Warming effects on grassland soil microbial communities are amplified in cool months.

Global warming modulates soil respiration (RS) via microbial decomposition, which is seasonally dependent. Yet, the magnitude and direction of this modulation remain unclear, partly owing to the lack of knowledge on how microorganisms respond to seasonal changes. Here, we investigated the temporal dynamics of soil microbial communities over 12 consecutive months under experimental warming in a tallgrass prairie ecosystem. The interplay between warming and time altered (p < 0.05) the taxonomic and functional compositions of microbial communities. During the cool months (January to February and October to December), warming induced a soil microbiome with a higher genomic potential for carbon decomposition, community-level ribosomal RNA operon (rrn) copy numbers, and microbial metabolic quotients, suggesting that warming stimulated fast-growing microorganisms that enhanced carbon decomposition. Modeling analyses further showed that warming reduced the temperature sensitivity of microbial carbon use efficiency (CUE) by 28.7% when monthly average temperature was low, resulting in lower microbial CUE and higher heterotrophic respiration (Rh) potentials. Structural equation modeling showed that warming modulated both Rh and RS directly by altering soil temperature and indirectly by influencing microbial community traits, soil moisture, nitrate content, soil pH, and gross primary productivity. The modulation of Rh by warming was more pronounced in cooler months compared to warmer ones. Together, our findings reveal distinct warming-induced effects on microbial functional traits in cool months, challenging the norm of soil sampling only in the peak growing season, and advancing our mechanistic understanding of the seasonal pattern of RS and Rh sensitivity to warming.

Microbial responses to long-term warming differ across soil microenvironments.

Soil carbon loss is likely to increase due to climate warming, but microbiomes and microenvironments may dampen this effect. In a 30-year warming experiment, physical protection within soil aggregates affected the thermal responses of soil microbiomes and carbon dynamics. In this study, we combined metagenomic analysis with physical characterization of soil aggregates to explore mechanisms by which microbial communities respond to climate warming across different soil microenvironments. Long-term warming decreased the relative abundances of genes involved in degrading labile compounds (e.g. cellulose), but increased those genes involved in degrading recalcitrant compounds (e.g. lignin) across aggregate sizes. These changes were observed in most phyla of bacteria, especially for Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, and Planctomycetes. Microbial community composition was considerably altered by warming, leading to declined diversity for bacteria and fungi but not for archaea. Microbial functional genes, diversity, and community composition differed between macroaggregates and microaggregates, indicating the essential role of physical protection in controlling microbial community dynamics. Our findings suggest that microbes have the capacity to employ various strategies to acclimate or adapt to climate change (e.g. warming, heat stress) by shifting functional gene abundances and community structures in varying microenvironments, as regulated by soil physical protection.

Regional microbial biogeography linked to soil respiration.

The relationships between α-diversity and ecosystem functioning (BEF) have been extensively examined. However, it remains unknown how spatial heterogeneity of microbial community, i.e., microbial ß-diversity within a region, shapes ecosystem functioning. Here, we examined microbial community compositions and soil respiration (Rs) along an elevation gradient of 853-4420 m a.s.l. in the southeastern Tibetan Plateau, which is renowned as one of the world's biodiversity hotspots. There were significant distance-decay relationships for both bacterial and fungal communities. Stochastic processes played a dominant role in shaping bacterial and fungal community compositions, while soil temperature was the most important environmental factor that affected microbial communities. We evaluated BEF relationships based on α-diversity measured by species richness and ß-diversity measured by community dispersions, revealing significantly positive correlations between microbial ß-diversities and Rs. These correlations became stronger with increasing sample size, differing from those between microbial α-diversities and Rs. Using Structural Equation Modeling (SEM), we found that soil temperature, soil moisture, and total nitrogen were the most important edaphic properties in explaining Rs. Meanwhile, stochastic processes (e.g., homogenous dispersal and dispersal limitation) significantly mediated effects between microbial ß-diversities and Rs. Microbial α-diversity poorly explained Rs, directly or indirectly. In a nutshell, we identified a previously unknown BEF relationship between microbial ß-diversity and Rs. By complementing common practices to examine BEF with α-diversity, we demonstrate that a focus on ß-diversity could be leveraged to explain Rs.

