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Protists are a diverse and understudied group of microbial eukaryotic organisms especially in terrestrial environments. Advances in molecular methods are increasing our understanding of the distribution and functions of these creatures; however, there is a vast array of choices researchers make including barcoding genes, primer pairs, PCR settings, and bioinformatic options that can impact the outcome of protist community surveys. Here, we tested four commonly used primer pairs targeting the V4 and V9 regions of the 18S rRNA gene using different PCR annealing temperatures and processed the sequences with different bioinformatic parameters in 10 diverse soils to evaluate how primer pair, amplification parameters, and bioinformatic choices influence the composition and richness of protist and non-protist taxa using Illumina sequencing. Our results showed that annealing temperature influenced sequencing depth and protist taxon richness for most primer pairs, and that merging forward and reverse sequencing reads for the V4 primer pairs dramatically reduced the number of sequences and taxon richness of protists. The data sets of primers that targeted the same 18S rRNA gene region (e.g., V4 or V9) had similar protist community compositions; however, data sets from primers targeting the V4 18S rRNA gene region detected a greater number of protist taxa compared to those prepared with primers targeting the V9 18S rRNA region. There was limited overlap of protist taxa between data sets targeting the two different gene regions (80/549 taxa). Together, we show that laboratory and bioinformatic choices can substantially affect the results and conclusions about protist diversity and community composition using metabarcoding.IMPORTANCEEcosystem functioning is driven by the activity and interactions of the microbial community, in both aquatic and terrestrial environments. Protists are a group of highly diverse, mostly unicellular microbes whose identity and roles in terrestrial ecosystem ecology have been largely ignored until recently. This study highlights the importance of choices researchers make, such as primer pair, on the results and conclusions about protist diversity and community composition in soils. In order to better understand the roles protist taxa play in terrestrial ecosystems, biases in methodological and analytical choices should be understood and acknowledged., Competing Interests: The authors declare no conflict of interest.

The central carbon (C) metabolic network is responsible for most of the production of energy and biosynthesis in microorganisms and is therefore key to a mechanistic understanding of microbial life in soil communities. Many upland soil communities have shown a relatively high C flux through the pentose phosphate (PP) or the Entner-Doudoroff (ED) pathway, thought to be related to oxidative damage control. We tested the hypothesis that the metabolic organization of the central C metabolic network differed between two ecosystems, an anoxic marsh soil and oxic upland soil, and would be affected by altering oxygen concentrations. We expected there to be high PP/ED pathway activity under high oxygen concentrations and in oxic soils and low PP/ED activity in reduced oxygen concentrations and in marsh soil. Although we found high PP/ED activity in the upland soil and low activity in the marsh soil, lowering the oxygen concentration for the upland soil did not reduce the relative PP/ED pathway activity as hypothesized, nor did increasing the oxygen concentration in the marsh soil increase the PP/ED pathway activity. We speculate that the high PP/ED activity in the upland soil, even when exposed to low oxygen concentrations, was related to a high demand for NADPH for biosynthesis, thus reflecting higher microbial growth rates in C-rich soils than in C-poor sediments. Further studies are needed to explain the observed metabolic diversity among soil ecosystems and determine whether it is related to microbial growth rates.IMPORTANCEWe observed that the organization of the central carbon (C) metabolic processes differed between oxic and anoxic soil. However, we also found that the pentose phosphate pathway/Entner-Doudoroff (PP/ED) pathway activity remained high after reducing the oxygen concentration for the upland soil and did not increase in response to an increase in oxygen concentration in the marsh soil. These observations contradicted the hypothesis that oxidative stress is a main driver for high PP/ED activity in soil communities. We suggest that the high PP/ED activity and NADPH production reflect higher anabolic activities and growth rates in the upland soil compared to the anaerobic marsh soil. A greater understanding of the molecular and biochemical processes in soil communities is needed to develop a mechanistic perspective on microbial activities and their relationship to soil C and nutrient cycling. Such an increased mechanistic perspective is ecologically relevant, given that the central carbon metabolic network is intimately tied to the energy metabolism of microbes, the efficiency of new microbial biomass production, and soil organic matter formation., Competing Interests: The authors declare no conflict of interest.

Soils store more carbon than other terrestrial ecosystems 1,2 . How soil organic carbon (SOC) forms and persists remains uncertain 1,3 , which makes it challenging to understand how it will respond to climatic change 3,4 . It has been suggested that soil microorganisms play an important role in SOC formation, preservation and loss 5-7 . Although microorganisms affect the accumulation and loss of soil organic matter through many pathways 4,6,8-11 , microbial carbon use efficiency (CUE) is an integrative metric that can capture the balance of these processes 12,13 . Although CUE has the potential to act as a predictor of variation in SOC storage, the role of CUE in SOC persistence remains unresolved 7,14,15 . Here we examine the relationship between CUE and the preservation of SOC, and interactions with climate, vegetation and edaphic properties, using a combination of global-scale datasets, a microbial-process explicit model, data assimilation, deep learning and meta-analysis. We find that CUE is at least four times as important as other evaluated factors, such as carbon input, decomposition or vertical transport, in determining SOC storage and its spatial variation across the globe. In addition, CUE shows a positive correlation with SOC content. Our findings point to microbial CUE as a major determinant of global SOC storage. Understanding the microbial processes underlying CUE and their environmental dependence may help the prediction of SOC feedback to a changing climate., (© 2023. The Author(s).)

The growth and physiology of soil microorganisms, which play vital roles in biogeochemical cycling, are shaped by both current and historical soil environmental conditions. Here, we developed and applied a genome-resolved metagenomic implementation of quantitative stable isotope probing (qSIP) with an H 2 O labeling experiment to identify actively growing soil microorganisms and their genomic capacities. qSIP enabled measurement of taxon-specific growth because isotopic incorporation into microbial DNA requires production of new genome copies. We studied three Mediterranean grassland soils across a rainfall gradient to evaluate the hypothesis that historic precipitation levels are an important factor controlling trait selection. We used qSIP-informed genome-resolved metagenomics to resolve the active subset of soil community members and identify their characteristic ecophysiological traits. Higher year-round precipitation levels correlated with higher activity and growth rates of flagellar motile microorganisms. In addition to heavily isotopically labeled bacteria, we identified abundant isotope-labeled phages, suggesting phage-induced cell lysis likely contributed to necromass production at all three sites. Further, there was a positive correlation between phage activity and the activity of putative phage hosts. Contrary to our expectations, the capacity to decompose the diverse complex carbohydrates common in soil organic matter or oxidize methanol and carbon monoxide were broadly distributed across active and inactive bacteria in all three soils, implying that these traits are not highly selected for by historical precipitation. 18 Soil moisture is a critical factor that strongly shapes the lifestyle of soil organisms by changing access to nutrients, controlling oxygen diffusion, and regulating the potential for mobility. We identified active microorganisms in three grassland soils with similar mineral contexts, yet different historic rainfall inputs, by adding water labeled with a stable isotope and tracking that isotope in DNA of growing microbes. By examining the genomes of active and inactive microorganisms, we identified functions that are enriched in growing organisms, and showed that different functions were selected for in different soils. Wetter soil had higher activity of motile organisms, but activity of pathways for degradation of soil organic carbon compounds, including simple carbon substrates, were comparable for all three soils. We identified many labeled, and thus active bacteriophages (viruses that infect bacteria), implying that the cells they killed contributed to soil organic matter. The activity of these bacteriophages was significantly correlated with activity of their hosts.IMPORTANCE Soil moisture is a critical factor that strongly shapes the lifestyle of soil organisms by changing access to nutrients, controlling oxygen diffusion, and regulating the potential for mobility. We identified active microorganisms in three grassland soils with similar mineral contexts, yet different historic rainfall inputs, by adding water labeled with a stable isotope and tracking that isotope in DNA of growing microbes. By examining the genomes of active and inactive microorganisms, we identified functions that are enriched in growing organisms, and showed that different functions were selected for in different soils. Wetter soil had higher activity of motile organisms, but activity of pathways for degradation of soil organic carbon compounds, including simple carbon substrates, were comparable for all three soils. We identified many labeled, and thus active bacteriophages (viruses that infect bacteria), implying that the cells they killed contributed to soil organic matter. The activity of these bacteriophages was significantly correlated with activity of their hosts.

