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Soil protists, the major predator of bacteria and fungi, shape the taxonomic and functional structure of soil microbiome via trophic regulation. However, how trophic interactions between protists and their prey influence microbially mediated soil organic carbon turnover remains largely unknown. Here, we investigated the protistan communities and microbial trophic interactions across different aggregates‐size fractions in agricultural soil with long‐term fertilization regimes. Our results showed that aggregate sizes significantly influenced the protistan community and microbial hierarchical interactions. Bacterivores were the predominant protistan functional group and were more abundant in macroaggregates and silt + clay than in microaggregates, while omnivores showed an opposite distribution pattern. Furthermore, partial least square path modeling revealed positive impacts of omnivores on the C‐decomposition genes and soil organic matter (SOM) contents, while bacterivores displayed negative impacts. Microbial trophic interactions were intensive in macroaggregates and silt + clay but were restricted in microaggregates, as indicated by the intensity of protistan‐bacterial associations and network complexity and connectivity. Cercozoan taxa were consistently identified as the keystone species in SOM degradation‐related ecological clusters in macroaggregates and silt + clay, indicating the critical roles of protists in SOM degradation by regulating bacterial and fungal taxa. Chemical fertilization had a positive effect on soil C sequestration through suppressing SOM degradation‐related ecological clusters in macroaggregate and silt + clay. Conversely, the associations between the trophic interactions and SOM contents were decoupled in microaggregates, suggesting limited microbial contributions to SOM turnovers. Our study demonstrates the importance of protists‐driven trophic interactions on soil C cycling in agricultural ecosystems. [ABSTRACT FROM AUTHOR]

Purpose: Unraveling the drivers of the nitrogen cycling functional guilds variation in response to fertilizer management is a major goal in agroecosystem study. Nitrospira-like nitrite oxidizing bacteria can oxidize nitrite to nitrate, which play a critical role in the biogeochemical nitrogen cycle. However, how the Nitrospira community structure and co-occurrence patterns in a long-term inorganic and organic fertilization regimes within different soil aggregates have been largely unexplored. Materials and methods: The soil samples were collected from a long-term (39-year) inorganic and organic fertilized agricultural ecosystem, including no-fertilizer (CK); mineral-fertilizer (NPK); manure-fertilizer (M); nitrogen-fertilizer (N); and M + N (MN). Three sizes of soil aggregate were manually fractionated by soil wet sieving technique with a series of meshes: (i) 2000–250 μm (macroaggregates), (ii) 250–53 μm (microaggregates), and (iii) < 53 μm fractions (silt and clay). We performed qPCR and high-throughput-sequencing to assess the Nitrospira abundance and community structure, respectively. Results and discussion: Organic fertilization (M and MN) efficiently increased soil potential nitrite oxidation activity (PNO), which was higher in the < 53 μm than 250–53 μm and followed the 2000–250 μm fraction, than other treatments. All the fertilizer treatments resulted in a similar pattern, where the 250–53-μm fraction had significantly greater Nitrospira abundance than the 2000–250 μm and < 53 μm fractions. The alpha diversity of Nitrosipra did not change in response to the fertilization treatments and soil aggregate sizes. Nitrospira community shifted in fertilization types, while not in aggregate sizes. Nitrospira community composition and abundance significantly correlated with PNO. Network analysis suggested that organic fertilizations (M and MN) promote the complexity of Nitrospira networks and possess higher connectivity than inorganic fertilization networks. Conclusions: Our data suggested that fertilization strongly affected the Nitrospira community structure and co-occurrence patterns, which may implicate the importance of Nitrospira function in the nitrite oxidation process in soils with organic fertilization. [ABSTRACT FROM AUTHOR]

