which is necessary to calculate microbial growth from 18 O incorporation in genomic DNA. We tested isotopic equilibration patterns in three natural and six artificially constructed soils covering a wide range of soil texture and soil organic matter content. We then measured microbial growth, respiration and carbon use efficiency (CUE) in three natural soils (either air-dry or moist). The proposed 18 O-vapor equilibration method provided similar results as the current method of liquid 18 O-water addition when used for moist soils. However, when applied to air-dry soils the liquid 18 O-water addition method overestimated growth by up to 250%, respiration by up to 500%, and underestimated CUE by up to 40%. We finally describe the new insights into biogeochemical cycling of C that the new method can help uncover, and we consider a range of questions regarding microbial physiology and its response to global change that can now be addressed.

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.

Novel high-throughput approach to determine key processes of soil organic nitrogen cycling: Gross protein depolymerization and microbial amino acid uptake.

Proteins comprise the largest soil N reservoir but cannot be taken up directly by microorganisms and plants due to size constraints and stabilization of proteins in organo-mineral associations. Therefore the cleavage of this high molecular weight organic N to smaller soluble compounds as amino acids is a key step in the terrestrial N cycle. In the last years two isotope pool dilution approaches have been successfully established to measure gross rates of protein depolymerization and microbial amino acid uptake in soils. However, both require laborious sample preparation and analyses, which limits sample throughput. Therefore, we here present a novel isotope pool dilution approach based on the addition of 15N-labeled amino acids to soils and subsequent concentration and 15N analysis by the oxidation of α-amino groups to NO2 - and further reduction to N2O, followed by purge-and-trap isotope ratio mass spectrometry (PT-IRMS). We applied this method in mesocosm experiments with forest and meadow soils as well as with a cropland soil amended with either organic C (cellulose) or organic N (bovine serum albumin). To measure direct organic N mineralization to NH4 +, the latter was captured in acid traps and analyzed by an elemental analyzer coupled to an isotope ratio mass spectrometer (EA-IRMS). Our results demonstrate that the proposed method provides fast and precise measurements of at%15N even at low amino acid concentrations, allows high sample throughput and enables parallel estimations of instantaneous organic N mineralization rates.

Environmental effects on soil microbial nitrogen use efficiency are controlled by allocation of organic nitrogen to microbial growth and regulate gross N mineralization.

Microbial nitrogen use efficiency (NUE) is the efficiency by which microbes allocate organic N acquired to biomass formation relative to the N in excess of microbial demand released through N mineralization. Microbial NUE thus is critical to estimate the capacity of soil microbes to retain N in soils and thereby affects inorganic N availability to plants and ecosystem N losses. However, how soil temperature and soil moisture/O2 affect microbial NUE to date is not clear. Therefore, two independent incubation experiments were conducted with soils from three land uses (cropland, grassland and forest) on two bedrocks (silicate and limestone). Soils were exposed to 5, 15 and 25 °C overnight at 60% water holding capacity (WHC) or acclimated to 30 and 60% WHC at 21% O2 and to 90% WHC at 1% O2 over one week at 20 °C. Microbial NUE was measured as microbial growth over microbial organic N uptake (the sum of growth N demand and gross N mineralization). Microbial NUE responded positively to temperature increases with Q10 values ranging from 1.30 ± 0.11 to 2.48 ± 0.67. This was due to exponentially increasing microbial growth rates with incubation temperature while gross N mineralization rates were relatively insensitive to temperature increases (Q10 values 0.66 ± 0.30 to 1.63 ± 0.15). Under oxic conditions (21% O2), microbial NUE as well as gross N mineralization were not stimulated by the increase in soil moisture from 30 to 60% WHC. Under suboxic conditions (90% WHC and 1% O2), microbial NUE markedly declined as microbial growth rates were strongly negatively affected due to increasing microbial energy limitation. In contrast, gross N mineralization rates increased strongly as organic N uptake became in excess of microbial growth N demand. Therefore, in the moisture/O2 experiment microbial NUE was mainly regulated by the shift in O2 status (to suboxic conditions) and less affected by increasing water availability per se. These temperature and moisture/O2 effects on microbial organic N metabolism were consistent across the soils differing in bedrock and land use. Overall it has been demonstrated that microbial NUE was controlled by microbial growth, and that NUE controlled gross N mineralization as an overflow metabolism when energy (C) became limiting or N in excess in soils. This study thereby greatly contributes to the understanding of short-term environmental responses of microbial community N metabolism and the regulation of microbial organic-inorganic N transformations in soils.

Wide-spread limitation of soil organic nitrogen transformations by substrate availability and not by extracellular enzyme content.