Contribution of Microorganisms with the Clade II Nitrous Oxide Reductase to Suppression of Surface Emissions of Nitrous Oxide.

The sources and sinks of nitrous oxide, as control emissions to the atmosphere, are generally poorly constrained for most environmental systems. Initial depth-resolved analysis of nitrous oxide flux from observation wells and the proximal surface within a nitrate contaminated aquifer system revealed high subsurface production but little escape from the surface. To better understand the environmental controls of production and emission at this site, we used a combination of isotopic, geochemical, and molecular analyses to show that chemodenitrification and bacterial denitrification are major sources of nitrous oxide in this subsurface, where low DO, low pH, and high nitrate are correlated with significant nitrous oxide production. Depth-resolved metagenomes showed that consumption of nitrous oxide near the surface was correlated with an enrichment of Clade II nitrous oxide reducers, consistent with a growing appreciation of their importance in controlling release of nitrous oxide to the atmosphere. Our work also provides evidence for the reduction of nitrous oxide at a pH of 4, well below the generally accepted limit of pH 5.

Legume rhizodeposition promotes nitrogen fixation by soil microbiota under crop diversification.

Biological nitrogen fixation by free-living bacteria and rhizobial symbiosis with legumes plays a key role in sustainable crop production. Here, we study how different crop combinations influence the interaction between peanut plants and their rhizosphere microbiota via metabolite deposition and functional responses of free-living and symbiotic nitrogen-fixing bacteria. Based on a long-term (8 year) diversified cropping field experiment, we find that peanut co-cultured with maize and oilseed rape lead to specific changes in peanut rhizosphere metabolite profiles and bacterial functions and nodulation. Flavonoids and coumarins accumulate due to the activation of phenylpropanoid biosynthesis pathways in peanuts. These changes enhance the growth and nitrogen fixation activity of free-living bacterial isolates, and root nodulation by symbiotic Bradyrhizobium isolates. Peanut plant root metabolites interact with Bradyrhizobium isolates contributing to initiate nodulation. Our findings demonstrate that tailored intercropping could be used to improve soil nitrogen availability through changes in the rhizosphere microbiome and its functions.

Warming Leads to Changes in Soil Organic Carbon Molecules Due to Decreased Mineral Protection.

Climate change affects the content and composition of soil organic carbon (SOC). However, warming-induced changes in the SOC compounds remain unknown. Using nuclear magnetic resonance spectroscopy, molecular mixing models, and Fourier transform ion cyclotron resonance mass spectrometry, we analyzed the variations and relationships in molecular compounds in Mollisol with 10-56 g C kg-1 soil-1 by translocating soils under six climate regimes. We found that increased temperature and precipitation were negatively correlated with carbohydrate versus lipid and lignin versus protein. The former was consistent across soils with varying SOC contents, but the latter decreased as the SOC content increased. The carbohydrate-lipid correlations were related to dithionite-citrate-extractable Fe, while the lignin-protein correlations were linked to changes in moisture and pyrophosphate-extractable Fe/Al. Our findings indicate that the reduction in the mineral protection of SOC is associated with molecular alterations in SOC under warming conditions.

Reproducible responses of geochemical and microbial successional patterns in the subsurface to carbon source amendment.

Carbon amendments designed to remediate environmental contamination lead to substantial perturbations when injected into the subsurface. For the remediation of uranium contamination, carbon amendments promote reducing conditions to allow microorganisms to reduce uranium to an insoluble, less mobile state. However, the reproducibility of these amendments and underlying microbial community assembly mechanisms have rarely been investigated in the field. In this study, two injections of emulsified vegetable oil were performed in 2009 and 2017 to immobilize uranium in the groundwater at Oak Ridge, TN, USA. Our objectives were to determine whether and how the injections resulted in similar abiotic and biotic responses and their underlying community assembly mechanisms. Both injections caused similar geochemical and microbial succession. Uranium, nitrate, and sulfate concentrations in the groundwater dropped following the injection, and specific microbial taxa responded at roughly the same time points in both injections, including Geobacter, Desulfovibrio, and members of the phylum Comamonadaceae, all of which are well established in uranium, nitrate, and sulfate reduction. Both injections induced a transition from relatively stochastic to more deterministic assembly of microbial taxonomic and phylogenetic community structures based on 16S rRNA gene analysis. We conclude that geochemical and microbial successions after biostimulation are reproducible, likely owing to the selection of similar phylogenetic groups in response to EVO injection.

