Metabolic potential of Nitrososphaera-associated clades.

Soil ammonia-oxidizing archaea (AOA) play a crucial role in converting ammonia to nitrite, thereby mobilizing reactive nitrogen species into their soluble form, with a significant impact on nitrogen losses from terrestrial soils. Yet, our knowledge regarding their diversity and functions remains limited. In this study, we reconstructed 97 high-quality AOA metagenome-assembled genomes (MAGs) from 180 soil samples collected in Central Germany during 2014-2019 summers. These MAGs were affiliated with the order Nitrososphaerales (NS) and clustered into four family-level clades (NS-α/γ/δ/ε). Among these MAGs, 75 belonged to the most abundant but least understood δ-clade. Within the δ-clade, the amoA genes in three MAGs from neutral soils showed a 99.5% similarity to the fosmid clone 54d9, which has served as representative of the δ-clade for the past two decades since even today no cultivated representatives are available. 72 MAGs constituted a distinct δ sub-clade, and their abundance and expression activity were more than twice that of other MAGs in slightly acidic soils. Unlike the less abundant clades (α, γ, and ε), the δ-MAGs possessed multiple highly expressed intracellular and extracellular carbohydrate-active enzymes responsible for carbohydrate binding (CBM32) and degradation (GH5), along with highly expressed genes involved in ammonia oxidation. Together, these results suggest metabolic versatility of uncultured soil AOA and a potential mixotrophic or chemolithoheterotrophic lifestyle among 54d9-like AOA.

Time-shifted expression of acetoclastic and methylotrophic methanogenesis by a single Methanosarcina genomospecies predominates the methanogen dynamics in Philippine rice field soil.

BACKGROUND: The final step in the anaerobic decomposition of biopolymers is methanogenesis. Rice field soils are a major anthropogenic source of methane, with straw commonly used as a fertilizer in rice farming. Here, we aimed to decipher the structural and functional responses of the methanogenic community to rice straw addition during an extended anoxic incubation (120 days) of Philippine paddy soil. The research combined process measurements, quantitative real-time PCR and RT-PCR of particular biomarkers (16S rRNA, mcrA), and meta-omics (environmental genomics and transcriptomics). RESULTS: The analysis methods collectively revealed two major bacterial and methanogenic activity phases: early (days 7 to 21) and late (days 28 to 60) community responses, separated by a significant transient decline in microbial gene and transcript abundances and CH4 production rate. The two methanogenic activity phases corresponded to the greatest rRNA and mRNA abundances of the Methanosarcinaceae but differed in the methanogenic pathways expressed. While three genetically distinct Methanosarcina populations contributed to acetoclastic methanogenesis during the early activity phase, the late activity phase was defined by methylotrophic methanogenesis performed by a single Methanosarcina genomospecies. Closely related to Methanosarcina sp. MSH10X1, mapping of environmental transcripts onto metagenome-assembled genomes (MAGs) and population-specific reference genomes revealed this genomospecies as the key player in acetoclastic and methylotrophic methanogenesis. The anaerobic food web was driven by a complex bacterial community, with Geobacteraceae and Peptococcaceae being putative candidates for a functional interplay with Methanosarcina. Members of the Methanocellaceae were the key players in hydrogenotrophic methanogenesis, while the acetoclastic activity of Methanotrichaceae members was detectable only during the very late community response. CONCLUSIONS: The predominant but time-shifted expression of acetoclastic and methylotrophic methanogenesis by a single Methanosarcina genomospecies represents a novel finding that expands our hitherto knowledge of the methanogenic pathways being highly expressed in paddy soils. Video Abstract.

Straw mulch improves soil carbon and nitrogen cycle by mediating microbial community structure and function in the maize field.