Proteins constitute the single largest soil organic nitrogen (SON) reservoir and its decomposition drives terrestrial N availability. Protein cleavage by extracellular enzymes is the rate limiting step in the soil organic N cycle and can be controlled by extracellular enzyme production or protein availability/stabilization in soil. Both controls can be affected by geology and land use, as well as be vulnerable to changes in soil temperature and moisture/O2. To explore major controls of soil gross protein depolymerization we sampled six soils from two soil parent materials (calcareous and silicate), where each soil type included three land uses (cropland, pasture and forest). Soil samples were subjected to three temperature treatments (5, 15, 25 °C at 60% water-holding capacity (WHC) and aerobic conditions) or three soil moisture/O2 treatments (30 and 60% WHC at 21% O2, 90% WHC at 1% O2, at 20 °C) in short-term experiments. Samples were incubated for one day in the temperature experiment and for one week in the moisture/O2 experiment. Gross protein depolymerization rates were measured by a novel 15N isotope pool dilution approach. The low temperature sensitivity of gross protein depolymerization, the lack of relationship with protease activity and strong effects of soil texture and pH demonstrate that this process is constrained by organo-mineral associations and not by soil enzyme content. This also became apparent from the inverse effects in calcareous and silicate soils caused by water saturation/O2 limitation. We highlight that the specific soil mineralogy influenced the response of gross depolymerization rates to water saturation/O2 limitation, causing (I) increasing gross depolymerization rates due to release of adsorbed proteins by reductive dissolution of Fe- and Mn-oxyhydroxides in calcareous soils and (II) decreasing gross depolymerization rates due to mobilization of coagulating and toxic Al3+ compounds in silicate soils.

Growth explains microbial carbon use efficiency across soils differing in land use and geology.

The ratio of carbon (C) that is invested into microbial growth to organic C taken up is known as microbial carbon use efficiency (CUE), which is influenced by environmental factors such as soil temperature and soil moisture. How microbes will physiologically react to short-term environmental changes is not well understood, primarily due to methodological restrictions. Here we report on two independent laboratory experiments to explore short-term temperature and soil moisture effects on soil microbial physiology (i.e. respiration, growth, CUE, and microbial biomass turnover): (i) a temperature experiment with 1-day pre-incubation at 5, 15 and 25 °C at 60% water holding capacity (WHC), and (ii) a soil moisture/oxygen (O2) experiment with 7-day pre-incubation at 20 °C at 30%, 60% WHC (both at 21% O2) and 90% WHC at 1% O2. Experiments were conducted with soils from arable, pasture and forest sites derived from both silicate and limestone bedrocks. We found that microbial CUE responded heterogeneously though overall positively to short-term temperature changes, and decreased significantly under high moisture level (90% WHC)/suboxic conditions due to strong decreases in microbial growth. Microbial biomass turnover time decreased dramatically with increasing temperature, and increased significantly at high moisture level (90% WHC)/suboxic conditions. Our findings reveal that the responses of microbial CUE and microbial biomass turnover to short-term temperature and moisture/O2 changes depended mainly on microbial growth responses and less on respiration responses to the environmental cues, which were consistent across soils differing in land use and geology.

Soil multifunctionality is affected by the soil environment and by microbial community composition and diversity.

Microorganisms are critical in mediating carbon (C) and nitrogen (N) cycling processes in soils. Yet, it has long been debated whether the processes underlying biogeochemical cycles are affected by the composition and diversity of the soil microbial community or not. The composition and diversity of soil microbial communities can be influenced by various environmental factors, which in turn are known to impact biogeochemical processes. The objectives of this study were to test effects of multiple edaphic drivers individually and represented as the multivariate soil environment interacting with microbial community composition and diversity, and concomitantly on multiple soil functions (i.e. soil enzyme activities, soil C and N processes). We employed high-throughput sequencing (Illumina MiSeq) to analyze bacterial/archaeal and fungal community composition by targeting the 16S rRNA gene and the ITS1 region of soils collected from three land uses (cropland, grassland and forest) deriving from two bedrock forms (silicate and limestone). Based on this data set we explored single and combined effects of edaphic variables on soil microbial community structure and diversity, as well as on soil enzyme activities and several soil C and N processes. We found that both bacterial/archaeal and fungal communities were shaped by the same edaphic factors, with most single edaphic variables and the combined soil environment representation exerting stronger effects on bacterial/archaeal communities than on fungal communities, as demonstrated by (partial) Mantel tests. We also found similar edaphic controls on the bacterial/archaeal/fungal richness and diversity. Soil C processes were only directly affected by the soil environment but not affected by microbial community composition. In contrast, soil N processes were significantly related to bacterial/archaeal community composition and bacterial/archaeal/fungal richness/diversity but not directly affected by the soil environment. This indicates direct control of the soil environment on soil C processes and indirect control of the soil environment on soil N processes by structuring the microbial communities. The study further highlights the importance of edaphic drivers and microbial communities (i.e. composition and diversity) on important soil C and N processes.

Application of stable-isotope labelling techniques for the detection of active diazotrophs.

