Soil Biology and Biochemistry

Soil viruses are increasingly recognized as components of microbial communities that may alter how communities may function, yet the frequency and functional distribution of virus-encoded metabolic genes in soils remain poorly understood. Here, the Global Soil Virus Atlas gene catalog, comprising 1,432,147 viral genes from 1,223 soil samples across 13 ecosystem types, was analyzed to quantify the distribution of virus-encoded functional annotations and to estimate their representation in matched total metagenomic inventories. Functional annotations were assessed across KEGG Orthology, Pfam, and CAZy and grouped into carbon cycling, nitrogen cycling, and antibiotic resistance-associated categories. Only 1,903 viral genes (0.13%) had functional annotations. Carbon cycling dominated the annotated repertoire (1,840 genes; 96.5%), whereas nitrogen cycling (33 genes; 1.7%) and antibiotic resistance-associated functions (30 genes; 1.6%) were rare. Within carbon-cycling annotations, chitinase-associated genes were the most frequent named category (628 genes), followed by hemicellulase-associated functions. To estimate the representation of viral genes within broader metagenomic functional inventories, viral and total metagenomic annotations were compared across six exactly matched JGI studies. Viral contributions were usually low, with four of six studies showing less than 1% viral representation in targeted functions, but reached 9.86% for chitinase in one study. Together, these results show that virus-encoded metabolic genes are globally sparse in soils but are non-randomly concentrated in carbon-active CAZyme-linked functions, indicating that analyses restricted to microbial genes may underestimate predicted functional potential for selected degradation traits. IMPORTANCESoil metagenomic studies usually interpret functional potential from microbial genes alone, even though soil viruses can also encode metabolic functions. In a global soil viral gene catalog, functionally annotated viral genes were rare overall, but the detectable signal was strongly concentrated in carbon-active CAZyme-linked functions, especially chitinase-associated annotations. Because viral diversity and activity can decouple from microbial responses to environmental gradients, virus-encoded functional genes may disproportionately affect gene-centric estimates of selected functions under stress or seasonal constraint (Zheng et al., 2022; Merges et al., 2023). A targeted comparison of viral and total metagenomic functional annotations further showed that viral contributions were usually small but could become non-trivial for selected carbon-degradation traits. These results identify viral CAZyme-associated functions as the clearest current case in which ignoring viruses may bias gene-centric estimates of predicted functional potential in soils. More broadly, the study provides a quantitative baseline for evaluating when virus-encoded genes are likely to matter for environmental metagenomic interpretation.

Society needs to capture gigatons of carbon dioxide from the atmosphere annually and then store it long-term to limit and ultimately reverse the effects of climate change. Bringing lost carbon back into agricultural soils should be a priority as it brings the added benefit of improving soil properties. Linking soil organic carbon (SOC) fractions of different stability with soil microbial composition can help understand and subsequently manage SOC storage. Here we develop a pipeline for evaluating the effects of microbial management on SOC content using rapid and low-cost SOC fractionation and metagenomics approaches. We tested the methods in a wheat pot trial inoculated with 17 individual endophytic fungal isolates. Two fungi increased total SOC in the area under the plant stem by ~15%. The fractionation assay showed that the medium stability soil aggregate carbon fraction (AggC) was increased by one of these fungi (+21%) and the chemically recalcitrant proportion (bleach oxidation) of AggC by the other (+35%). Both fungi increased mineral-associated organic carbon (MAOC), the long-term SOC storage, by ~10%. We used rapid, portable, low-cost, whole metagenome long read sequencing to detect a shift in the microbial composition for one of the fungi-inoculated treatments. This treatment showed a more diverse microbial community and a higher quantity of DNA in soil. The results emphasise the link between composition and abundance of soil microorganisms with soil carbon formation. Our dual carbon fractional and metagenomic analysis pipeline can be used to further test the effects of microbial management and ultimately to model the soil factors that influence SOC storage, such as nutrient and water availability, starting SOC content, soil texture and aggregation.