Diversity and distribution of biosynthetic gene clusters in agricultural soil microbiomes.

Bacterial secondary metabolites serve as an important source of molecules for drug discovery. They also play an important function in mediating the interactions of microbial producers with their living environment and surrounding organisms. However, little is known about the genetic novelty, distribution, and community-level impacts of soil bacterial biosynthetic potential on a large geographic scale. Here, we constructed the first catalog of 11,149 biosynthetic gene clusters (BGCs) from agricultural soils across China and unearthed hidden biosynthetic potential for new natural product discovery from the not-yet-cultivated soil bacteria. Notably, we revealed soil pH as the strongest environmental driver of BGC biogeography and predicted that soil acidification and global climate change could damage the biosynthetic potential of the soil microbiome. The co-occurrence network of bacterial genomes revealed two BGC-rich species, i.e., Nocardia niigatensis from Actinobacteriota and PSRF01 from Acidobacteriota, as the module hub and connector, respectively, indicating their keystone positions in the soil microbial communities. We also uncovered a dominant role of BGC-inferred biotic interactions over environmental drivers in structuring the soil microbiome. Overall, this study achieved novel insights into the BGC landscape in agricultural soils of China, substantially expanding our understanding of the diversity and novelty of bacterial secondary metabolism and the potential role of secondary metabolites in microbiota assembly.IMPORTANCEBacterial secondary metabolites not only serve as the foundation for numerous therapeutics (e.g., antibiotics and anticancer drugs), but they also play critical ecological roles in mediating microbial interactions (e.g., competition and communication). However, our knowledge of bacterial secondary metabolism is limited to only a small fraction of cultured strains, thus restricting our comprehensive understanding of their diversity, novelty, and potential ecological roles in soil ecosystems. Here, we used culture-independent metagenomics to explore biosynthetic potentials in agricultural soils of China. Our analyses revealed a high degree of genetic diversity and novelty within biosynthetic gene clusters in agricultural soil environments, offering valuable insights for biochemists seeking to synthesize novel bioactive products. Furthermore, we uncovered the pivotal role of BGC-rich species in microbial communities and the significant relationship between BGC richness and microbial phylogenetic turnover. This information emphasizes the importance of biosynthetic potential in the assembly of microbial communities.

BASALT refines binning from metagenomic data and increases resolution of genome-resolved metagenomic analysis.

Metagenomic binning is an essential technique for genome-resolved characterization of uncultured microorganisms in various ecosystems but hampered by the low efficiency of binning tools in adequately recovering metagenome-assembled genomes (MAGs). Here, we introduce BASALT (Binning Across a Series of Assemblies Toolkit) for binning and refinement of short- and long-read sequencing data. BASALT employs multiple binners with multiple thresholds to produce initial bins, then utilizes neural networks to identify core sequences to remove redundant bins and refine non-redundant bins. Using the same assemblies generated from Critical Assessment of Metagenome Interpretation (CAMI) datasets, BASALT produces up to twice as many MAGs as VAMB, DASTool, or metaWRAP. Processing assemblies from a lake sediment dataset, BASALT produces ~30% more MAGs than metaWRAP, including 21 unique class-level prokaryotic lineages. Functional annotations reveal that BASALT can retrieve 47.6% more non-redundant opening-reading frames than metaWRAP. These results highlight the robust handling of metagenomic sequencing data of BASALT.

Effects of operational parameters on bacterial communities in Hong Kong and global wastewater treatment plants.