Nutrient amendment diminished bacterial functional diversity, consolidating carbon flow through fewer bacterial taxa. Here, we show strong differences in the bacterial taxa responsible for respiration from four ecosystems, indicating the potential for taxon-specific control over soil carbon cycling. Trends in functional diversity, defined as the richness of bacteria contributing to carbon flux and their equitability of carbon use, paralleled trends in taxonomic diversity although functional diversity was lower overall. Among genera common to all ecosystems, Bradyrhizobium, the Acidobacteria genus RB41, and Streptomyces together composed 45-57% of carbon flow through bacterial productivity and respiration. Bacteria that utilized the most carbon amendment (glucose) were also those that utilized the most native soil carbon, suggesting that the behavior of key soil taxa may influence carbon balance. Mapping carbon flow through different microbial taxa as demonstrated here is crucial in developing taxon-sensitive soil carbon models that may reduce the uncertainty in climate change projections.

Microbial activity increases after rewetting dry soil, resulting in a pulse of carbon mineralization and nutrient availability. The biogeochemical responses to wet-up are reasonably well understood and known to be microbially mediated. Yet, the population level dynamics, and the resulting changes in microbial community patterns, are not well understood as ecological phenomena. Here, we used sequencing of 16S rRNA genes coupled with heavy water (H 2 O) DNA quantitative stable isotope probing to estimate population-specific rates of growth and mortality in response to a simulated wet-up event in a California annual grassland soil. Bacterial growth and mortality responded rapidly to wet-up, within 3 h, and continued throughout the 168 h incubation, with patterns of sequential growth observed at the phylum level. Of the 37 phyla detected in the prewet community, growth was found in 18 phyla while mortality was measured in 26 phyla. Rapid growth and mortality rates were measurable within 3 h of wet-up but had contrasting characteristics; growth at 3 h was dominated by select taxa within the Proteobacteria and Firmicutes, whereas mortality was taxonomically widespread. Furthermore, across the community, mortality exhibited density-independence, consistent with the indiscriminate shock resulting from dry-down and wet-up, whereas growth was density-dependent, consistent with control by competition or predation. Total aggregated growth across the community was highly correlated with total soil CO 18 O) DNA quantitative stable isotope probing to estimate population-specific rates of growth and mortality in response to a simulated wet-up event in a California annual grassland soil. Bacterial growth and mortality responded rapidly to wet-up, within 3 h, and continued throughout the 168 h incubation, with patterns of sequential growth observed at the phylum level. Of the 37 phyla detected in the prewet community, growth was found in 18 phyla while mortality was measured in 26 phyla. Rapid growth and mortality rates were measurable within 3 h of wet-up but had contrasting characteristics; growth at 3 h was dominated by select taxa within the Proteobacteria and Firmicutes, whereas mortality was taxonomically widespread. Furthermore, across the community, mortality exhibited density-independence, consistent with the indiscriminate shock resulting from dry-down and wet-up, whereas growth was density-dependent, consistent with control by competition or predation. Total aggregated growth across the community was highly correlated with total soil CO 2 production. Together, these results illustrate how previously "invisible" population responses can translate quantitatively to emergent observations of ecosystem-scale biogeochemistry.

Growth of soil microorganisms is often described as carbon limited, and adding labile carbon to soil often results in a transient and large increase in respiration. In contrast, soil microbial biomass changes little, suggesting that growth and respiration are decoupled in response to a carbon pulse. Alternatively, measuring bulk responses of the entire community (total respiration and biomass) could mask ecologically important variation among taxa in response to the added carbon. Here, we assessed taxon-specific variation in cellular growth (measured as DNA synthesis) and metabolic activity (measured as rRNA synthesis) following glucose addition to soil using quantitative stable isotope probing with H 2 O. We found that glucose addition altered rates of DNA and rRNA synthesis, but the effects were strongly taxon specific: glucose stimulated growth and rRNA transcription for some taxa, and suppressed these for others. These contrasting taxon-specific responses could explain the small and transient changes in total soil microbial biomass. Responses to glucose were not well predicted by a priori assignments of taxa into copiotrophic or oligotrophic categories. Across all taxa, rates of DNA and rRNA synthesis changed in parallel, indicating that growth and activity were coupled, and the degree of coupling was unaffected by glucose addition. This pattern argues against the idea that labile carbon addition causes a large reduction in metabolic growth efficiency; rather, the large pulse of respiration observed with labile substrate addition is more likely to be the result of rapid turnover of microbial biomass, possibly due to trophic interactions. Our results support a strong connection between rRNA synthesis and bacterial growth, and indicate that taxon-specific responses among soil bacteria can buffer responses at the scale of the whole community.18 O. We found that glucose addition altered rates of DNA and rRNA synthesis, but the effects were strongly taxon specific: glucose stimulated growth and rRNA transcription for some taxa, and suppressed these for others. These contrasting taxon-specific responses could explain the small and transient changes in total soil microbial biomass. Responses to glucose were not well predicted by a priori assignments of taxa into copiotrophic or oligotrophic categories. Across all taxa, rates of DNA and rRNA synthesis changed in parallel, indicating that growth and activity were coupled, and the degree of coupling was unaffected by glucose addition. This pattern argues against the idea that labile carbon addition causes a large reduction in metabolic growth efficiency; rather, the large pulse of respiration observed with labile substrate addition is more likely to be the result of rapid turnover of microbial biomass, possibly due to trophic interactions. Our results support a strong connection between rRNA synthesis and bacterial growth, and indicate that taxon-specific responses among soil bacteria can buffer responses at the scale of the whole community., (© 2019 by the Ecological Society of America.)

Relationships between microbial genes and performance are often evaluated in the laboratory in pure cultures, with little validation in nature. Here, we show that genomic traits related to laboratory measurements of maximum growth potential failed to predict the growth rates of bacteria in unamended soil, but successfully predicted growth responses to resource pulses: growth increased with 16S rRNA gene copy number and declined with genome size after substrate addition to soils, responses that were repeated in four different ecosystems. Genome size best predicted growth rate in response to addition of glucose alone; adding ammonium with glucose weakened the relationship, and the relationship was absent in nutrient-replete pure cultures, consistent with the idea that reduced genome size is a mechanism of nutrient conservation. Our findings demonstrate that genomic traits of soil bacteria can map to their ecological performance in nature, but the mapping is poor under native soil conditions, where genomic traits related to stress tolerance may prove more predictive. These results remind that phenotype depends on environmental context, underscoring the importance of verifying proposed schemes of trait-based strategies through direct measurement of performance in nature, an important and currently missing foundation for translating microbial processes from genes to ecosystems.