Wildfires cause immediate changes in above and belowground carbon (C) stocks in boreal forest ecosystems with long-term repercussions for C cycling. Understanding the role of soil microbes in mediating post-fire C cycling and recovery is an important step to predicting how these ecosystems will respond to novel wildfire regimes caused by climate change. Wildfires can cause large shifts in soil bacterial and fungal community composition that can persist for years post-fire. Less is known about the effects of fire on soil microbial community function, such as C use efficiency (CUE). In this study, we measured the effects of burning on substrate-specific CUE using a laboratory incubation of boreal forest soils. We amended burned and unburned soils with either 13C-labelled ground pine roots or glucose and measured the amount of added substrate C that was incorporated into microbial biomass C versus respired as CO2 in order to calculate CUE. Burning caused a decrease in the amount of soil microbial biomass and respiration derived from soil organic C. Glucose-specific CUE declined with burning, driven by a decrease in glucose-derived microbial biomass. This decrease in glucose-specific CUE following burning correlated with an increase in weighted mean predicted 16S rRNA gene copy number, raising the possibility of using copy number as a proxy for post-fire CUE in boreal forest soils. Overall, pine-specific CUE was lower than glucose-specific CUE, likely reflecting the difference in chemical complexity between the two substrates; burning had a much smaller effect on pine-specific CUE, highlighting the variability of CUE between substrates in burned soils.

Viral-induced microbial mortality has been proposed as a major contributor in shaping microbial community structure and function, soil carbon (C) accrual and mobilization of plant available nutrients. Yet, how soil viruses influence soil organic C (SOC) turnover and sequestration remains unknown. Here, we performed microcosm experiments with two distinct soils from grassland (GL) and agricultural (AG) sites and interrogated the roles of soil viruses in driving microbial community succession, SOC transformation and sequestration. The results show that soil viruses affected microbial C use efficiency and reduced respiration in microbial communities obtained from both GL and AG soils. Soil viruses affected microbial successional trajectories (via predation of dominant populations) and functional gene profiles triggering a significant decrease in CO2 and N2O emissions. The impact of soil viruses on microbial community composition in GL microcosms was much less pronounced compared with that in AG microcosms, suggesting contrasting virus-host interaction patterns under different environmental settings. Viral infection significantly enhanced microbial necromass accumulation thereby increasing SOC and total nitrogen (TN) content. The results implicate viral-mediated microbial mortality as a key factor influencing the distribution of C between mineralization and soil C storage pathways. We proposed "viral loop" to explain the crucial function of soil viruses in SOC turnover and sequestration.

AO_SCPLOWBSTRACTC_SCPLOWClimate-smart land management practices that replace shallow-rooted annual crop systems with deeply-rooted perennial plants can contribute to soil carbon sequestration. However, deep soil carbon accrual may be influenced by active microbial biomass and their capacity to assimilate fresh carbon at depth. Incorporating active microbial biomass, dormancy and growth in microbially-explicit models can improve our ability to predict soils capacity to store carbon. But, so far, the microbial parameters that are needed for such modeling are poorly constrained, especially in deep soil layers. Here, we investigated whether a change in crop rooting depth affects microbial growth kinetics in deep soils compared to surface soils. We used a lab incubation experiment and growth kinetics model to estimate how microbial parameters vary along 240 cm of soil depth in profiles under shallow- (soy) and deeply-rooted plants (switchgrass) 11 years after plant cover conversion. We also assessed resource origin and availability (total organic carbon, 14C, dissolved organic carbon, specific UV absorbance, total nitrogen, total dissolved nitrogen) along the soil profiles to examine associations between soil chemical and biological parameters. Even though root biomass was higher and rooting depth was deeper under switchgrass than soy, resource availability and microbial growth parameters were generally similar between vegetation types. Instead, depth significantly influenced soil chemical and biological parameters. For example, resource availability, and total and relative active microbial biomass decreased with soil depth. Decreases in the relative active microbial biomass coincided with increased lag time (response time to external carbon inputs) along the soil profiles. Even at a depth of 210-240 cm, microbial communities were activated to grow by added resources within a day. Maximum specific growth rate decreased to a depth of 90 cm and then remained consistent in deeper layers. Our findings show that > 10 years of vegetation and rooting depth changes may not be long enough to alter microbial growth parameters, and suggest that at least a portion of the microbial community in deep soils can grow rapidly in response to added resources. Our study determined microbial growth parameters that can be used in microbially-explicit models to simulate carbon dynamics in deep soil layers.