Wastewater treatment plants (WWTPs) are indispensable biotechnology facilities for modern cities and play an essential role in modern urban infrastructure by employing microorganisms to remove pollutants in wastewater, thus protecting public health and the environment. This study conducted a 13-month bacterial community survey of six full-scale WWTPs in Hong Kong with samples of influent, activated sludge (AS), and effluent to explore their synchronism and asynchronism of bacterial community. Besides, we compared AS results of six Hong Kong WWTPs with data from 1,186 AS amplicon data in 269 global WWTPs and a 9-year metagenomic sequencing survey of a Hong Kong WWTP. Our results showed the compositions of bacterial communities varied and the bacterial community structure of AS had obvious differences across Hong Kong WWTPs. The co-occurrence analysis identified 40 pairs of relationships that existed among Hong Kong WWTPs to show solid associations between two species and stochastic processes took large proportions for the bacterial community assembly of six WWTPs. The abundance and distribution of the functional bacteria in worldwide and Hong Kong WWTPs were examined and compared, and we found that ammonia-oxidizing bacteria had more diversity than nitrite-oxidizing bacteria. Besides, Hong Kong WWTPs could make great contributions to the genome mining of microbial dark matter in the global "wanted list." Operational parameters had important effects on OTUs' abundance, such as the temperature to the genera of Tetrasphaera, Gordonia and Nitrospira. All these results obtained from this study can deepen our understanding of the microbial ecology in WWTPs and provide foundations for further studies. IMPORTANCE: Wastewater treatment plants (WWTPs) are an indispensable component of modern cities, as they can remove pollutants in wastewater to prevent anthropogenic activities. Activated sludge (AS) is a fundamental wastewater treatment process and it harbors a highly complex microbial community that forms the main components and contains functional groups. Unveiling "who is there" is a long-term goal of the research on AS microbiology. High-throughput sequencing provides insights into the inventory diversity of microbial communities to an unprecedented level of detail. At present, the analysis of communities in WWTPs usually comes from a specific WWTP and lacks comparisons and verification among different WWTPs. The wide-scale and long-term sampling project and research in this study could help us evaluate the AS community more accurately to find the similarities and different results for different WWTPs in Hong Kong and other regions of the world.

Experimental warming accelerates positive soil priming in a temperate grassland ecosystem.

Unravelling biosphere feedback mechanisms is crucial for predicting the impacts of global warming. Soil priming, an effect of fresh plant-derived carbon (C) on native soil organic carbon (SOC) decomposition, is a key feedback mechanism that could release large amounts of soil C into the atmosphere. However, the impacts of climate warming on soil priming remain elusive. Here, we show that experimental warming accelerates soil priming by 12.7% in a temperate grassland. Warming alters bacterial communities, with 38% of unique active phylotypes detected under warming. The functional genes essential for soil C decomposition are also stimulated, which could be linked to priming effects. We incorporate lab-derived information into an ecosystem model showing that model parameter uncertainty can be reduced by 32-37%. Model simulations from 2010 to 2016 indicate an increase in soil C decomposition under warming, with a 9.1% rise in priming-induced CO2 emissions. If our findings can be generalized to other ecosystems over an extended period of time, soil priming could play an important role in terrestrial C cycle feedbacks and climate change.

Community assembly of organisms regulates soil microbial functional potential through dual mechanisms.

Unraveling the influence of community assembly processes on soil ecosystem functioning presents a major challenge in the field of theoretical ecology, as it has received limited attention. Here, we used a series of long-term experiments spanning over 25 years to explore the assembly processes of bacterial, fungal, protist, and nematode communities using high-throughput sequencing. We characterized the soil microbial functional potential by the abundance of microbial genes associated with carbon, nitrogen, phosphorus, and sulfur cycling using GeoChip-based functional gene profiling, and determined how the assembly processes of organism groups regulate soil microbial functional potential through community diversity and network stability. Our results indicated that balanced fertilization (NPK) treatment improved the stochastic assembly of bacterial, fungal, and protist communities compared to phosphorus-deficient fertilization (NK) treatment. However, there was a nonsignificant increase in the normalized stochasticity ratio of the nematode community in response to fertilization across sites. Our findings emphasized that soil environmental factors influenced the assembly processes of the biotic community, which regulated soil microbial functional potential through dual mechanisms. One mechanism indicated that the high phosphorus levels and low soil nutrient stoichiometry may increase the stochasticity of bacterial, fungal, and protist communities and the determinism of the nematode community under NPK treatment, ultimately enhancing soil microbial functional potential by reinforcing the network stability of the biotic community. The other mechanism indicated that the low phosphorus levels and high soil nutrient stoichiometry may increase the stochastic process of the bacterial community and the determinism of the fungal, protist, and nematode communities under NK treatment, thereby enhancing soil microbial functional potential by improving the ß-diversity of the biotic community. Taken together, these results provide valuable insights into the mechanisms underlying the assembly processes of the biotic community that regulate ecosystem functioning.

