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.

Soil microorganisms carry out many processes that are fundamental to soil functions. Among the millions of microbial cells present in a gram of soil, however, less than 2% are commonly estimated to be active at any point in time. Because the respiratory response of a bulk soil to carbon substrate addition would be expected to reflect the number of active cells, we hypothesized a positive correlation between active cells and soil respiration rates during substrate-induced respiration (SIR) assays. To test this, we monitored respiration and active cell counts during 24-h incubations of agricultural soil subsamples after treating with two carbon substrates or a water-only control. We enumerated active cells with the Bioorthogonal Non-canonical Amino Acid Tagging (BONCAT) method. BONCAT provides a labeled amino acid for active cells to incorporate into newly synthesized proteins, which can then be tagged with a fluorescent dye to enable enumeration by flow cytometry. Both respiration rates and active cell counts increased over time and were positively correlated with each other after 6 h of incubation. After 24 h, increases in active cells were proportionally greater than increases in respiration. Additionally, carbon-amended soils had higher respiration rates than water-only soils with similar active cell counts, suggesting differences in carbon use efficiency. Our study documents for the first time the respiratory response from in-situ microbial activation induced by substrate amendment of soil within 6 h, a short enough timescale to exclude most cell replication. This study also demonstrates that the correlation between active cell numbers and respiration is substrate-dependent. IMPORTANCEWhile many critical ecosystem services provided by soil are known to rely on microbial activity, the soil microbial community largely remains a black box. While respiration is a common indicator of bulk soil microbial activity, this study demonstrates that the relationship between respiration and the number of active cells differs based on available carbon substrates. Advancing knowledge in this area will both enable better interpretation of biological soil tests by land managers and inform researchers modeling contributions of soil microbial respiration to global carbon dynamics.

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.

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

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

Microbial analysis at the micro scale of soil is essential to the overall understanding of microbial organization and interactions, and necessary for a better understanding of soil ecosystem functioning. While bacterial communities have been extensively described, little is known about the organization of fungal communities as well as functional potentials at scales relevant to microbial interactions. Fungal and bacterial communities and changes in nitrogen cycling potentials in the pristine Rothamsted Park Grass soil (bulk soil) as well as in its particle size sub-fractions (PSFs; > 250 m, 250-63 m, 63-20 m, 20-2 m, < 2 m and supernatant) were studied. The potential for nitrogen reduction was found elevated in bigger aggregates. The relative abundance of Basidiomycota deceased with decreasing particle size, Ascomycota showed an increase and Mucoromycota became more prominent in particles less than 20 m. Bacterial community structures changed below 20 m at the scale where microbes operate. Strikingly, only members of two bacterial and one fungal phyla (Proteobacteria, Bacteroidota and Ascomycota, respectively) were washed-off the soil during fractionation and accumulated in the supernatant fraction where most of the detected bacterial genera (e.g., Pseudomonas, Massilia, Mucilaginibacter, Edaphobaculum, Duganella, Janthinobacterium and Variovorax) were previously associated with exopolysaccharide production and biofilm formation. Overall, the applied method shows potential to study soil microbial communities at micro scales which might be useful in studies focusing on the role of specific fungal taxa in soil structure formation as well as research on how and by whom biofilm-like structures are distributed and organized in soil. ImportanceIntensive exploitation of soils has led to increasing environmental concerns such as pollution, erosion, emission of greenhouse gases and, in general, the weakening of its ecosystem services that are mainly regulated by microbial activity. Microbial activity and metabolism drive the formation of soil aggregates, ranging in size from a few micrometres to several millimetres. Understanding biological mechanisms related to aggregate size classes can provide insight into large-scale processes, but most research has focused on macroaggregates. Here, we investigated the microbial community and its functional changes at these smaller scales that are clearly more relevant for assessing microbial activity. We demonstrated that fungal communities are more sensitive to bigger size classes than bacteria, suggesting their dominant role in soil structure formation and turnover. We also identified preferential niches for reductive processes within the nitrogen cycle and a selection of specific taxa by analysing the water used for the wet-fractionation approach.

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.