Summary: Soil aggregates, with complex spatial and nutritional heterogeneity, are clearly important for regulating microbial community ecology and biogeochemistry in soils. However, how the taxonomic composition and functional attributes of N‐cycling‐microbes within different soil particle‐size fractions under a long‐term fertilization treatment remains largely unknown. Here, we examined the composition and metabolic potential for urease activity, nitrification, N2O production and reduction of the microbial communities attached to different sized soil particles (2000–250, 250–53 and <53 μm) using a functional gene microarray (GeoChip) and functional assays. We found that urease activity and nitrification were higher in <53 μm fractions, whereas N2O production and reduction rates were greater in 2000–250 and 250–53 μm across different fertilizer regimes. The abundance of key N‐cycling genes involved in anammox, ammonification, assimilatory and dissimilatory N reduction, denitrification, nitrification and N2‐fixation detected by GeoChip increased as soil aggregate size decreased; and the particular key genes abundance (e.g., ureC, amoA, narG, nirS/K) and their corresponding activity were uncoupled. Aggregate fraction exerted significant impacts on N‐cycling microbial taxonomic composition, which was significantly shaped by soil nutrition. Taken together, these findings indicate the important roles of soil aggregates in differentiating N‐cycling metabolic potential and taxonomic composition, and provide empirical evidence that nitrogen metabolism potential and community are uncoupled due to aggregate heterogeneity. [ABSTRACT FROM AUTHOR]

Ammonia oxidizers (ammonia‐oxidizing archaea (AOA) and bacteria (AOB)), nitrite oxidizers (e.g., Nitrobacter and Nitrospira) and comammox Nitrospira play important roles in the nitrogen cycle in agroecosystems, yet their activities and abundance in soil microhabitats under environmental disturbances remain unclear. In this study, we collected samples from treatment plots in a field experiment established in 2010 in a lime concretion black soil in central China to investigate the effects of varying inorganic and organic fertilizer managements in a wheat–maize crop rotation, in order to investigate the impacts on the abundance and activities of ammonia and nitrite oxidizers inhabiting different microenvironments in soil, represented by three soil aggregate size fractions. The treatments were: control without fertilizer (CK), chemical fertilization (NPK), NPK and straw (NPKS), NPKS and pig manure (NPKSP) or cow manure (NPKSC). The potential nitrite oxidation activity (PNO) and soil nutrients (soil carbon, nitrogen and phosphorus content) were the highest in the microaggregates, followed by those in the macroaggregates and silt+clay fractions in all the plots. Both the ammonia and nitrite oxidizers were more abundant in the macroaggregates and microaggregates than in the silt+clay fractions. A two‐way analysis of variance (ANOVA) indicated that the soil aggregate size had a significant influence on the abundance of ammonia and nitrite oxidizers and the soil nutrients. We also found a significant positive correlation between PNO and the nitrite oxidizer and AOA abundance. In addition, the abundances of the ammonia oxidizers (AOA and AOB) and nitrite oxidizers (Nitrobacter and Nitrospira) were positively correlated with each other. Overall, the soil aggregates with a higher inorganic nutrient and organic carbon content may provide a better protective microenvironment to guard against environmental disturbances and facilitate the inhabitation of ammonia and nitrite oxidizers, which cooperate in the nitrogen cycling process. Highlights: Nitrifying guilds and their activities were investigated under soil aggregates in a Vertisol.Soil aggregates of different sizes provide spatially heterogeneous habitats for nitrifying guilds.Ammonia‐ and nitrite‐oxidizer abundances and their activities are controlled by soil aggregate size.Soil aggregates with higher nutrients may provide a better protective habitat for nitrifying guilds. [ABSTRACT FROM AUTHOR]

Soil aggregates are often considered the basic structural elements of soils. Aggregates of different size vary in their ability to retain or transfer heavy metals in the environment. Here, after incubation of a sieved (<2 mm) topsoil with copper, bulk soil was separated into four aggregate-size fractions and their adsorption characteristics for Cu were determined. By combining nano-scale secondary ion mass spectrometry and C-1s Near Edge X-ray Absorption Fine Structure Spectroscopy, we found that copper tends to bind onto organic matter in the <2 μm and 20–63 μm aggregates. Surprisingly, Cu correlated with carboxyl-C in the <2 μm aggregates but with alkyl-C in the 20–63 μm aggregates. This is the first attempt to visualize the spatial distribution of copper in aggregate size fractions. These direct observations can help improve the understanding of interactions between heavy metals and various soil components. [ABSTRACT FROM AUTHOR]