The continuously increasing concentration of atmospheric CO 2 has considerably altered ecosystem functioning. However, few studies have examined the long-term (i.e. over a decade) effect of elevated CO 2 on soil microbial communities. Using 16S rRNA gene amplicons and a GeoChip microarray, we investigated soil microbial communities from a Californian annual grassland after 14 years of experimentally elevated CO 2 (275 ppm higher than ambient). Both taxonomic and functional gene compositions of the soil microbial community were modified by elevated CO 2 . There was decrease in relative abundance for taxa with higher ribosomal RNA operon (rrn) copy number under elevated CO 2 , which is a functional trait that responds positively to resource availability in culture. In contrast, taxa with lower rrn copy number were increased by elevated CO 2 . As a consequence, the abundance-weighted average rrn copy number of significantly changed OTUs declined from 2.27 at ambient CO 2 to 2.01 at elevated CO 2 . The nitrogen (N) fixation gene nifH and the ammonium-oxidizing gene amoA significantly decreased under elevated CO 2 by 12.6% and 6.1%, respectively. Concomitantly, nitrifying enzyme activity decreased by 48.3% under elevated CO 2 , albeit this change was not significant. There was also a substantial but insignificant decrease in available soil N, with both nitrate (NO 3 - ) (-15.4%) declining. Further, a large number of microbial genes related to carbon (C) degradation were also affected by elevated CO 4 + ) (-15.4%) declining. Further, a large number of microbial genes related to carbon (C) degradation were also affected by elevated CO 2 , whereas those related to C fixation remained largely unchanged. The overall changes in microbial communities and soil N pools induced by long-term elevated CO 2 suggest constrained microbial N decomposition, thereby slowing the potential maximum growth rate of the microbial community., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Most soil bacterial taxa are thought to be dormant, or inactive, yet the extent to which they synthetize new rRNA is poorly understood. We analyzed 18 O composition of RNA extracted from soil incubated with H 2 18 O, peaking at a mean of 23.6 ± 6.8 atom percent excess (APE) 18 O, peaking at a mean of 23.6 ± 6.8 atom percent excess (APE) 18 O after eight days of incubation, suggesting some ribonucleotides in soil were more than eight days old. Microbial taxa varied in the degree they incorporated 18 O into their rRNA over time and there was no correlation between the APE 18 O of bacterial rRNA and their rRNA to DNA ratios, suggesting that the ratios were not appropriate to measure ribonucleotide synthesis. Our study indicates that, on average, 94% of soil taxa produced new rRNA and therefore were metabolically active.

Growing bacteria have a high concentration of ribosomes to ensure sufficient protein synthesis, which is necessary for genome replication and cellular division. To elucidate whether metabolic activity of soil microorganisms is coupled with growth, we investigated the relationship between rRNA and DNA synthesis in a soil bacterial community using quantitative stable isotope probing (qSIP) with H 2 18 O. Most soil bacterial taxa were metabolically active and grew, and there was no significant difference between the isotopic composition of DNA and RNA extracted from soil incubated with H 2 O content of DNA and rRNA of taxa, with a slope statistically indistinguishable from 1 (slope = 0.96; 95% confidence interval [CI], 0.90 to 1.02), indicated that few taxa made new rRNA without synthesizing new DNA. There was no correlation between rRNA-to-DNA ratios obtained from sequencing libraries and the atom percent excess (APE) 18 O values of DNA or rRNA, suggesting that the ratio of rRNA to DNA is a poor indicator of microbial growth or rRNA synthesis. Our results support the notion that metabolic activity is strongly coupled to cellular division and suggest that nondividing taxa do not dominate soil metabolic activity. 18 O content of DNA and rRNA of taxa, with a slope statistically indistinguishable from 1 (slope = 0.96; 95% confidence interval [CI], 0.90 to 1.02), indicated that few taxa made new rRNA without synthesizing new DNA. There was no correlation between rRNA-to-DNA ratios obtained from sequencing libraries and the atom percent excess (APE) 18 O values of DNA or rRNA, suggesting that the ratio of rRNA to DNA is a poor indicator of microbial growth or rRNA synthesis. Our results support the notion that metabolic activity is strongly coupled to cellular division and suggest that nondividing taxa do not dominate soil metabolic activity. IMPORTANCE O. A majority of the populations that make new rRNA also grow, which argues against the common paradigm that most soil taxa are dormant. Additionally, our results indicate that relative sequence abundance-based RNA-to-DNA ratios, which are frequently used for identifying active microbial populations in the environment, underestimate the number of metabolically active taxa within soil microbial communities.2 18 O, we show that most soil taxa are metabolically active and grow because their nucleic acids are significantly labeled with 18 O. A majority of the populations that make new rRNA also grow, which argues against the common paradigm that most soil taxa are dormant. Additionally, our results indicate that relative sequence abundance-based RNA-to-DNA ratios, which are frequently used for identifying active microbial populations in the environment, underestimate the number of metabolically active taxa within soil microbial communities., (Copyright © 2018 American Society for Microbiology.)

Microorganisms perform most decomposition on Earth, mediating carbon (C) loss from ecosystems, and thereby influencing climate. Yet, how variation in the identity and composition of microbial communities influences ecosystem C balance is far from clear. Using quantitative stable isotope probing of DNA, we show how individual bacterial taxa influence soil C cycling following the addition of labile C (glucose). Specifically, we show that increased decomposition of soil C in response to added glucose (positive priming) occurs as a phylogenetically diverse group of taxa, accounting for a large proportion of the bacterial community, shift toward additional soil C use for growth. Our findings suggest that many microbial taxa exhibit C use plasticity, as most taxa altered their use of glucose and soil organic matter depending upon environmental conditions. In contrast, bacteria that exhibit other responses to glucose (reduced growth or reliance on glucose for additional growth) clustered strongly by phylogeny. These results suggest that positive priming is likely the prototypical response of bacteria to sustained labile C addition, consistent with the widespread occurrence of the positive priming effect in nature.

Many studies have assessed the responses of soil microbial functional groups to increases in atmospheric CO2 or N deposition alone and more rarely in combination. However, the effects of elevated CO2 and N on the (de)coupling between different microbial functional groups (e.g., different groups of nitrifiers) have been barely studied, despite potential consequences for ecosystem functioning. Here, we investigated the short-term combined effects of elevated CO2 and N supply on the abundances of the four main microbial groups involved in soil nitrification: ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and nitrite-oxidizing bacteria (belonging to the genera Nitrobacter and Nitrospira) in grassland mesocosms. AOB and AOA abundances responded differently to the treatments: N addition increased AOB abundance, but did not alter AOA abundance. Nitrobacter and Nitrospira abundances also showed contrasted responses to the treatments: N addition increased Nitrobacter abundance, but decreased Nitrospira abundance. Our results support the idea of a niche differentiation between AOB and AOA, and between Nitrobacter and Nitrospira. AOB and Nitrobacter were both promoted at high N and C conditions (and low soil water content for Nitrobacter), while AOA and Nitrospira were favored at low N and C conditions (and high soil water content for Nitrospira). In addition, Nitrobacter abundance was positively correlated to AOB abundance and Nitrospira abundance to AOA abundance. Our results suggest that the couplings between ammonia and nitrite oxidizers are influenced by soil N availability. Multiple environmental changes may thus elicit rapid and contrasted responses between and among the soil ammonia and nitrite oxidizers due to their different ecological requirements.

Native soil carbon (C) can be lost in response to fresh C inputs, a phenomenon observed for decades yet still not understood. Using dual-stable isotope probing, we show that changes in the diversity and composition of two functional bacterial groups occur with this 'priming' effect. A single-substrate pulse suppressed native soil C loss and reduced bacterial diversity, whereas repeated substrate pulses stimulated native soil C loss and increased diversity. Increased diversity after repeated C amendments contrasts with resource competition theory, and may be explained by increased predation as evidenced by a decrease in bacterial 16S rRNA gene copies. Our results suggest that biodiversity and composition of the soil microbial community change in concert with its functioning, with consequences for native soil C stability.