Investigating active participants in the fixation of dinitrogen gas is vital as N is often a limiting factor for primary production. Biological nitrogen fixation is performed by a diverse guild of bacteria and archaea (diazotrophs), which can be free-living or symbionts. Free-living diazotrophs are widely distributed in the environment, yet our knowledge about their identity and ecophysiology is still limited. A major challenge in investigating this guild is inferring activity from genetic data as this process is highly regulated. To address this challenge, we evaluated and improved several 15 N-based methods for detecting N2 fixation activity (with a focus on soil samples) and studying active diazotrophs. We compared the acetylene reduction assay and the 15 N2 tracer method and demonstrated that the latter is more sensitive in samples with low activity. Additionally, tracing 15 N into microbial RNA provides much higher sensitivity compared to bulk soil analysis. Active soil diazotrophs were identified with a 15 N-RNA-SIP approach optimized for environmental samples and benchmarked to 15 N-DNA-SIP. Lastly, we investigated the feasibility of using SIP-Raman microspectroscopy for detecting 15 N-labelled cells. Taken together, these tools allow identifying and investigating active free-living diazotrophs in a highly sensitive manner in diverse environments, from bulk to the single-cell level.

pH-Dependent Bioavailability, Speciation, and Phytotoxicity of Tungsten (W) in Soil Affect Growth and Molybdoenzyme Activity of Nodulated Soybeans.

Increasing use of tungsten (W)-based products opened new pathways for W into environmental systems. Due to its chemical alikeness with molybdenum (Mo), W is expected to behave similarly to its "twin element", Mo; however, our knowledge of the behavior of W in the plant-soil environment remains inadequate. The aim of this study was to investigate plant growth as well as W and nutrient uptake depending on soil chemical properties such as soil pH and texture. Soybean ( Glycine max cv. Primus) was grown on two acidic soils differing in soil texture that were either kept at their natural soil pH (pH of 4.5-5) or limed (pH of ≥7) and amended with increasing concentrations of metallic W (control and 500 and 5000 mg kg-1). In addition, the activity of molybdoenzymes involved in N assimilation (nitrate reductase) and symbiotic N2 fixation (nitrogenase) was also investigated. Our results showed that the risk of W entering the food web was significantly greater in high-pH soils due to increased solubility of mainly monomeric W. The effect of soil texture on W solubility and phytoavailability was less pronounced compared to soil pH. Particularly at intermediate W additions (W 500 mg kg-1), symbiotic nitrogen fixation was able to compensate for reduced leaf nitrate reductase activity. When W soil solution concentrations became too toxic (W 5000 mg kg-1), nodulation was more strongly inhibited than nitrogenase activity in the few nodules formed, suggesting a more-efficient detoxification and compartmentalization mechanism in nodules than in soybean leaves. The increasing presence of polymeric W species observed in low-pH soils spiked with high W concentrations resulted in decreased W uptake. Simultaneously, polymeric W species had an overall negative effect on nutrient assimilation and plant growth, suggesting a greater phytotoxicity of W polymers. Our study demonstrates the importance of accounting for soil pH in risk assessment studies of W in the plant-soil environment, something that has been completely neglected in the past.

Significant release and microbial utilization of amino sugars and D-amino acid enantiomers from microbial cell wall decomposition in soils.

Amino sugars and D-amino acid enantiomers are major components of bacterial and fungal cell walls (i.e. peptidoglycan and chitin) and are often used as biomarkers of microbial residue turnover in soils. However, little is known about the in situ decomposition rates of microbial cell wall residues and how soil physicochemical properties affect this process. In this study, we investigated the in situ gross production and consumption rates of free amino sugars (glucosamine and muramic acid) and amino acids (meso-diaminopimelic acid, l-alanine, and d-alanine) by a novel isotope pool dilution assay using 15N-labeled amino compounds. Soils were obtained from six sites differing in land management (cropland, pasture, and forest) and bedrock (silicate and limestone) and incubated at three temperatures (5, 15, and 25 °C). Free glucosamine released during the decomposition of peptidoglycan and chitin contributed significantly to the extractable soil organic nitrogen pool. Gross production and consumption rates of glucosamine were higher than those of individual amino acids, i.e. L- and D-alanine. Muramic acid had a longer mean residence time (68 h compared to 2.7 h for glucosamine, L- and D-alanine) and made a negligible contribution to soil organic nitrogen fluxes, indicating that free muramic acid was not a major decomposition product of peptidoglycan in soils. Meso-diaminopimelic acid and D-alanine exhibited comparable gross production and consumption rates with L-alanine. These amino acids can be used as indicators to estimate the decomposition of peptidoglycan from bacterial cell wall residues. We found that chitin decomposition was greater in silicate soils, while peptidoglycan decomposition dominated in limestone soils. Glucosamine production rates were not correlated with soil total amino sugars, microbial community structure, or hydrolytic enzyme activities, but were highest in soils with low pH and high sand content, indicating that soil texture and soil pH may strongly influence the decomposition of amino sugar polymers. In contrast, mDAP, L- and D-alanine gross production and consumption rates were positively correlated with soil pH and clay content, due to greater depolymerization of peptidoglycan stem peptides in limestone soils. This isotope pool dilution approach strongly improves our understanding of the mechanisms and environmental controls on microbial cell wall decomposition in soils.