This study was conducted to investigate the capability of the microbial community characteristics and soil variables to promote carbon and nitrogen cycles in maize fields under straw mulch. We covered the surface soil of the maize field with different amounts of wheat straw (0 kg/ha, 2,250 kg/ha, and 4,500 kg/ha) and used 16S rRNA and ITS sequencing, Biology ECO-plate, traditional enzymology, TOC analyzer, and HPLC to measure bacterial and fungal community composition and functions, characteristics of microbial carbon source metabolism, carbon and nitrogen fraction, enzyme activity, and organic acid content in the maize rhizosphere and non-rhizosphere. The results indicated that short-term straw mulch insignificantly affected the alpha diversity of bacterial and fungal communities whereas significantly influenced their beta diversity. The results of functional prediction revealed that straw mulch considerably boosted the relative abundances of bacteria belonging to chemoheterotrophy, aerobic chemoheterotrophy, ureolysis, and nitrogen fixation and inhibited fermentation and nitrate reduction in maize rhizosphere soil. These processes primarily drove the C and N cycles in soil. Straw mulch also improved fungal saprotrophs by raising the proportion of Chaetomiaceae and Chaetosphaeriaceae. The Biology ECO-plate results illustrated that straw mulch weakened the metabolism capacity of microbial labile carbon resources. As a result, the labile C and N fractions were raised under straw mulch. Our results also showed that straw mulch primarily regulated the microbial community structure in rhizosphere soil by significantly decreasing Firmicutes and Ascomycota relative abundance while increasing Basidiomycota. The fungal community structure is more than bacterial for affecting soil microbial biomass carbon, readily oxidizable organic carbon, dissolved organic carbon, available nitrogen, ammonium, and nitrate directly and indirectly through malic acid content and cellulase, protease, and amylase activity. Overall, our findings imply that straw mulch might influence the bacterial and fungal community structures, thereby boosting the production of labile C and N components and accelerating the C and N cycle in maize fields.

Extreme summers impact cropland and grassland soil microbiomes.

The increasing frequency of extreme weather events highlights the need to understand how soil microbiomes respond to such disturbances. Here, metagenomics was used to investigate the effects of future climate scenarios (+0.6 °C warming and altered precipitation) on soil microbiomes during the summers of 2014-2019. Unexpectedly, Central Europe experienced extreme heatwaves and droughts during 2018-2019, causing significant impacts on the structure, assembly, and function of soil microbiomes. Specifically, the relative abundance of Actinobacteria (bacteria), Eurotiales (fungi), and Vilmaviridae (viruses) was significantly increased in both cropland and grassland. The contribution of homogeneous selection to bacterial community assembly increased significantly from 40.0% in normal summers to 51.9% in extreme summers. Moreover, genes associated with microbial antioxidant (Ni-SOD), cell wall biosynthesis (glmSMU, murABCDEF), heat shock proteins (GroES/GroEL, Hsp40), and sporulation (spoIID, spoVK) were identified as potential contributors to drought-enriched taxa, and their expressions were confirmed by metatranscriptomics in 2022. The impact of extreme summers was further evident in the taxonomic profiles of 721 recovered metagenome-assembled genomes (MAGs). Annotation of contigs and MAGs suggested that Actinobacteria may have a competitive advantage in extreme summers due to the biosynthesis of geosmin and 2-methylisoborneol. Future climate scenarios caused a similar pattern of changes in microbial communities as extreme summers, but to a much lesser extent. Soil microbiomes in grassland showed greater resilience to climate change than those in cropland. Overall, this study provides a comprehensive framework for understanding the response of soil microbiomes to extreme summers.

Salt tolerance-based niche differentiation of soil ammonia oxidizers.

Ammonia oxidizers are key players in the global nitrogen cycle, yet little is known about their ecological performances and adaptation strategies for growth in saline terrestrial ecosystems. This study combined 13C-DNA stable-isotope probing (SIP) microcosms with amplicon and shotgun sequencing to reveal the composition and genomic adaptations of active ammonia oxidizers in a saline-sodic (solonetz) soil with high salinity and pH (20.9 cmolc exchangeable Na+ kg-1 soil and pH 9.64). Both ammonia-oxidizing archaea (AOA) and bacteria (AOB) exhibited strong nitrification activities, although AOB performed most of the ammonia oxidation observed in the solonetz soil and in the farmland soil converted from solonetz soil. Members of the Nitrosococcus, which are more often associated with aquatic habitats, were identified as the dominant ammonia oxidizers in the solonetz soil with the first direct labeling evidence, while members of the Nitrosospira were the dominant ammonia oxidizers in the farmland soil, which had much lower salinity and pH. Metagenomic analysis of "Candidatus Nitrosococcus sp. Sol14", a new species within the Nitrosococcus lineage, revealed multiple genomic adaptations predicted to facilitate osmotic and pH homeostasis in this extreme habitat, including direct Na+ extrusion/H+ import and the ability to increase intracellular osmotic pressure by accumulating compatible solutes. Comparative genomic analysis revealed that variation in salt-tolerance mechanisms was the primary driver for the niche differentiation of ammonia oxidizers in saline-sodic soils. These results demonstrate how ammonia oxidizers can adapt to saline-sodic soil with excessive Na+ content and provide new insights on the nitrogen cycle in extreme terrestrial ecosystems.