Despite the importance of mineral-associated organic matter (MAOM) in long-term soil carbon (C) and nitrogen (N) persistence, and the significant contribution of fungal necromass to this pool, the factors controlling the formation of fungal-derived MAOM remain unclear. This study investigated how fungal necromass chemistry, specifically melanin, interacts with soil mineral properties and microbial communities to influence MAOM formation and persistence. We cultured the fungus Hyaloscypha bicolor to produce {superscript 1}3C- and {superscript 1}{square}N-labeled necromass with varying melanin content (high or low) and incubated it in both live and sterile soils collected from six Indiana forests that differed in their clay and iron oxide (FeOx) content. After 38 days, we found that seven times more fungal-derived N was incorporated into MAOM than fungal-derived C, with fungal N comprising 20% of the MAOM-N pool. Low melanin necromass formed more MAOM-C than high melanin necromass, although site-level differences in overall MAOM formation were substantial. Soil clay and FeOx content were strong predictors of MAOM formation, explaining [~]60% and [~]68% of the variation in MAOM-C and MAOM-N, respectively. However, microbial communities significantly influenced MAOM formation, with MAOM-C formation enhanced and MAOM-N formation reduced in sterile soils. Furthermore, the relative abundance of fungal saprotrophs was negatively correlated, and bacterial richness was positively correlated with MAOM formation, and these relationships were influenced by necromass melanin content. This study reveals that microbial communities and soil properties interactively mediate the incorporation of fungal necromass C and N into MAOM, with microbes differentially influencing C and N incorporation, and these processes being further modulated by necromass melanization.

Biodiversity loss and climate change are two of the most alarming threats to ecosystem functioning and stability. These factors are, however, typically investigated separately, overlooking potential interactions between species extinctions and climate change on ecosystems. Here, we evaluated how different temperature regimes impact the relationship between microbial diversity and ecosystem functioning, in terms of temperature sensitivity of carbon (C) cycling functions. We hypothesized that more diverse soil communities promote the stability of C cycling functions under both temperature regimes. We did not observe a ubiquitous response of all C-cycling processes to temperature increases within the distinct community diversities. While growth was stable with increasing temperatures among the diversity levels, respiration rates increased more strongly at low diversity levels than high diversity levels at higher temperatures. Carbon use efficiency, which integrates both growth and respiration, tended to decrease with temperature at lower levels of diversity. Jointly, these results demonstrate the importance of belowground soil community diversity for maintaining C-cycling thermal response under changing climate.

Relationships among variables in ecological systems are inherently scale-dependent, especially in heterogeneous systems. Yet it remains to be examined whether relationships among variables vary across observation scales in soil. Generally, it is desirable that observation scale matches the intrinsic scale of a process or pattern. Millimetre-sized soil aggregates are closer to the intrinsic scale of microbial communities than traditionally studied bulk soil samples, making them more suitable for studying potential links between microbial communities and their environment. To explore the effect of observation scale on relationships among soil parameters, we measured bacterial, archaeal, and fungal taxa richness and density, organic matter properties (e.g., carbon and nitrogen content, stoichiometric and isotopic ratios), and soil water content in individual aggregates and aliquots of homogenised soil cores, bulk soil samples, in two soil layers. We analysed pairwise correlations among these variables and assessed whether individual aggregates systematically differed from bulk soil samples. Organic matter properties were more strongly correlated in bulk soil samples, consistent with the idea that increasing the sample volume reduces noise. In contrast, microbial community and organic matter properties showed weaker correlations in bulk soil samples than aggregates in topsoil. In addition, we found that aggregates and bulk soil samples differed systematically in individual microbial and organic matter properties, particularly in the topsoil. Our study demonstrates that relationships among variables in soil are spatial scale-dependent. Aggregates offer valuable insights into microbial communities in soil, complementing bulk soil samples, and are useful for studying links between microbial communities and their environment.