Bacterial adhesion to minerals in soils and sediments is of fundamental importance in mineral weathering and formation, soil aggregate stability, organic matter degradation and the fate of pollutants. Bacterial surface properties are considered to govern adhesion, and these properties likely change as a function of bacterial growth phase. However, the effect of growth stage on bacterial adhesion to clay minerals remains unclear. This work examined the influence of growth phase on the adhesion of Pseudomonas putida to kaolinite-coated coverslips. Fluorescence microscopy, together with a bacterial viability stain, was used to directly quantify surface cell density and viability of adhered P. putida . In situ attenuated total reflectance Fourier transform infrared spectroscopy was applied to yield molecular information about the characteristics of the bacteria, and the adsorption and desorption kinetics. Stationary-phase cells exhibited a higher adsorption density on kaolinite surfaces than mid-exponential-phase cells under static deposition conditions. Compared with the mid-exponential-phase cells, the stationary-phase cells displayed higher saturation coverage, and we fitted the results using a pseudo-first-order kinetics equation. The greater extent of adhesion of the stationary-phase cells was probably due to their smaller cell size and less negative surface charges compared with the cells from other growth stages, which resulted in deeper secondary energy minima and lower energy barriers for adhesion. The results from this study suggest that growth phase may strongly influence cell mobility and biofilm formation in aqueous geochemical environments. [ABSTRACT FROM AUTHOR]

Soil aggregates provide spatially heterogeneous microhabitats for microorganisms and their activities. In metal contaminated soil, the microbial community is shaped by both metal stress and aggregate sizes. However, it remains unclear whether and how aggregate sizes affect the legacy effect of metal pollution on microbial communities. Here, we investigated the legacy effect of long-term copper (Cu) pollution on the distribution patterns of the bacterial and protistan communities in soil aggregates. Our results showed that soil aggregates of different sizes possessed distinct bacterial and protistan communities under Cu-polluted and unpolluted conditions (aggregate effects), as reflected by distinct α-diversity and community compositions. The bacterial and protistan communities in micro- and macro-aggregates were more responsive to Cu pollution than those in mega-aggregates, primarily because micro- and macro-aggregates contained a greater number of Cu susceptible bacterial and protistan species. Ciliophora and Chlorophyta displayed great sensitivity to Cu, especially in small aggregate-size fractions, and could serve as a bio-indicator of Cu pollution. Furthermore, the magnitude of aggregate effects on microbial communities was weakened in Cu-polluted soils compared to unpolluted soils. With decreasing aggregate sizes, the co-occurrence networks between bacteria and protists became more complex, connected and compact. Robustness analysis showed that phagotrophic protists played essential roles in sustaining the network stability, especially in mega-aggregates. Our findings highlight the asymmetrical changes in microorganisms in aggregates under long-term Cu pollution, providing a novel insight into the legacy effects of metal pollution on soil microbiomes at the micro-level. • The distribution of bacteria and protists in Cu-polluted and unpolluted soil aggregates was distinct. • The Cu legacy effect on the bacterial and protist communities was strong in aggregates of small size. • Cu pollution lessened aggregate size-induced effects on the bacterial and protistan communities. • Phagotrophs were crucial in sustaining the network stability in large aggregates. [ABSTRACT FROM AUTHOR]