Metagenomic insights into nitrogen and phosphorus cycling at the soil aggregate scale driven by organic material amendments.

The soil microbiome, existing as interconnected communities closely associated with soil aggregates, is the key driver in nutrient cycling. However, the underlying genomic information encoding the machinery of the soil microbiome involved in nutrient cycling at the soil aggregate scale is barely known. Here comparative metagenomics and genome binning were applied to investigate microbial functional profiles at the soil aggregate scale under different organic material amendments in a long-term field experiment. Soil samples were sieved into large macroaggregates (>2 mm), macroaggregates (0.25-2 mm) and microaggregates (<0.25 mm). Microbial taxonomic and functional alpha diversity were significantly correlated to soil NO3- and SOC. The highest abundance of nasB, nirK, and amoA genes, which are responsible for denitrification and ammonia oxidizers driving nitrification, was observed in microaggregates. Both manure and peat treatments significantly decreased the abundance of napA and nrfA that encode enzymes involved in dissimilatory nitrate reduction to ammonium (DNRA). As a biomarker for soil inorganic P solubilization, the relative abundance of gcd was significantly increased in macroaggregates and large macroaggregates. Three nearly complete genomes of Nitrososphaeraceae (AOA) and seven bacterial genomes were shown to harbor a series of genes involved in nitrification and P solubilization, respectively. Our study provides comprehensive insights into the microbial genetic potential for DNRA and P-solubilizing activity across different soil aggregate fractions and fertilization regimes.

Seasonality affects function and complexity but not diversity of the rhizosphere microbiome in European temperate grassland.

Knowledge on how grassland microbiota responds on gene expression level to winter-summer change of seasons is poor. Here, we used a combination of quantitative PCR-based assays and metatranscriptomics to assess the impact of seasonality on the rhizospheric microbiota in temperate European grassland. Bacteria dominated, being at least one order of magnitude more abundant than fungi. Despite a fivefold summer increase in bacterial community size, season had nearly no effect on microbiome diversity. It, however, had a marked impact on taxon-specific gene expression, with 668 genes significantly differing in relative transcript abundance between winter and summer samples. Acidobacteria, Bacteroidetes, Planctomycetes, and Proteobacteria showed a greater relative gene expression activity in winter, while mRNA of Actinobacteria and Fungi was, relative to other taxa, significantly enriched in summer. On functional level, mRNA involved in protein turnover (e.g., transcription and translation) and cell maintenance (e.g., chaperones that protect against cell freezing damage such as GroEL and Hsp20) were highly enriched in winter. By contrast, mRNA involved in central carbon and amino acid metabolisms had a greater abundance in summer. Among carbohydrate-active enzymes, transcripts of GH36 family (hemicellulases) were highly enriched in winter, while those encoding GH3 family (cellulases) showed increased abundance in summer. The seasonal differences in plant polymer breakdown were linked to a significantly greater microbial network complexity in winter than in summer. Conceptually, the winter-summer change in microbiome functioning can be well explained by a shift from stress-tolerator to high-yield life history strategy.

A fast chemical oxidation method for predicting the long-term mineralization of biochar in soils.

Biochar stability determines the effectiveness of biochar's functions such as carbon sequestration, soil structure improvement, soil fertility enhancement and soil pollution remediation. However, a fast method for accurately predicting biochar long-term stability in soil remains elusive. Here, firstly, an incubation experiment was conducted on mineralization dynamics of different 13C-labelled biochars over 368 days to explore their actual mineralization in soils and establish their mineralization model. Thereafter, ten treatments of fast chemical oxidation methods using K2Cr2O7 (0.1 M) with different H+ concentrations and oxidation times were applied to the biochars to reveal which method best matches the mineralization of biochar in soils. Results showed that the percentage of biochar­carbon oxidized by the solution containing 0.1 M K2Cr2O7 and 0.2 M H+ at 100 °C for 2 h was in accordance with the one that potentially would be mineralized in soils at a 100-year scale (R2 > 0.99; REMS = 2.53; RD = 15.3). The results provided a chemical oxidation method that was robust, effective, low cost and highly available for measuring the long-term stability of biochar in soils.