Climate change is predicted to have a negative effect on the grasslands of the United States and will be detrimental to the economy and environment. The changing precipitation levels would also have an effect on the structural and functional potential of associated soil microbiome communities, which in turn will regulate the health of the plants during stressful conditions. In this study, we applied metagenomics analyses to capture the responses of the bacterial populations under drier soil conditions. We collected soil from two sites (dry and wet) at the Konza Long-Term Ecological Research field station in Kansas, which had characteristic features of the native prairies. Soil drying resulted in a significant shift in the bacterial population at the community level. Following that, fifteen bacterial genomes were short-listed based on the availability in the public database, higher relative abundance in dry soils than in wet, and also according to their contributions in drier soil. The potential microbial mechanisms were elucidated when an in-depth analysis of the functional genes was performed. Translation elongation factor EF-Tu, thiamine biosynthesis protein, and catalase were identified as a part of the overall stress functional responses in the bacterial population in this study. We speculate that these identified bacterial populations are important for maintaining the health of the soil under dry conditions. Genes and/or pathways found in this study provide insights into microbial mechanisms that these bacterial populations might employ to resist challenging drought conditions.

Carbon Use Efficiency (CUE) is a popular concept for measuring the efficiency of biomass production in different biological systems and, is frequently employed to understand effects of microbial processes on soil carbon dynamics. CUE in soil microbes is often measured through respiration-based studies, especially through the addition of a labile carbon substrate such as glucose. Therefore, exploring the response of microbial respiration to availability of labile substrates is crucial to understand microbial CUE in soils. In this work, we build upon a cellular model of the Warburg effect, where cells simultaneously utilize inefficient aerobic glycolysis/fermentation and efficient oxidative phosphorylation pathways for energy synthesis even at high oxygen availability, to predict microbial community response to various levels of substrate availability. We test our predictions systematically using a series of substrate-induced respiration (SIR) experiments to demonstrate prevalence of the Warburg effect in soil microbial communities. We further discuss the relevance of the underlying metabolic processes behind the Warburg effect in interpreting soil microbial CUE.

Soil microbes play a critical role in carbon (C) cycling, however, the influence of microbial life strategies and their interactions on C mineralization remains poorly understood. This study aimed to investigate how r- and K-strategist bacteria influence glucose mineralization using an artificial soil system, focusing on Bacillus subtilis and Streptomyces cinnamoneus. Through a 14-day incubation experiment, we found that B. subtilis exhibited rapid and high respiration rates, while S. cinnamoneus showed slower, delayed respiration rates, supporting their respective r/K classification. In co-culture treatments, cumulative glucose mineralization converged with B. subtilis monoculture levels and positively correlated with its relative abundance. These findings demonstrate that artificial soil systems effectively reveal how microbial interactions drive C dynamics, offering a controlled approach to elucidate mechanisms underlying soil C cycling.

The present study challenges the traditional view that respiration of organic carbon to CO2 is exclusively an intracellular process, revealing that organic compound respiration can occur spontaneously in an extracellular context in soils. Using 1H nuclear magnetic resonance spectroscopy to analyse the dynamics of the sterile soil exometabolomes alongside C-CO2 flux analyses and sterile soil fuel cells, we show that soil catalysts facilitate a diverse array of substrate-driven reactions, leading to the complete oxidation of organic compounds to CO2 with O2 consumption. Our results indicate that soil particles are capable of transferring electrons from substrates to the final acceptor, sustaining metabolic processes independently of living cells. Notably, some soil catalysts and induced respiration remain stable for over six years. Our results support the coexistence of cellular and non-cellular metabolic pathways in soil respiration.