Rising atmospheric carbon dioxide (CO₂) could alter the carbon (C) and nitrogen (N) content of ecosystems, yet the magnitude of these effects are not well known. We examined C and N budgets of a subtropical woodland after 11 yr of exposure to elevated CO₂. We used open-top chambers to manipulate CO₂ during regrowth after fire, and measured C, N and tracer (15) N in ecosystem components throughout the experiment. Elevated CO₂ increased plant C and tended to increase plant N but did not significantly increase whole-system C or N. Elevated CO₂ increased soil microbial activity and labile soil C, but more slowly cycling soil C pools tended to decline. Recovery of a long-term (15) N tracer indicated that CO₂ exposure increased N losses and altered N distribution, with no effect on N inputs. Increased plant C accrual was accompanied by higher soil microbial activity and increased C losses from soil, yielding no statistically detectable effect of elevated CO₂ on net ecosystem C uptake. These findings challenge the treatment of terrestrial ecosystems responses to elevated CO₂ in current biogeochemical models, where the effect of elevated CO₂ on ecosystem C balance is described as enhanced photosynthesis and plant growth with decomposition as a first-order response., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

Six terrestrial ecosystems in the USA were exposed to elevated atmospheric CO(2) in single or multifactorial experiments for more than a decade to assess potential impacts. We retrospectively assessed soil bacterial community responses in all six-field experiments and found ecosystem-specific and common patterns of soil bacterial community response to elevated CO(2) . Soil bacterial composition differed greatly across the six ecosystems. No common effect of elevated atmospheric CO(2) on bacterial biomass, richness and community composition across all of the ecosystems was identified, although significant responses were detected in individual ecosystems. The most striking common trend across the sites was a decrease of up to 3.5-fold in the relative abundance of Acidobacteria Group 1 bacteria in soils exposed to elevated CO(2) or other climate factors. The Acidobacteria Group 1 response observed in exploratory 16S rRNA gene clone library surveys was validated in one ecosystem by 100-fold deeper sequencing and semi-quantitative PCR assays. Collectively, the 16S rRNA gene sequencing approach revealed influences of elevated CO(2) on multiple ecosystems. Although few common trends across the ecosystems were detected in the small surveys, the trends may be harbingers of more substantive changes in less abundant, more sensitive taxa that can only be detected by deeper surveys. Representative bacterial 16S rRNA gene clone sequences were deposited in GenBank with Accession No. JQ366086–JQ387568., (Published 2012. This article is a U.S. Government work and is in the public domain in the USA.)

Elevated atmospheric CO(2) generally increases plant productivity and subsequently increases the availability of cellulose in soil to microbial decomposers. As key cellulose degraders, soil fungi are likely to be one of the most impacted and responsive microbial groups to elevated atmospheric CO(2). To investigate the impacts of ecosystem type and elevated atmospheric CO(2) on cellulolytic fungal communities, we sequenced 10,677 cbhI gene fragments encoding the catalytic subunit of cellobiohydrolase I, across five distinct terrestrial ecosystem experiments after a decade of exposure to elevated CO(2). The cbhI composition of each ecosystem was distinct, as supported by weighted Unifrac analyses (all P-values; < 0.001), with few operational taxonomic units (OTUs) being shared across ecosystems. Using a 114-member cbhI sequence database compiled from known fungi, less than 1% of the environmental sequences could be classified at the family level indicating that cellulolytic fungi in situ are likely dominated by novel fungi or known fungi that are not yet recognized as cellulose degraders. Shifts in fungal cbhI composition and richness that were correlated with elevated CO(2) exposure varied across the ecosystems. In aspen plantation and desert creosote bush soils, cbhI gene richness was significantly higher after exposure to elevated CO(2) (550 µmol mol(-1)) than under ambient CO(2) (360 µmol mol(-1) CO(2)). In contrast, while the richness was not altered, the relative abundance of dominant OTUs in desert soil crusts was significantly shifted. This suggests that responses are complex, vary across different ecosystems and, in at least one case, are OTU-specific. Collectively, our results document the complexity of cellulolytic fungal communities in multiple terrestrial ecosystems and the variability of their responses to long-term exposure to elevated atmospheric CO(2)., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Organic carbon (C) and nitrogen (N) are essential for heterotrophic soil microorganisms, and their bioavailability strongly influences ecosystem C and N cycling. We show here that the natural (15)N abundance of the soil microbial biomass is affected by both the availability of C and N and ecosystem N processing. Microbial (15)N enrichment correlated negatively with the C : N ratio of the soil soluble fraction and positively with net N mineralization for ecosystems spanning semiarid, temperate and tropical climates, grassland and forests, and over four million years of ecosystem development. In addition, during soil incubation, large increases in microbial (15)N enrichment corresponded to high net N mineralization rates. These results support the idea that the N isotope composition of an organism is determined by the balance between N assimilation and dissimilation. Thus, (15)N enrichment of the soil microbial biomass integrates the effects of C and N availability on microbial metabolism and ecosystem processes.

Increased carbon storage in ecosystems due to elevated CO(2) may help stabilize atmospheric CO(2) concentrations and slow global warming. Many field studies have found that elevated CO(2) leads to higher carbon assimilation by plants, and others suggest that this can lead to higher carbon storage in soils, the largest and most stable terrestrial carbon pool. Here we show that 6 years of experimental CO(2) doubling reduced soil carbon in a scrub-oak ecosystem despite higher plant growth, offsetting approximately 52% of the additional carbon that had accumulated at elevated CO(2) in aboveground and coarse root biomass. The decline in soil carbon was driven by changes in soil microbial composition and activity. Soils exposed to elevated CO(2) had higher relative abundances of fungi and higher activities of a soil carbon-degrading enzyme, which led to more rapid rates of soil organic matter degradation than soils exposed to ambient CO(2). The isotopic composition of microbial fatty acids confirmed that elevated CO(2) increased microbial utilization of soil organic matter. These results show how elevated CO(2), by altering soil microbial communities, can cause a potential carbon sink to become a carbon source.

BackgroundAnthropogenic activities have increased the inputs of atmospheric reactive nitrogen (N) into terrestrial ecosystems, affecting soil carbon stability and microbial communities. Previous studies have primarily examined the effects of nitrogen deposition on microbial taxonomy, enzymatic activities, and functional processes. Here, we examined various functional traits of soil microbial communities and how these traits are interrelated in a Mediterranean-type grassland administrated with 14 years of 7 g m-2 year-1 of N amendment, based on estimated atmospheric N deposition in areas within California, USA, by the end of the twenty-first century.ResultsSoil microbial communities were significantly altered by N deposition. Consistent with higher aboveground plant biomass and litter, fast-growing bacteria, assessed by abundance-weighted average rRNA operon copy number, were favored in N deposited soils. The relative abundances of genes associated with labile carbon (C) degradation (e.g., amyA and cda) were also increased. In contrast, the relative abundances of functional genes associated with the degradation of more recalcitrant C (e.g., mannanase and chitinase) were either unchanged or decreased. Compared with the ambient control, N deposition significantly reduced network complexity, such as average degree and connectedness. The network for N deposited samples contained only genes associated with C degradation, suggesting that C degradation genes became more intensely connected under N deposition.ConclusionsWe propose a conceptual model to summarize the mechanisms of how changes in above- and belowground ecosystems by long-term N deposition collectively lead to more soil C accumulation. Video Abstract.