Impacts of Mo application on biological nitrogen fixation and diazotrophic communities in a flooded rice-soil system.

Molybdenum (Mo) deficiency in the farmland of China may limit biological nitrogen fixation (BNF), however, the impact of Mo application on BNF capacities and diazotrophic communities in rice-soil systems is unclear. In this experiment, treatments in a 6.7 atom% 15N2-labelling field-based growth chamber for 74 days and treatments in a 99 atom% 15N2-labelling microcosm experiment for 40 days combined with 16S rRNA gene sequencing and DNA-stable isotope probing (SIP) were used to investigate the impacts of Mo application on BNF and diazotrophic communities. Our results showed that under the condition that no nitrogen (N) fertilizer was applied, Mo application (500 g sodium molybdate ha-1) significantly increased N2 fixation in a rice-Inceptisol system, from 22.3 to 53.1 kg N ha-1. Mo application significantly increased the number of nifH gene copies and the relative abundance of cyanobacteria in both growth chamber and microcosm experiments. Among cyanobacteria, the relative abundances of the most abundant genera Leptolyngbya and Microcoleus were significantly increased by Mo application. 15N2-DNA-SIP further demonstrated that Leptolyngbya and Microcoleus incorporated 15N2. Mo application greatly increased BNF in Mo-deficient paddy field (≤0.068 mg kg-1) and stimulated the growth of cyanobacteria. These results indicated that Mo application in Mo-deficient paddy field could be a useful measure to increase soil N input under no N fertilization.

Metatranscriptomics reveals a differential temperature effect on the structural and functional organization of the anaerobic food web in rice field soil.

BACKGROUND: The expected increase in global surface temperature due to climate change may have a tremendous effect on the structure and function of the anaerobic food web in flooded rice field soil. Here, we used the metatranscriptomic analysis of total RNA to gain a system-level understanding of this temperature effect on the methanogenic food web. RESULTS: Mesophilic (30 °C) and thermophilic (45 °C) food web communities had a modular structure. Family-specific rRNA dynamics indicated that each network module represents a particular function within the food webs. Temperature had a differential effect on all the functional activities, including polymer hydrolysis, syntrophic oxidation of key intermediates, and methanogenesis. This was further evidenced by the temporal expression patterns of total bacterial and archaeal mRNA and of transcripts encoding carbohydrate-active enzymes (CAZymes). At 30 °C, various bacterial phyla contributed to polymer hydrolysis, with Firmicutes decreasing and non-Firmicutes (e.g., Bacteroidetes, Ignavibacteriae) increasing with incubation time. At 45 °C, CAZyme expression was solely dominated by the Firmicutes but, depending on polymer and incubation time, varied on family level. The structural and functional community dynamics corresponded well to process measurements (acetate, propionate, methane). At both temperatures, a major change in food web functionality was linked to the transition from the early to late stage. The mesophilic food web was characterized by gradual polymer breakdown that governed acetoclastic methanogenesis (Methanosarcinaceae) and, with polymer hydrolysis becoming the rate-limiting step, syntrophic propionate oxidation (Christensenellaceae, Peptococcaceae). The thermophilic food web had two activity stages characterized first by polymer hydrolysis and followed by syntrophic oxidation of acetate (Thermoanaerobacteraceae, Heliobacteriaceae, clade OPB54). Hydrogenotrophic Methanocellaceae were the syntrophic methanogen partner, but their population structure differed between the temperatures. Thermophilic temperature promoted proliferation of a new Methanocella ecotype. CONCLUSIONS: Temperature had a differential effect on the structural and functional continuum in which the methanogenic food web operates. This temperature-induced change in food web functionality may not only be a near-future scenario for rice paddies but also for natural wetlands in the tropics and subtropics.