Novel order-level lineage of ammonia-oxidizing archaea widespread in marine and terrestrial environments.

Ammonia-oxidizing archaea (AOA) are among the most ubiquitous and abundant archaea on Earth, widely distributed in marine, terrestrial, and geothermal ecosystems. However, the genomic diversity, biogeography, and evolutionary process of AOA populations in subsurface environments are vastly understudied compared to those in marine and soil systems. Here, we report a novel AOA order Candidatus (Ca.) Nitrosomirales which forms a sister lineage to the thermophilic Ca. Nitrosocaldales. Metagenomic and 16S rRNA gene-read mapping demonstrates the abundant presence of Nitrosomirales AOA in various groundwater environments and their widespread distribution across a range of geothermal, terrestrial, and marine habitats. Terrestrial Nitrosomirales AOA show the genetic capacity of using formate as a source of reductant and using nitrate as an alternative electron acceptor. Nitrosomirales AOA appear to have acquired key metabolic genes and operons from other mesophilic populations via horizontal gene transfer, including genes encoding urease, nitrite reductase, and V-type ATPase. The additional metabolic versatility conferred by acquired functions may have facilitated their radiation into a variety of subsurface, marine, and soil environments. We also provide evidence that each of the four AOA orders spans both marine and terrestrial habitats, which suggests a more complex evolutionary history for major AOA lineages than previously proposed. Together, these findings establish a robust phylogenomic framework of AOA and provide new insights into the ecology and adaptation of this globally abundant functional guild.

Microbially mediated mechanisms underlie soil carbon accrual by conservation agriculture under decade-long warming.

Increasing soil organic carbon (SOC) in croplands by switching from conventional to conservation management may be hampered by stimulated microbial decomposition under warming. Here, we test the interactive effects of agricultural management and warming on SOC persistence and underlying microbial mechanisms in a decade-long controlled experiment on a wheat-maize cropping system. Warming increased SOC content and accelerated fungal community temporal turnover under conservation agriculture (no tillage, chopped crop residue), but not under conventional agriculture (annual tillage, crop residue removed). Microbial carbon use efficiency (CUE) and growth increased linearly over time, with stronger positive warming effects after 5 years under conservation agriculture. According to structural equation models, these increases arose from greater carbon inputs from the crops, which indirectly controlled microbial CUE via changes in fungal communities. As a result, fungal necromass increased from 28 to 53%, emerging as the strongest predictor of SOC content. Collectively, our results demonstrate how management and climatic factors can interact to alter microbial community composition, physiology and functions and, in turn, SOC formation and accrual in croplands.

Environmental stress mediates groundwater microbial community assembly.

Community assembly describes how different ecological processes shape microbial community composition and structure. How environmental factors impact community assembly remains elusive. Here we sampled microbial communities and >200 biogeochemical variables in groundwater at the Oak Ridge Field Research Center, a former nuclear waste disposal site, and developed a theoretical framework to conceptualize the relationships between community assembly processes and environmental stresses. We found that stochastic assembly processes were critical (>60% on average) in shaping community structure, but their relative importance decreased as stress increased. Dispersal limitation and 'drift' related to random birth and death had negative correlations with stresses, whereas the selection processes leading to dissimilar communities increased with stresses, primarily related to pH, cobalt and molybdenum. Assembly mechanisms also varied greatly among different phylogenetic groups. Our findings highlight the importance of microbial dispersal limitation and environmental heterogeneity in ecosystem restoration and management.

Ammonia-oxidizing bacteria and archaea exhibit differential nitrogen source preferences.