The functioning, health, and productivity of soil are intimately tied to a complex network of interactions, particularly in plant root-associated rhizosphere soil. We conducted a stable-isotope-informed, genome-resolved metagenomic study to trace carbon from Avena fatua grown in a 13CO2 atmosphere into soil. We collected paired rhizosphere and nonrhizosphere soil at 6 and 9 weeks of plant growth and extracted DNA that was then separated by density using ultracentrifugation. Thirty-two fractions from each of five samples were grouped by density, sequenced, assembled, and binned to generate 55 unique bacterial genomes that were ≥70% complete. We also identified complete 18S rRNA sequences of several 13C-enriched microeukaryotic bacterivores and fungi. We generated 10 circularized bacteriophage (phage) genomes, some of which were the most labeled entities in the rhizosphere, suggesting that phage may be important agents of turnover of plant-derived C in soil. CRISPR locus targeting connected one of these phage to a Burkholderiales host predicted to be a plant pathogen. Another highly labeled phage is predicted to replicate in a Catenulispora sp., a possible plant growth-promoting bacterium. We searched the genome bins for traits known to be used in interactions involving bacteria, microeukaryotes, and plant roots and found DNA from heavily 13C-labeled bacterial genes thought to be involved in modulating plant signaling hormones, plant pathogenicity, and defense against microeukaryote grazing. Stable-isotope-informed, genome-resolved metagenomics indicated that phage can be important agents of turnover of plant-derived carbon in soil. IMPORTANCE Plants grow in intimate association with soil microbial communities; these microbes can facilitate the availability of essential resources to plants. Thus, plant productivity commonly depends on interactions with rhizosphere bacteria, viruses, and eukaryotes. Our work is significant because we identified the organisms that took up plant-derived organic C in rhizosphere soil and determined that many of the active bacteria are plant pathogens or can impact plant growth via hormone modulation. Further, by showing that bacteriophage accumulate CO2-derived carbon, we demonstrated their vital roles in redistribution of plant-derived C into the soil environment through bacterial cell lysis. The use of stable-isotope probing (SIP) to identify consumption (or lack thereof) of root-derived C by key microbial community members within highly complex microbial communities opens the way for assessing manipulations of bacteria and phage with potentially beneficial and detrimental traits, ultimately providing a path to improved plant health and soil carbon storage.

Fire is a crucial event regulating the structure and functioning of many ecosystems. Yet few studies have focused on how fire affects taxonomic and functional diversities of soil microbial communities, along with changes in plant communities and soil carbon (C) and nitrogen (N) dynamics. Here, we analyze these effects in a grassland ecosystem 9 months after an experimental fire at the Jasper Ridge Global Change Experiment site in California, USA. Fire altered soil microbial communities considerably, with community assembly process analysis showing that environmental selection pressure was higher in burned sites. However, a small subset of highly connected taxa was able to withstand the disturbance. In addition, fire decreased the relative abundances of most functional genes associated with C degradation and N cycling, implicating a slowdown of microbial processes linked to soil C and N dynamics. In contrast, fire stimulated above- and belowground plant growth, likely enhancing plant-microbe competition for soil inorganic N, which was reduced by a factor of about 2. To synthesize those findings, we performed structural equation modeling, which showed that plants but not microbial communities were responsible for significantly higher soil respiration rates in burned sites. Together, our results demonstrate that fire 'reboots' the grassland ecosystem by differentially regulating plant and soil microbial communities, leading to significant changes in soil C and N dynamics.

The carbon stored in soil exceeds that of plant biomass and atmospheric carbon and its stability can impact global climate. Growth of decomposer microorganisms mediates both the accrual and loss of soil carbon. Growth is sensitive to temperature and given the vast biological diversity of soil microorganisms, the response of decomposer growth rates to warming may be strongly idiosyncratic, varying among taxa, making ecosystem predictions difficult. Here, we show that 15 years of warming by transplanting plant–soil mesocosms down in elevation, strongly reduced the growth rates of soil microorganisms, measured in the field using undisturbed soil. The magnitude of the response to warming varied among microbial taxa. However, the direction of the response—reduced growth—was universal and warming explained twofold more variation than did the sum of taxonomic identity and its interaction with warming. For this ecosystem, most of the growth responses to warming could be explained without taxon‐specific information, suggesting that in some cases microbial responses measured in aggregate may be adequate for climate modeling. Long‐term experimental warming also reduced soil carbon content, likely a consequence of a warming‐induced increase in decomposition, as warming‐induced changes in plant productivity were negligible. The loss of soil carbon and decreased microbial biomass with warming may explain the reduced growth of the microbial community, more than the direct effects of temperature on growth. These findings show that direct and indirect effects of long‐term warming can reduce growth rates of soil microbes, which may have important feedbacks to global warming. [ABSTRACT FROM AUTHOR]

Summary: Soils are among the most biodiverse habitats on earth and while the species composition of microbial communities can influence decomposition rates and pathways, the functional significance of many microbial species and phylogenetic groups remains unknown. If bacteria exhibit phylogenetic organization in their function, this could enable ecologically meaningful classification of bacterial clades. Here, we show non‐random phylogenetic organization in the rates of relative carbon assimilation for both rapidly mineralized substrates (amino acids and glucose) assimilated by many microbial taxa and slowly mineralized substrates (lipids and cellulose) assimilated by relatively few microbial taxa. When mapped onto bacterial phylogeny using ancestral character estimation this phylogenetic organization enabled the identification of clades involved in the decomposition of specific soil organic matter substrates. Phylogenetic organization in substrate assimilation could provide a basis for predicting the functional attributes of uncharacterized microbial taxa and understanding the significance of microbial community composition for soil organic matter decomposition. [ABSTRACT FROM AUTHOR]

Growing bacteria have a high concentration of ribosomes to ensure suf- ficient protein synthesis, which is necessary for genome replication and cellular division. To elucidate whether metabolic activity of soil microorganisms is coupled with growth, we investigated the relationship between rRNA and DNA synthesis in a soil bacterial community using quantitative stable isotope probing (qSIP) with H218O. Most soil bacterial taxa were metabolically active and grew, and there was no significant difference between the isotopic composition of DNA and RNA extracted from soil incubated with H218O. The positive correlation between 18O content of DNA and rRNA of taxa, with a slope statistically indistinguishable from 1 (slope = 0.96; 95% confidence interval [CI], 0.90 to 1.02), indicated that few taxa made new rRNA without synthesizing new DNA. There was no correlation between rRNA-to-DNA ratios obtained from sequencing libraries and the atom percent excess (APE) 18O values of DNA or rRNA, suggesting that the ratio of rRNA to DNA is a poor indicator of microbial growth or rRNA synthesis. Our results support the notion that metabolic activity is strongly coupled to cellular division and suggest that nondividing taxa do not dominate soil metabolic activity. IMPORTANCE Using quantitative stable isotope probing of microbial RNA and DNA with H218O, we show that most soil taxa are metabolically active and grow because their nucleic acids are significantly labeled with 18O. A majority of the populations that make new rRNA also grow, which argues against the common paradigm that most soil taxa are dormant. Additionally, our results indicate that relative sequence abundance-based RNA-to-DNA ratios, which are frequently used for identifying active microbial populations in the environment, underestimate the number of metabolically active taxa within soil microbial communities. [ABSTRACT FROM AUTHOR]

Soil microbial diversity is huge and a few grams of soil contain more bacterial taxa than there are bird species on Earth. This high diversity often makes predicting the responses of soil bacteria to environmental change intractable and restricts our capacity to predict the responses of soil functions to global change. Here, using a long-term field experiment in a California grassland, we studied the main and interactive effects of three global change factors (increased atmospheric CO2 concentration, precipitation and nitrogen addition, and all their factorial combinations, based on global change scenarios for central California) on the potential activity, abundance and dominant taxa of soil nitrite-oxidizing bacteria (NOB). Using a trait-based model, we then tested whether categorizing NOB into a few functional groups unified by physiological traits enables understanding and predicting how soil NOB respond to global environmental change. Contrasted responses to global change treatments were observed between three main NOB functional types. In particular, putatively mixotrophic Nitrobacter, rare under most treatments, became dominant under the 'High CO2CNitrogenCPrecipitation' treatment. The mechanistic trait-based model, which simulated ecological niches of NOB types consistent with previous ecophysiological reports, helped predicting the observed effects of global change on NOB and elucidating the underlying biotic and abiotic controls. Our results are a starting point for representing the overwhelming diversity of soil bacteria by a few functional types that can be incorporated into models of terrestrial ecosystems and biogeochemical processes. [ABSTRACT FROM AUTHOR]