Soil microbial growth and respiration play a critical role for soil organic carbon dynamics. Yet, we lack understanding of the main controls of soil microbial carbon metabolism at large scales. Here, we investigated whether and how the chemical composition of microbially available organic matter affects soil microbial carbon metabolism across soil systems. We linked soil microbial growth and respiration rates as well as carbon use efficiency (quantified with 18O stable isotope probing) to the chemical composition of extractable organic matter (characterized with reversed-phase liquid chromatography coupled to Fourier-transform ion cyclotron resonance mass spectrometry) along a geoclimatic gradient of 33 Chilean temperate grassland soils. We found that biomass-normalized rates of growth and respiration were primarily positively linked to aliphatics such as carbohydrate-, proteinaceous- and amino sugar-like compounds, and secondarily to unsaturated lignin-like compounds. Respiration was positively linked to compounds with carbon in a reduced oxidation state, suggesting carbon-conserving catabolism, while growth appeared unrelated to the oxidation state of carbon. This suggests that other mechanisms than mere energetic constraints control microbial growth rates in aerated soils. Our findings demonstrate that information on the chemical composition of bioavailable organic matter can provide insights into the processes that govern the fate of carbon across different ecosystems. Key pointsO_LIWe investigated if bulk soil microbial growth (18O stable isotope probing) and respiration is linked to the chemical composition of extractable organic matter (LC-FT-ICR MS) along a geoclimatic gradient of temperate grasslands. C_LIO_LIHigher rates of microbial carbon turnover were positively linked to aliphatic and unsaturated compounds. C_LIO_LISpecific (i.e., biomass-normalized) respiration was positively linked to compounds with carbon in a reduced oxidation state, suggesting carbon-conserving catabolism. C_LIO_LISpecific growth was unrelated to the oxidation states of substrate carbon, suggesting that soil microbial substrate use for anabolism may not be determined by direct energetic constraints. C_LI

The soil microbiome is key to plant health and nutrient acquisition, and viruses likely play important but largely unknown roles in these processes. To interrogate bulk and rhizosphere soil viral biogeography, we collected samples over a tomato growing season in California from an experiment testing arbuscular mycorrhizal fungi (AMF) treatment. We generated 78 viromes, 16S rRNA gene, and ITS1 amplicon datasets, and 33 rhizosphere metatranscriptomes. Of 67,038 DNA viral species genomes (vOTUs), 25% were previously identified, predominantely in agricultural systems, suggesting habitat filtering and greater viral homogeneity across agricultural compared to natural soils globally. Rhizospheres had significantly higher DNA viral richness than bulk soils, whereas no significant richness differences were observed for other biota. 60% of vOTUs were shared between compartments, compared to only 21-23% of bacterial and fungal taxa. Although bulk soil viral biogeography resembled that of prokaryotes, with significant structuring by moisture content, greater virome similarity between high-moisture bulk soils and rhizospheres suggests that conditions with high host activity selected for similar viral communities. In rhizospheres, while bacterial and fungal communities differed most over time, DNA and RNA viral communities differed most by sampling location, matching prokaryotic transcriptional patterns and further implicating host activity in viral biogeography. Similarly, AMF treatment induced changes in the prokaryotic transcriptome but, across biota, only significantly affected DNA viral communities. Overall, results indicate strong viral responses to spatiotemporally localized conditions, with viral biogeography reflecting both dispersal opportunities (high between neighboring bulk and rhizosphere soils, low across fields) and selection via local host activity.

The interactions between plants and soil microorganisms are fundamental for ecosystem functioning. However, it remains unclear if seasonality of plant growth impacts plant-microbial interactions, such as by inducing shifts in the microbial community composition, their biomass, or changes in the microbial uptake of plant-derived carbon. Here, we investigate the stability of microbial biomass of different functional groups and their net assimilation of plant-derived carbon over an entire growing season. Using a C3-C4 vegetation change experiment, and taking advantage of natural abundances of 13C, we measured the plant-derived carbon in lipid biomarkers of soil microorganisms in rhizosphere and non-rhizosphere soil. We found that temporal and spatial stability was higher in bacterial than in fungal biomass, while the high temporal stability of all bacterial groups even increased in close proximity to roots. Moreover, differences in the association to plants, i.e., symbionts vs. free-living microorganisms, tend to determine the stability in the uptake of plant-derived carbon. Our results indicate, the inputs of plant-derived carbon over the growing season did not result in a shift in the microbial community composition, but instead, functional groups that are not in obligate symbiosis with plants showed a varying use of soil- and plant-derived carbon.