Reducing the environmental impact of Canadian field crop agriculture, including the reliance on conventional synthesised fertilisers, are key societal targets for establishing long-term sustainable practices. Municipal biosolids (MSB) are an abundant, residual organic material, rich in phosphate, nitrogen and other oligo-nutrients, that could be used in conjunction with conventional fertilisers to decrease their use. Though MBS have previously been shown to be an effective fertiliser substitute for different crops, including corn and soybean, there remain key knowledge gaps concerning the impact of MBS on the resident soil bacterial communities in agro-ecosystems. We hypothesised that the MBS fertiliser amendment would not significantly impact the structure or function of the soil bacterial communities, nor contribute to the spread of human pathogenic bacteria, in corn or soybean agricultural systems. In field experiments, fertiliser regimes for both crops were amended with MBS, and compared to corn and soybean plots with standard fertiliser treatments. We repeated this across four different agricultural sites in Quebec, over 2021 and 2022. We sampled MBS-treated, and untreated soils, and identified the composition of the soil bacterial communities via 16S rRNA metabarcoding. We found no indication that the MBS fertiliser amendment altered the structure of the soil bacterial communities, but rather that the soil type and crop identities were the most significant factors in structuring the bacterial communities. Moreover, there was no evidence that the MBS-treated soils experienced a shift in functions, nor contributed potential human bacterial pathogens over the two years of our study. Our analysis indicates that not only can MBS function as substitutes for conventional, synthesised fertilisers, but that they also do not disrupt the structure, or function, of the resident soil bacterial communities in the short term. Finally, we suggest that the use of MBS in agro-ecosystems poses no greater concern to the public than existing soil bacterial communities. HighlightsO_LIMunicipal biosolids may represent a sustainable fertiliser substitute C_LIO_LIBut, the impact of biosolids on soil bacteria in agricultural fields is unknown C_LIO_LIUsing 16S rRNA metabarcoding we analysed community structure and functions C_LIO_LIWe found no disruption of soil bacterial communities fertilised with biosolids C_LIO_LIBiosolids are safe, sustainable fertilisers with little impact on soil bacteria C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/571735v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1b9ca2corg.highwire.dtl.DTLVardef@8818d2org.highwire.dtl.DTLVardef@1158864org.highwire.dtl.DTLVardef@ad952f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Interactions between plants and microorganisms strongly affect ecosystem functioning as processes of plant productivity, litter decomposition and nutrient cycling are controlled by both organisms. Though two-sided interactions between plants and microorganisms and between microorganisms and litter decomposition are areas of major scientific research, our understanding of the three-sided interactions of plant-derived carbon flow into the soil microbial community and their follow-on effects on ecosystem processes like litter decomposition and plant nutrient uptake remains limited. Therefore, we performed a greenhouse experiment with two plant communities differing in their ability to associate with arbuscular mycorrhizal fungi (AMF). By applying a 13CO2 pulse label to the plant communities and adding various 15N labelled substrate types to ingrowth cores, we simultaneously traced the flow of plant-derived carbon into soil microbial communities and the return of mineralized nitrogen back to the plant communities. We observed that net 13C assimilation by the rhizosphere microbial communities and their community composition not only depended on plant-AMF association but also type of substrate being decomposed. AMF-association resulted in lower net 13C investment into the decomposer community than absence of the association for similar 15N uptake. This effect was driven by a reduced carbon flow to fungal and bacterial saprotrophs and a simultaneous increase of carbon flow to AMF. Additionally, in presence of AMF association CN flux also depended on the type of substrate being decomposed. Lower net 13C assimilation was observed for decomposition of plant-derived and microorganism-derived substrates whereas opposite was true for inorganic nitrogen. Interestingly, the decomposer communities assembled in the rhizosphere were structured by both the plant community and substrate amendments which suggests existence of functional overlap between the two soil contexts. Moreover, we present preliminary evidence that AMF association helps plants access nutrients that are locked in bacterial and plant necromass at a lower carbon cost. Therefore, we conclude that a better understanding of ecosystem processes like decomposition can only be achieved when the whole plant-microorganism-litter context is investigated.