Ammonia-oxidizing microorganisms (AOM) contribute to one of the largest nitrogen fluxes in the global nitrogen budget. Four distinct lineages of AOM: ammonia-oxidizing archaea (AOA), beta- and gamma-proteobacterial ammonia-oxidizing bacteria (ß-AOB and γ-AOB) and complete ammonia oxidizers (comammox), are thought to compete for ammonia as their primary nitrogen substrate. In addition, many AOM species can utilize urea as an alternative energy and nitrogen source through hydrolysis to ammonia. How the coordination of ammonia and urea metabolism in AOM influences their ecology remains poorly understood. Here we use stable isotope tracing, kinetics and transcriptomics experiments to show that representatives of the AOM lineages employ distinct regulatory strategies for ammonia or urea utilization, thereby minimizing direct substrate competition. The tested AOA and comammox species preferentially used ammonia over urea, while ß-AOB favoured urea utilization, repressed ammonia transport in the presence of urea and showed higher affinity for urea than for ammonia. Characterized γ-AOB co-utilized both substrates. These results reveal contrasting niche adaptation and coexistence patterns among the major AOM lineages.

Multiple spatial scales of bacterial and fungal structural and functional traits affect carbon mineralization.

Studying the functional heterogeneity of soil microorganisms at different spatial scales and linking it to soil carbon mineralization is crucial for predicting the response of soil carbon stability to environmental changes and human disturbance. Here, a total of 429 soil samples were collected from typical paddy fields in China, and the bacterial and fungal communities as well as functional genes related to carbon mineralization in the soil were analysed using MiSeq sequencing and GeoChip gene microarray technology. We postulate that CO2 emissions resulting from bacterial and fungal carbon mineralization are contingent upon their respective carbon consumption strategies, which rely on the regulation of interactions between biodiversity and functional genes. Our results showed that the spatial turnover of the fungal community was 2-4 times that of the bacterial community from hundreds of meters to thousands of kilometres. The effect of spatial scale exerted a greater impact on the composition rather than the functional characteristics of the microbial community. Furthermore, based on the establishment of functional networks at different spatial scales, we observed that both bacteria and fungi within the top 10 taxa associated with carbon mineralization exhibited a prevalence of generalist species at the regional scale. This study emphasizes the significance of spatial scaling patterns in soil bacterial and fungal carbon degradation functions, deepening our understanding of how the relationship between microbial decomposers and soil heterogeneity impacts carbon mineralization and subsequent greenhouse gas emissions.

Effects of error, chimera, bias, and GC content on the accuracy of amplicon sequencing.

IMPORTANCE: Amplicon sequencing of targeted genes is the predominant approach to estimate the membership and structure of microbial communities. However, accurate reconstruction of community composition is difficult due to sequencing errors, and other methodological biases and effective approaches to overcome these challenges are essential. Using a mock community of 33 phylogenetically diverse strains, this study evaluated the effect of GC content on sequencing results and tested different approaches to improve overall sequencing accuracy while characterizing the pros and cons of popular amplicon sequence data processing approaches. The sequencing results from this study can serve as a benchmarking data set for future algorithmic improvements. Furthermore, the new insights on sequencing error, chimera formation, and GC bias from this study will help enhance the quality of amplicon sequencing studies and support the development of new data analysis approaches.

Root microbiota confers rice resistance to aluminium toxicity and phosphorus deficiency in acidic soils.

Aluminium (Al) toxicity impedes crop growth in acidic soils and is considered the second largest abiotic stress after drought for crops worldwide. Despite remarkable progress in understanding Al resistance in plants, it is still unknown whether and how the soil microbiota confers Al resistance to crops. Here we found that a synthetic community composed of highly Al-resistant bacterial strains isolated from the rice rhizosphere increased rice yield by 26.36% in acidic fields. The synthetic community harvested rhizodeposited carbon for successful proliferation and mitigated soil acidification and Al toxicity through extracellular protonation. The functional coordination between plants and microbes offers a promising way to increase the usage of legacy phosphorus in topsoil. These findings highlight the potential of microbial tools for advancing sustainable agriculture in acidic soils.