Bacteria grow and transform elements at different rates, and as yet, quantifying this variation in the environment is difficult. Determining isotope enrichment with fine taxonomic resolution after exposure to isotope tracers could help, but there are few suitable techniques. We propose a modification to stable isotope probing (SIP) that enables the isotopic composition of DNA from individual bacterial taxa after exposure to isotope tracers to be determined. In our modification, after isopycnic centrifugation, DNA is collected in multiple density fractions, and each fraction is sequenced separately. Taxon-specific density curves are produced for labeled and nonlabeled treatments, from which the shift in density for each individual taxon in response to isotope labeling is calculated. Expressing each taxon's density shift relative to that taxon's density measured without isotope enrichment accounts for the influence of nucleic acid composition on density and isolates the influence of isotope tracer assimilation. The shift in density translates quantitatively to isotopic enrichment. Because this revision to SIP allows quantitative measurements of isotope enrichment, we propose to call it quantitative stable isotope probing (qSIP). We demonstrated qSIP using soil incubations, in which soil bacteria exhibited strong taxonomic variations in 18O and 13C composition after exposure to [18O]water or [13C]glucose. The addition of glucose increased the assimilation of 18O into DNA from [18O]water. However, the increase in 18O assimilation was greater than expected based on utilization of glucose-derived carbon alone, because the addition of glucose indirectly stimulated bacteria to utilize other substrates for growth. This example illustrates the benefit of a quantitative approach to stable isotope probing. [ABSTRACT FROM AUTHOR]

Rising atmospheric carbon dioxide ( CO2) could alter the carbon (C) and nitrogen (N) content of ecosystems, yet the magnitude of these effects are not well known. We examined C and N budgets of a subtropical woodland after 11 yr of exposure to elevated CO2., We used open-top chambers to manipulate CO2 during regrowth after fire, and measured C, N and tracer 15N in ecosystem components throughout the experiment., Elevated CO2 increased plant C and tended to increase plant N but did not significantly increase whole-system C or N. Elevated CO2 increased soil microbial activity and labile soil C, but more slowly cycling soil C pools tended to decline. Recovery of a long-term 15N tracer indicated that CO2 exposure increased N losses and altered N distribution, with no effect on N inputs., Increased plant C accrual was accompanied by higher soil microbial activity and increased C losses from soil, yielding no statistically detectable effect of elevated CO2 on net ecosystem C uptake. These findings challenge the treatment of terrestrial ecosystems responses to elevated CO2 in current biogeochemical models, where the effect of elevated CO2 on ecosystem C balance is described as enhanced photosynthesis and plant growth with decomposition as a first-order response. [ABSTRACT FROM AUTHOR]

Organic carbon (C) and nitrogen (N) are essential for heterotrophic soil microorganisms, and their bioavailability strongly influences ecosystem C and N cycling. We show here that the natural 15N abundance of the soil microbial biomass is affected by both the availability of C and N and ecosystem N processing. Microbial 15N enrichment correlated negatively with the C : N ratio of the soil soluble fraction and positively with net N mineralization for ecosystems spanning semiarid, temperate and tropical climates, grassland and forests, and over four million years of ecosystem development. In addition, during soil incubation, large increases in microbial 15N enrichment corresponded to high net N mineralization rates. These results support the idea that the N isotope composition of an organism is determined by the balance between N assimilation and dissimilation. Thus, 15N enrichment of the soil microbial biomass integrates the effects of C and N availability on microbial metabolism and ecosystem processes. [ABSTRACT FROM AUTHOR]

Increases in Arctic temperatures have thawed permafrost and accelerated tundra soil microbial activity, releasing greenhouse gases that amplify climate warming. Warming over time has also accelerated shrub encroachment in the tundra, altering plant input abundance and quality, and causing further changes to soil microbial processes. To better understand the effects of increased temperature and the accumulated effects of climate change on soil bacterial activity, we quantified the growth responses of individual bacterial taxa to short-term warming (3 months) and long-term warming (29 years) in moist acidic tussock tundra. Intact soil was assayed in the field for 30 days using 18O-labeled water, from which taxon-specific rates of 18O incorporation into DNA were estimated as a proxy for growth. Experimental treatments warmed the soil by approximately 1.5°C. Short-term warming increased average relative growth rates across the assemblage by 36%, and this increase was attributable to emergent growing taxa not detected in other treatments that doubled the diversity of growing bacteria. However, long-term warming increased average relative growth rates by 151%, and this was largely attributable to taxa that co-occurred in the ambient temperature controls. There was also coherence in relative growth rates within broad taxonomic levels with orders tending to have similar growth rates in all treatments. Growth responses tended to be neutral in short-term warming and positive in long-term warming for most taxa and phylogenetic groups co-occurring across treatments regardless of phylogeny. Taken together, growing bacteria responded distinctly to short-term and long-term warming, and taxa growing in each treatment exhibited deep phylogenetic organization. IMPORTANCE Soil carbon stocks in the tundra and underlying permafrost have become increasingly vulnerable to microbial decomposition due to climate change. The microbial responses to Arctic warming must be understood in order to predict the effects of future microbial activity on carbon balance in a warming Arctic. In response to our warming treatments, tundra soil bacteria grew faster, consistent with increased rates of decomposition and carbon flux to the atmosphere. Our findings suggest that bacterial growth rates may continue to increase in the coming decades as faster growth is driven by the accumulated effects of long-term warming. Observed phylogenetic organization of bacterial growth rates may also permit taxonomy-based predictions of bacterial responses to climate change and inclusion into ecosystem models. [ABSTRACT FROM AUTHOR]

Fertilizer inputs affect plant uptake of native soil nitrogen (N), yet the underlying mechanisms remain elusive. To increase mechanistic insight into this phenomenon, we evaluated the effect of fertilizer addition on mineralization (in the absence of plants) and plant uptake of native soil N. We synthesized 43 isotope tracer ( 15 N) studies and estimated the effects of fertilizer addition using meta -analysis. We found that organic fertilizer tended to reduce native soil N mineralization (−99 kg ha −1 year −1 ; p = 0.09) while inorganic fertilizer tended to increase N priming (58 kg ha −1 year −1 ; p = 0.17). In contrast, both organic and inorganic fertilizers significantly increased plant uptake of native soil N (179 and 107 kg ha −1 year −1 ). Organic fertilizer had greater effect on plant uptake than on mineralization of native soil N (p < 0.001), but inorganic fertilizer had similar effects. Fertilizer effects on mineralization and plant uptake of native soil N were not influenced by study location (laboratory or field) and duration, soil texture, carbon and N content, and pH. Fertilizer addition variably affected native soil N mineralization but consistently increased plant uptake of native soil N. The positive effect of organic fertilizer on plant uptake of native soil N can not be explained by its negative effect on native soil N mineralization, suggesting that increased plant uptake of native soil N was caused mostly by plant-mediated mechanisms (e.g., increased root growth, rhizosphere N priming) rather than by soil microbe-mediated mechanisms. [ABSTRACT FROM AUTHOR]

Nutrient limitation has been shown to reduce bacterial genome size and influence nucleotide composition; however, much of this work has been conducted in marine systems and the factors which shape soil bacterial genomic traits remain largely unknown. Here we determined average genome size, GC content, codon usage, and amino acid content from 398 soil metagenomes across a broad geographic range and used machine-learning to determine the environmental parameters that most strongly explain the distribution of these traits. We found that genomic trait averages were most related to pH, which we suggest is primarily due to the correlation of pH with several environmental parameters, particularly soil carbon content. Low pH soils had higher carbon to nitrogen ratios (C:N) and tended to have communities with lower GC content and larger genomes, potentially a response to increased physiological stress and a requirement for metabolic diversity. Conversely, communities in high pH and low soil C:N had smaller genomes and higher GC content—indicating potential resource driven selection against AT base pairs, which have a higher C:N than GC base pairs. Similarly, we found that nutrient conservation also applied to amino acid stoichiometry, where bacteria in soils with low C:N ratios tended to code for amino acids with lower C:N. Together, these relationships point towards fundamental mechanisms that underpin genome size, and nucleotide and amino acid selection in soil bacteria. • Community averaged genomic traits of 398 soil metagenomes and environmental data. • Average genome size and GC-% negatively correlated, consistent with C limitation. • Correlation between soil C:N and predicted amino acid mean C:N. • Machine learning approach indicated pH strongest driver of genomic traits. [ABSTRACT FROM AUTHOR]