Decomposition of organic matter is a key process in soils contributing to carbon and nutrient cycling. To identify management strategies for agroecosystems that reduce nutrient losses while maximizing plant growth, it is important to understand which parameters determine decomposition rates. This study therefore investigated how the presence of winter wheat (Triticum aestivum var. Asory) affects decomposition in a controlled Ecotron setup with two soil types with varying organic matter content across three simulated climates (2013, 2068, 2085). Using the tea bag index, interstitial soil pore water analyses, microbial biomass quantification, bacterial and fungal gene abundance, and soil respiration measurements, we tested the hypotheses that plant exudates would enhance decomposition rate and microbial biomass, while plant nitrogen uptake would deplete soil nitrate, potentially mitigated by fertilization. Contrary to expectations, decomposition rates were lower in planted than in unplanted soils, suggesting resource competition between plants and microbes. No significant differences were observed in microbial biomass or respiration due to plant presence, and fertilization effects on nitrate or microbial mineralization were undetectable, likely due to rapid turnover of organic molecules including uptake by plants and microbes. Mechanistically, fungi and soil humidity were more important for decomposition than bacteria or temperature. The findings corroborate climate impacts on decomposition but also indicate microbial resilience and highlight the potential of management strategies like cover crops, adjusted planting dates and crop residual management which can contribute to healthy soils by sustaining carbon and nutrient cycling.

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.

Soil microbes play pivotal roles in the multifunctioning of terrestrial ecosystems. In the context of global changes, there is an urgent need to protect soil microbial diversity, which relies on determining the abiotic and biotic factors that influence the diversity, composition, and assemblage of soil microbiota. A large number of informative studies have reported edaphic properties and climate factors as key drivers of soil bacteria microbiota. However, these studies were mainly conducted at the phylum level and based on a restricted number of non-microbial variables. In this study, we aimed to estimate the relative effects of abiotic and biotic factors in shaping soil bacterial communities at diverse taxonomic levels by focusing on 160 natural sites located in the southwest of France for which a large and unique set of non-microbial variables is available. After characterizing soil bacterial communities with the highly taxonomically resolving gyrB gene, we identified that in addition to pH, temperature, and precipitations, soil bacterial communities at the lowest taxonomic levels appear strongly structured by soil micronutrients, notably manganese. On the other hand, soil bacterial communities at the highest taxonomic levels appear strongly structured by the interplay between descriptors of plant communities and edaphic properties. Similar to previous observations on microbial pathogens, the strong and positive associations between soil bacterial species and the presence of particular plant species suggest host specificity for soil commensal bacteria. Altogether, a deeper characterization of both abiotic and biotic factors could help fuel programs designed for protecting and restoring soil ecosystem functions.

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.

Applying crop residues is a widely used strategy to increase soil organic matter (SOM) in arable soils because of its recorded effects on increasing microbial biomass and consequently necromass. However, fresh residue inputs could also "prime" the decomposition of native SOM, resulting in accelerated SOM depletion and greenhouse gas (GHG) emission. Increasing the botanical diversity of the crops grown in arable systems has been promoted to increase the delivery of multiple ecological functions, including increasing soil microbial biomass and SOM. Whether mixtures of fresh residues from different crops grown in polyculture contribute to soil carbon (C) pools to a greater extent than would be expected from applying individual residues (i.e., the mixture produces a non-additive synergistic effect) has not been systematically tested and is currently unknown. In this study, we used 13C isotope labelled cover crop residues (i.e., buckwheat, clover, radish, and sunflower) to track the fate of plant residue-derived C and C derived from the priming of SOM in treatments comprising a quaternary mixture of the residues and the average effect of the four individual residues one day after residue incorporation in a laboratory microcosm experiment. Our results indicate that, despite all treatments receiving the same amount of plant residue-derived C (1 mg-1 C g soil), the total microbial biomass in the treatment receiving the residue mixture was significantly greater, by 26% (3.69 {micro}g-1 C g), than the average microbial biomass observed in treatments receiving the four individual components of the mixture one day after applying crop residues. The greater microbial biomass C in the quaternary mixture, compared to average of the individual residue treatments, that can be attributed directly to the plant residue applied was also significantly greater, by 132% (3.61 {micro}g-1 C g). However, there was no evidence that the mixture resulted in any more priming of native SOM than average priming observed in the individual residue treatments. The soil microbial community structure, assessed using phospholipid fatty acid (PLFA) analysis, was significantly (P < 0.001) different in the soil receiving the residue mixture, compared to the average structures of the communities in soil receiving four individual residues. Differences in the biomass of fungi, general bacteria, and Gram-positive bacteria were responsible for the observed synergistic effect of crop residue mixtures on total microbial biomass and residue-derived microbial biomass, especially biomarkers 16:0, 18:2{omega}6 and 18:3{omega}3. Our study demonstrates that applying a mixture of crop residues increases soil microbial biomass to a greater extent than would be expected from applying individual residues and that this occurs either due to faster decomposition of the crop residues or greater carbon use efficiency (CUE), rather than priming the decomposition of native SOM. Therefore, growing crop polycultures (e.g., cover crop mixtures) and incorporating mixtures of the resulting crop residues into the soil could be an effective method to increase microbial biomass and ultimately C stocks in arable soils.