Microbial growth dynamics are deterministic of the fate of carbon in soil, responsible for the transformation of new carbon inputs and their stabilization on soil surfaces. Bacterial life history strategies are predictive of C substrate assimilation and growth response. High disturbance management practices such as tillage alter microbial community structure but have a poorly described impact on life histories that are central to C metabolism. We conducted a DNA stable isotope probing experiment using soil from a long-term field experiment with a 42-year legacy of no-till or annual moldboard plowing. We predicted that divergent legacies of disturbance would result in bacterial communities with distinct life histories, altering C assimilation dynamics. We incubated soil from each tillage regime with 13C-xylose and 13C-cellulose, two substrates that are components of plant litter and which differ in bioavailability. We identified 730 bacterial taxa that incorporated the labeled substrates and tracked their abundance in bulk microcosm soil over a 30 day period. Carbon addition rapidly altered bacterial community structure and function, with tilled soils demonstrating lower mineralization rates of each substrate. Xylose-assimilating taxa exhibited significantly lagged growth in tilled soils relative to no-till. We also found a higher number and diversity of late (day 30) cellulose incorporators in no-till soil, suggesting that minimal disturbance resulted in a longer residence time of 13C-cellulose in members of the bacterial community. We show that soil management practices shape the path of carbon through bacterial communities by altering dynamic growth responses and secondary incorporation of carbon. HighlightsO_LIDNA SIP identified divergent carbon dynamics resulting from tillage legacies C_LIO_LIXylose assimilation in plow-till soils was late and decoupled from mineralization C_LIO_LICellulose-C was assimilated later in no-till soils relative to plow-till C_LIO_LIGrowth responses of incorporator taxa differ by tillage and explain mineralization C_LI

Tree mortality in oak savannas is increasing under climate change, but its impact on microbial communities and soil carbon below the top 20 centimeters is relatively unknown. Deep tree roots, their ectomycorrhizal fungi, and associated bacteria may have a particularly important effect on landscape carbon storage, as they mediate the transfer of recently fixed plant carbon into deep soil and subsoil layers. To investigate how tree mortality impacts microbes and soil carbon, we sampled under living and recently dead Quercus douglasii trees in a California oak savanna, gathering depth-resolved soil cores to 45 cm below the surface. We captured finely resolved biological detail on these soil samples, comparing living (RNA-based) to potential and historical (DNA-based) microbial communities and assessing microbial biomass with phospholipid fatty acid analysis. Tree mortality greatly reduced the abundance of ectomycorrhizal fungi, particularly in subsoils. Fungal niches were more variable at depth under dead trees than under living ones, and RNA-based profiling captured substantially different communities than DNA, especially under living trees. However, tree mortality three years prior to our study did not impact the overall quantity of carbon stored in the soil. Tree mortality can have profound effects on the interactions between tree roots, mycorrhizal fungi, and soil bacteria, which may shift soil carbon dynamics over long time scales. Understanding the mechanisms of these interactions, and their time scales, will improve our ability to predict and manage soil carbon in savanna landscapes as drought and heat events kill more oaks in arid climates. HighlightsO_LIRibosomal RNA, from living cells, revealed different communities than from DNA. C_LIO_LIMicrobial functions changed more with depth and tree health than taxonomy. C_LIO_LIDifferences were most extreme below 20 cm depth. C_LIO_LIMicrobial population distributions changed under living and dead trees. C_LIO_LITotal microbial biomass and carbon were similar beneath living and dead trees. C_LI

Viruses are key regulators of terrestrial carbon, nitrogen, and phosphorus cycling, yet how environmental perturbations structure viral activity remains poorly resolved. Rewetting of seasonally dry soils triggers rapid microbial and viral responses, but the relationships between virion-associated and transcriptionally active viral communities, and the role of phosphorus in these dynamics, remain unclear. Here, we integrated viromics, metatranscriptomics, environmental DNA (eDNA), and amplicon sequencing to track viral succession and virus-host interactions over three weeks following soil rewetting, with and without phosphorus amendment. We identified 13,840 viral populations (vOTUs), of which 3,803 were transcriptionally active, representing ongoing infections. Wet-up significantly altered virion and transcriptionally active viral communities, while phosphorus selectively influenced prokaryotic and transcriptionally active viral communities but not virion composition. Virus-host linkages were predicted for 32% of vOTUs, with transcriptionally active bacteriophages infecting Actinomycetota increasing under phosphorus amendment. Following wet-up, virion abundance decreased [~]3-fold while virocells increased [~]5-fold, indicating a shift from viral persistence in dry soils to active infection. Phosphorus further enhanced virocell abundance. eDNA captured rapid viral turnover and revealed transient dynamics not resolved by viromes or metatranscriptomes alone. Together, these results demonstrate that soil viral communities are structured by distinct but complementary molecular pools that operate over different ecological timescales. Wet-up activates a reservoir of persistent virions, while phosphorus availability regulates infection dynamics and host-virus coupling. These findings highlight viruses as dynamic drivers of microbial turnover and nutrient cycling following environmental perturbation, advancing a more predictive understanding of soil ecosystem responses to changing resource availability.