Boreal peatlands are important global carbon reservoirs that are vulnerable to increasing CO 2 and associated warming. Soil microbes regulate the balance of carbon that is stored in peat or remineralized to CO 2 ; so characterizing microbial responses to warming and rising CO 2 is critical to predicting how peatlands will feed back to ongoing climate change. To address microbiome responses to changing climate, we examined taxon-specific bacterial growth under elevated CO 2 and across a warming gradient in a peatland using 18O-water quantitative stable isotope probing. Using in situ temperatures, we clustered the responses of bacterial taxa according to excess atom fraction 18O of their genomes, a proxy for growth. Many taxa that showed little to no growth across the temperature range under ambient CO 2 grew rapidly at certain temperatures under elevated CO 2 , highlighting a strong interplay between warming and CO 2 concentrations. The temperature of maximum growth for Proteobacteria shifted higher under elevated CO 2 , while that of Acidobacteria shifted lower. We found support for phylogenetic conservation of growth patterns among Acidobacteria and Proteobacteria under ambient, but not elevated CO 2. Our results suggest that certain taxa may be predisposed for growth under altered climate conditions, with a disproportionate influence on carbon cycling and peatland feedbacks to climate change. • Elevated CO 2 with increased temperature modifies bacterial growth. • Taxa that showed little to no growth under ambient CO 2 grew rapidly under eCO 2. • Proteobacteria and Actinobacteria appear poised to benefit from climate shifts. [ABSTRACT FROM AUTHOR]

Climate influences soil microbial composition and function, but the relative importance of a site's historic climate versus its more immediate environmental conditions is unclear. Using quantitative stable isotope probing (qSIP), we characterized actively growing soil microbial communities and soil properties in three California annual grasslands that span a rainfall gradient and have developed on similar parent material. The soils were assayed in the wet winter season, when environmental conditions are most similar across sites. Since growing populations might be expected to be most responsive to contemporary environmental conditions, we hypothesized that the structure of growing microbial communities would be more similar across the gradient than that of total communities (i.e., including non-growing populations). In addition, we hypothesized that population growth rates would be slowest in the driest site, reflecting a legacy effect of low soil moisture on microbial growth. Soils along the rainfall gradient differed in pH, texture, and cation exchange capacity, but not in total C, C:N or dominant minerals. The radiocarbon (14C) age of soil C (reflecting turnover time) increased with mean annual precipitation but soil respiration was uniformly modern, reflecting microbial reliance on recent C inputs across the sites. The structure of both total and growing microbial communities differed across sites. Across major microbial phyla, including the Actinobacteria, Acidobacteria, Bacteroidetes, Gemmatimonadetes and Proteobacteria, bacterial growth rates were consistently lower in the site with the lowest mean annual precipitation. Taxa that were growing at the dry site alone grew more slowly than taxa that grew at multiple sites. These results reflect the influence of climate history and point to the role of environmental filtering at the driest site in shaping its slower growing microbial community, possibly reflecting adaptation to repeated exposure to water stress. Lastly, across taxa, the growth rate of a taxon at one site was correlated with its growth rate in the other sites. This growth rate coherence is likely a consequence of genetically determined physiological traits and is consistent with the idea that evolutionary history constrains growth rate. • Climate strongly influences structure of growing soil microbial communities. • Repeated low water exposure reduces soil microbial growth across taxonomic levels. • Microbial growth rates are consistent between sites across a precipitation gradient. • Genetics and physiology constrain variation in taxon-specific growth rates. [ABSTRACT FROM AUTHOR]

The efficiency with which microbes use substrate (Carbon Use Efficiency or CUE) to make new microbial biomass is an important variable in soil and ecosystem C cycling models. It is generally assumed that CUE of microbial activity in soils is low, however measured values vary widely. It is hypothesized that high values of CUE observed in especially short-term incubations reflect the build-up of storage compounds in response to a sudden increase in substrate availability and are therefore not representative of CUE of microbial activity in unamended soil. To test this hypothesis, we measured the 13 CO 2 release from six position-specific 13 C-labeled glucose isotopomers in ponderosa pine and piñon-juniper soil. We compared this position-specific CO 2 production pattern with patterns expected for 1) balanced microbial growth (synthesis of all compounds needed to build new microbial cells) at a low, medium, or high CUE, and 2) synthesis of storage compounds (glycogen, tri-palmitoyl-glycerol, and polyhydroxybutyrate). Results of this study show that synthesis of storage compounds is not responsible for the observed high CUE. Instead, it is the position-specific CO 2 production expected for balanced growth and high CUE that best matches the observed CO 2 production pattern in these two soils. Comparison with published studies suggests that the amount of glucose added in this study is too low and the duration of the experiment too short to affect microbial metabolism. We conclude that the hypothesis of high CUE in undisturbed soil microbial communities remains viable and worthy of further testing. [ABSTRACT FROM AUTHOR]

Microbial necromass is an important source of stabilized organic matter in soil, yet the decomposition dynamics of necromass constituents have not been adequately characterized. This includes DNA, a nutrient-rich molecule that when released into the environment as extracellular DNA (eDNA) can be readily used by soil microorganisms. However, the ecological relevance of eDNA as a nutrient source for soil microorganisms is relatively unknown. To address these deficits, we performed a laboratory experiment wherein soils were amended with 13C-labeled eDNA and clay minerals known to interact with DNA (kaolinite and montmorillonite). The amount of eDNA-carbon remaining in the soil declined exponentially over time. Kaolinite amendment decreased eDNA decomposition rates and, after 30 days, retained a higher fraction of eDNA-carbon (~70% remaining) than control or montmorillonite soils (~40% remaining), indicating that clay mineral sorption can stabilize eDNA-derived carbon in soil. Sequencing of bacterial 16S rRNA genes showed that during the incubation the relative abundance of the added eDNA's sequence decreased by 98%, 92% and 99% in the control, montmorillonite, and kaolinite amended soils respectively. These results suggest that the fraction of eDNA-carbon that remained in the soil was incorporated into microbial biomass, firmly bound to soil constituents, or fragmented and no longer amenable to sequencing. In addition, the eDNA amendment affected the composition of the bacterial community. Specifically, the relative abundance of select phyla (Planctomycetes and TM7) and genera (e.g., Arthrobacter and Nocardioides) were elevated in soils that received eDNA, suggesting these groups may be particularly effective at degrading eDNA and using it for growth. Taken together, these results indicate that while eDNA is consumed by bacteria in soil, a fraction of eDNA material is resistant to decomposition, particularly when stabilized by soil minerals, suggesting a substantial amount of recalcitrant eDNA could accumulate over time. [ABSTRACT FROM AUTHOR]

Abstract: Tillage practices and straw management can affect soil microbial activities with consequences for soil organic carbon (C) dynamics. Microorganisms metabolize soil organic C and in doing so gain energy and building blocks for biosynthesis, and release CO2 to the atmosphere. Insight into the response of microbial metabolic processes and C use efficiency (CUE; microbial C produced per substrate C utilized) to management practices may therefore help to predict long term changes in soil C stocks. In this study, we assessed the effects of reduced (RT) and conventional tillage (CT) on the microbial central C metabolic network, using soil samples from a 12-year-old field experiment in an Irish winter wheat cropping system. Straw was removed from half of the RT and CT plots after harvest or incorporated into the soil in the other half, resulting in four treatment combinations. We added 1-13C and 2,3-13C pyruvate and 1-13C and U-13C glucose as metabolic tracer isotopomers to composite soil samples taken at two depths (0–15 cm and 15–30 cm) from each of the treatments and used the rate of position-specific respired 13CO2 to parameterize a metabolic model. Model outcomes were then used to calculate CUE of the microbial community. Whereas the composite samples differed in CUE, the changes were small, with values ranging between 0.757 and 0.783 across treatments and soil depth. Increases in CUE were associated with a reduced tricarboxylic acid cycle and reductive pentose phosphate pathway activity and increased consumption of metabolic intermediates for biosynthesis. Our results suggest that RT and straw incorporation do not substantially affect CUE. [Copyright &y& Elsevier]