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.

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.

Oxygen concentrations fluctuate in soil across time and space. Under anoxic conditions, the three main microbial metabolic pathways - denitrification, fermentation, and DNRA - compete for the same carbon (C) sources. Studies on denitrification in complex soil communities often rely on incubation experiments to determine how various factors affect the regulatory biology of denitrifying organisms and their N2O emissions. These experiments typically require an exogenous C source to stimulate measurable activity, and the choice of C source is critical as it should support denitrification while minimizing competition from fermentation and DNRA. This consideration is equally important for the enrichment and isolation of diverse denitrifying organisms and for bioaugmentation-based N2O-mitigation strategies. Here, we compared twelve C sources, including glutamic acid, acetate, an artificial root exudate cocktail (eight compounds, individually and in combination), and a clover extract. By combining high-resolution denitrification gas kinetics, metagenomic sequencing, and 15N isotope labelling, we aimed to find a C source(s) that (1) supports a diverse soil-derived denitrifying community and (2) limits the competition for C from alternative anaerobic pathways. Among the tested substrates, only the clover extract supported denitrification and maintained a complex denitrifying community. Yet, it also promoted fermentation and DNRA, revealing that a trade-off must exist between fostering denitrifier diversity and limiting growth of organisms using competing anaerobic pathways. More broadly, our results highlight that C source is a methodological fulcrum in controlled soil microbiome studies. It shapes community composition, drives metabolic processes, and ultimately determines the ecological relevance of experimental outcomes and the success of enrichments and soil bioaugmentation approaches.

Soil organic matter (SOM) consists of diverse biochemical constituents, spanning a spectrum of chemical complexity, and the relative abundance of these substrates influences microbial metabolism and soil carbon persistence. However, mechanistic controls governing these processes and how they may be affected by environmental change remains incomplete. This study aims to assess (1) the molecular-level changes that occur across stages of root decomposition, from undecayed plant root litter to 1-year decomposed root litter, to mineral SOM and (2) how these changes are altered by anthropogenic nitrogen (N) deposition by using SOM biochemical and microbiome datasets and a long-term field experiment. N deposition did not significantly alter undecomposed or 1-year decomposed root litter, but did alter decomposing microbial communities and mineral SOM biochemical composition, specifically in lignin- and lipid-derived compounds. Taken together, this restructuring of microbial communities and alteration of SOM biochemistry likely contributed to the previously observed reduction in SOM decomposition.

Climate change is expected to affect precipitation intensity and soil temperature and indirectly impact the release of leached dissolved organic carbon (LDOC) from leaf litter during the early stages of its decomposition, which could affect the health and function of forest soil ecosystem. Here, we experimentally investigate the spatially-explicit impact of LDOC on the forest soil microbiome and the associated biogeochemical processes. Homogenized soil columns were subjected to realistic artificial precipitation for 3 months with the initial level of LDOC adjusted by the number of times the leaf litter was flushed in preparation for the experiment. Hydrological and geochemical parameters (redox potential, pH, dissolved oxygen, soil moisture, matric potential, chemical speciation) were measured continuously as a function of time and depth. The same initial microbial community developed into distinct communities under different LDOC and above and below the water table. The LDOC from leaf litter increased the availability of carbon (C) and nitrogen (N) in porewater four-fold and two-fold respectively in the first two weeks. This resulted in the expansion of the anoxic zone above the water table and a decrease in the soil microbial metabolic potential for cellulolysis and N2 fixation in unsaturated soil along with an increase of soil microbial metabolic potential for fermentation at all depths. Finally, increased LDOC decreased the stability, phylogenetic diversity, and complexity of the soil microbiome, limiting its functional diversity. Thus, management of leaf litter should receive more attention due to its indirect role in the impact of climate change on the soil microbiome. HighlightsO_LIDecreased microbiome diversity and stability due to enhanced leaf litter leaching C_LIO_LIExpansion of the anoxic zone into the unsaturated zone due to increased organic carbon supply C_LIO_LIDecreased soil microbiome metabolic potential for cellulolysis and N2 fixation in unsaturated soil C_LIO_LIDepth-dependent response of microbial community to increased organic carbon availability C_LIO_LIImplications for soil response to climate change C_LI

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.