AbstractSoil microbes have sophisticated mechanisms to detect and respond to short pulses of C inputs, often involving changes in gene-expression. We studied gene transcription in a soil microbial community before, and 8, 24, and 48h after glucose addition (0.7 mg C g-1 dry soil) to understand how microbes react to periods of substrate excess and subsequent starvation. The relative transcript abundance of genes associated with energy metabolism and biosynthesis of amino acids, lipids, nucleotides, and cell wall components increased 8h after glucose addition. By 24 and 48h, the abundances of these transcripts reversed. Transcript abundance for genes associated with degradation of lipids, nucleotides, and (hetero)cyclic hydrocarbons decreased at 8h, but increased 24 and 48h after glucose addition. Simultaneously with a rise in transcripts for energy production and biosynthesis at 8h, transcription of regulatory genes for the exponential growth phase and ribosome assembly and maturation increased. In contrast, at 24 and 48h, transcript abundance for genes associated with ribosomal hibernation, sporulation, and regulation of the stationary phase increased, while transcripts for regulators for the exponential phase, and ribosome activation decreased. Based on changes in transcript abundance of phosphoenolpyruvate carboxylase and pyruvate carboxylase, it appeared that 8h after glucose addition glycolytic activity was high, however, gluconeogenesis returned at 24 and 48h. High levels of transcripts for nrtC-ntrB indicated N limitation 8 and 24h after glucose addition. Transcripts associated with Type VI Secretion Systems increased 24 and 48h after start of the experiment, suggesting a short lag between primary consumers and predatory bacteria. These results illustrate how metatranscriptome analysis can be used to study the ecophysiology of soil microbes providing details on the timing of exponential and stationary phase processes, coordination between anabolism and catabolism, and emerging nutrient limitations in natural soil communities. Research HighlightsO_LIWe studied gene transcription of a soil microbial community after glucose addition C_LIO_LITranscript abundances for biosynthesis and energy production initially increased, while those for degradation decreased C_LIO_LITranscripts of regulators and sporulation genes indicated start of stationary phase at 24h C_LIO_LINitrogen limitation induced transcription of nitrogen stress genes C_LI

Microbial nitrogen (N) transformations in soil, notably denitrification, result in the production of the potent greenhouse and ozone depleting gas nitrous oxide (N2O). Soil chemistry and microbiome composition impact N2O emission potential but the relative importance of these factors as determinants of N2O emission in denitrifying systems is rarely tested. In addition, previous linkages between microbiome composition and N2O emission potential rarely demonstrate causality. Here, we determined the relative impact of microbiome composition (i.e. soil extracted cells) and chemistry (i.e. water extractable chemicals) on N2O emission potential utilizing an anoxic cell based assay system. Cells and chemistry for assays were sourced from soils with contrasting N2O/N2O+N2 ratios, combined in various combinations and denitrification gas production was measured in response to nitrate addition. Average directionless effects of cell and chemical extract on N2O/N2O+N2 (Cell: {Delta}0.16, Chemical extract: {Delta}0.22) and total N2O hypothetically emitted (Cell: {Delta}2.62 mol-N, Chemical extract: {Delta}4.14 mol-N) indicated chemistry is the most important determinant of N2O emissions. Independent pH differences of just 0.6 points impacted N2O/N2O+N2 on par with independent chemical extract differences, supporting the dominance of this variable in previous studies. However, impacts on overall N2O hypothetically emitted were smaller suggesting that soil pH manipulation may not necessarily be a successful approach to mitigate emissions over a fixed time period. In addition, we observed increased N2O accumulation and emission potential at the end of incubations concomitant with predicted decreases in carbon availability suggesting that carbon limitation increases N2O emission transiently with the magnitude of emission dependent on the both chemical and microbiome controls.