Abstract: We used metabolic tracers and modeling to analyze the response of soil metabolism to a sudden change in temperature from 4 to 20°C. We hypothesized that intact soil microbial communities would exhibit shifts in pentose phosphate pathway and glycolysis activity in the same way as is regularly observed for individual microorganisms in pure culture. We also hypothesized that increased maintenance respiration at higher temperature would result in greater energy production and reduced carbon use efficiency (CUE). Two hours after temperature increase, respiration increased almost 10-fold. Although all metabolic processes were increased, the relative activity of metabolic processes, biosynthesis, and energy production changed. Pentose phosphate pathway was reduced (17–20%), while activities of specific steps in glycolysis (51%) and Krebs cycle (7–13%) were increased. In contrast, only small but significant changes in biosynthesis (+2%), ATP production (−3%) and CUE (+2%) were observed. In a second experiment, we compared the metabolic responses to temperature increases in soils from high and low elevation. The shift in activity from pentose phosphate pathway to glycolysis with higher temperature was confirmed in both soils, but the responses of Krebs cycle, biosynthesis, ATP production, and CUE were site dependent. Our results indicate that 1) in response to temperature, communities behave biochemically similarly to single species and, 2) our understanding of temperature effects on CUE, energy production and use for maintenance and growth processes is still incomplete. [Copyright &y& Elsevier]

Abstract: Most organic carbon (C) in soils eventually turns into CO2 after passing through microbial metabolic pathways, while providing cells with energy and biosynthetic precursors. Therefore, detailed insight into these metabolic processes may help elucidate mechanisms of soil C cycling processes. Here, we describe a modeling approach to quantify the C flux through metabolic pathways by adding 1-13C and 2,3-13C pyruvate and 1-13C and U-13C glucose as metabolic tracers to intact soil microbial communities. The model calculates, assuming steady-state conditions and glucose as the only substrate, the reaction rates through glycolysis, Krebs cycle, pentose phosphate pathway, anaplerotic activity through pyruvate carboxylase, and various biosynthesis reactions. The model assumes a known and constant microbial proportional precursor demand, estimated from literature data. The model is parameterized with experimentally determined ratios of 13CO2 production from pyruvate and glucose isotopologue pairs. Model sensitivity analysis shows that metabolic flux patterns are especially responsive to changes in experimentally determined 13CO2 ratios from pyruvate and glucose. Calculated fluxes are far less sensitive to assumptions concerning microbial chemical and community composition. The calculated metabolic flux pattern for a young volcanic soil indicates significant pentose phosphate pathway activity in excess of pentose precursor demand and significant anaplerotic activity. These C flux patterns can be used to calculate C use efficiency, energy production and consumption for growth and maintenance purposes, substrate consumption, nitrogen demand, oxygen consumption, and microbial C isotope composition. The metabolic labeling and modeling methods may improve our ability to study the biochemistry and ecophysiology of intact and undisturbed soil microbial communities. [Copyright &y& Elsevier]

Abstract: Soil microbial organisms are central to carbon (C) and nitrogen (N) transformations in soils, yet not much is known about the stable isotope composition of these essential regulators of element cycles. We investigated the relationship between C and N availability and stable C and N isotope composition of soil microbial biomass across a three million year old semiarid substrate age gradient in northern Arizona. The δ 15N of soil microbial biomass was on average 7.2‰ higher than that of soil total N for all substrate ages and 1.6‰ higher than that of extractable N, but not significantly different for the youngest and oldest sites. Microbial 15N enrichment relative to soil extractable and total N was low at the youngest site, increased to a maximum after 55,000 years, and then decreased slightly with age. The degree of 15N enrichment of microbial biomass correlated negatively with the C:N mass ratio of the soil extractable pool. The δ 13C signature of soil microbial biomass was 1.4‰ and 4.6‰ enriched relative to that of soil total and extractable pools respectively and showed significant differences between sites. However, microbial 13C enrichment was unrelated to measures of C and N availability. Our results confirm that 15N, but not 13C enrichment of soil microbial biomass reflects changes in C and N availability and N processing during long-term ecosystem development. [Copyright &y& Elsevier]

Microbes decompose soil organic matter (SOM), yet it is unclear how substrate inputs (i.e., stoichiometry) directly mediate microbial activities and community dynamics. We hypothesized that C+N input has the largest effect on microbial respiration and community structure, followed by C input and N input. Soils were collected from four ecosystems (grassland, piñon-juniper, ponderosa pine, mixed conifer) and amended with NH 4 NO 3 (N only; 100 μg g−1 wk−1), 13C-glucose (C only; 1000 μg g−1 wk−1), or C+N in a five-week laboratory incubation. We found that C+N input induced the greatest total respiration while C input induced the greatest SOM-derived respiration (i.e., priming effect) across ecosystems. Shifts in community composition were the largest with C+N input, followed by C input, and showed little response to N input. C only and C+N inputs increased both of the relative and absolute abundances of Actinobacteria and Proteobacteria (α, β, γ), but reduced the relative abundances of Verrucomicrobia and δ-Proteobacteria. C+N input increased the relative abundances of Bacillales, Rhizobiales, Burkholderiales and of 9 families, and reduced the relative abundances of Myxococcales and of 12 families, but showed little effect on the absolute abundances of these bacterial taxa. N input reduced the absolute abundances of Actinobacteria, Proteobacteria, and Verrucomicrobia but did not affect their relative abundances in the mixed conifer soil; by contrast, N input reduced relative abundances of δ-Proteobacteria and increased the relative abundances of γ-Proteobacteria but did not affect their absolute abundances in the ponderosa pine soil. We also found that substrate inputs were the main driver of SOM decomposition, microbial respiration and diversity, while soil ecosystem was the main driver of community composition and abundances of most bacterial phyla. Our work suggests that substrate stoichiometry has predictable effects on soil C cycling, microbial diversity and community composition, but has variable effects on microbial abundances, and that incorporating bacterial gene copies in abundance calculations can help more accurately estimate microbial responses across taxonomic levels and ecosystems. • Microbial responses to labile substate stoichiometry were studied in four ecosystems. • C only and C + N input increased respiration while N input showed little effect. • C only and C + N input altered microbial community composition and abundances. • Respiration was driven by substrate stoichiometry while communities by ecosystems. [ABSTRACT FROM AUTHOR]

Abstract: We report the first simultaneous measurements of δ 15N and δ 13C of DNA extracted from surface soils. The isotopic composition of DNA differed significantly among nine different soils. The δ 13C and δ 15N of DNA was correlated with δ 13C and δ 15N of soil, respectively, suggesting that the isotopic composition of DNA is strongly influenced by the isotopic composition of soil organic matter. However, in all samples DNA was enriched in 13C relative to soil, indicating microorganisms fractionated C during assimilation or preferentially used 13C enriched substrates. Enrichment of DNA in 15N relative to soil was not consistently observed, but there were significant differences between δ 15N of DNA and δ 15N of soil for three different sites, suggesting microorganisms are fractionating N or preferentially using N substrates at different rates across these contrasting ecosystems. There was a strong linear correlation between δ 15N of DNA and δ 15N of the microbial biomass, which indicated DNA was depleted in 15N relative to the microbial biomass by approximately 3.4‰. Our results show that accurate and precise isotopic measurements of C and N in DNA extracted from the soil are feasible, and that these analyses may provide powerful tools for elucidating C and N cycling processes through soil microorganisms. [Copyright &y& Elsevier]