Soil aggregates provide spatially heterogeneous microenvironments for soil microbiome and ecological processes. Revealing the nuances of assembly processes in aggregates is critical for understanding the fine-scale organization of the soil microbiome and associated biogeochemistry. However, the effect of aggregate sizes on microbial assemblies remains poorly explored. In this study, we investigated the mechanism underlying the assembly processes of microbial communities within three different aggregates sizes across five long-term fertilization experimental stations throughout eastern China. Our results showed that although geographic sites explained most of the variations in microbial composition in aggregates, aggregates of the same size selected phylogenetically closely related microbial species with similar ecological preferences for specific nutrient variables. Null model analysis showed that environmental selection and dispersal limitation primarily governed the assembly of aggregate bacterial and fungal communities, respectively. The relative importance of deterministic processes on microbial communities increased with the increasing aggregate size across all sites. Multiple stepwise regression analysis indicated that nutrient variables associated with community assembly are distinct in different aggregate-size fractions. Bacterial assembly is mainly affected by total carbon and soil organic carbon in macro- and micro-aggregates, whereas it is influenced by the C/P ratio in the silt + clay fraction. For fungal community assembly, the C/N ratio was the major factor in macroaggregate and silt + clay fractions, while N/P ratio had stronger effects in the microaggregates. Furthermore, fertilization effects on the microbial community assembly processes varied across aggregate sizes. Organic fertilization led to a decrease for bacteria while an increase for fungi in the sorting/dispersal effect ratio. The magnitude of such an impact was higher for macro- and micro-aggregates. GeoChip and enzymatic results further demonstrated the regulation of ecological clusters and functions by nutrient variables across aggregates. Our findings highlight that microbial assembly and functional traits in large aggregate-size fractions are susceptible to nutrient changes, and thus can act as potential biological indicators for sustainable management of agricultural soils. • Microbial communities in aggregates are adapted to nutrient variables. • Soil aggregate size modulates the balance of community assembly processes. • Community assembly are more susceptible to fertilization in large aggregates. • Microbes within large aggregate can act as biological indicators of soil management. [ABSTRACT FROM AUTHOR]

This study coupled a measurement of denitrifier gene abundances at the phylogroup level with denitrifying enzyme activities to show how long-term fertilization shaped the community of denitrifiers inhabiting the soil aggregates of various sizes. Three groups of nirK , including clade I (nirK -I), clade II (nirK -II), and group IIa (nirK -III), clade I nirS (nirS -I), and two clades of nosZ (nosZ -I and nosZ -II) were detectable. Most of these genes were increased by the fertilization with swine manure and subsequently diverged across aggregate size fractions. Both nirK-I and nirK - II were mainly driven by the available phosphorus (AP) and total nitrogen (TN), and nirK-III (group IIa) by pH. NirS-I was best predicted by total phosphorus and TN. For both nosZ-I and nosZ-II , TN and total carbon are the best predictors. Group IIa nirK and nirS-I nitrite reducers possibly possessed an advantage in the soil aggregates with a lower nutrient level than the other nitrite reducers, and nosZ-II N 2 O reducers were more competitive at a condition with a lower pH. The gross N 2 O production and the net N 2 O production were both stimulated by the fertilization with swine manure, and the former also significantly differed across different soil aggregate size fractions. Structure equation modeling analysis indicated that the Gross N 2 O production was dominantly explained by the nirK-I nitrite-reducers and total nitrogen, whereas the net N 2 O production by the total phosphorus and the sum of nosZ-II and nosZ-I N 2 O reducers, which were also associated with the Gross N 2 O production. Our results also highlighted that microaggregates with manure fertilization are likely hot spots for denitrifiers in the soil. • Phylogroup-specifc primers were used for detecting nir in soil aggregates. • Denitrifiers were enriched in the microaggregates from the manure applied plots. • Clade I nirK dominantly associated with Gross N 2 O production. • Both typical and atypical nosZ N 2 O reducers explained net N 2 O production. [ABSTRACT FROM AUTHOR]

Soil protists are major biotic forces driving microbial dynamics within aggregates. Interpretation of protistan spatial distribution and ecological processes in aggregates primarily relies on aggregate isolation methods. However, comparative studies illustrating how isolation methods influence protistan communities are lacking. Here, two isolation procedures, dry- and wet-sieving, were evaluated. The wet-sieving led to separation of dry mega-aggregates, which consequently altered aggregate size distribution and associated protistan communities. The variable influences of sieving methods on aggregate protists were dependent on soil type. However, the aggregate isolation method did not affect the prominent aggregate-effects on protistan communities and detection of dominant taxa. Collectively, our findings highlight the importance of isolation procedures for exploring the spatial heterogeneity of protists at the micro-level. • Aggregate isolation method affected protistan diversity and community composition. • Detection of dominant protistan taxa was not influenced by the sieving method. • Impacts of sieving method on protistan communities relied on soil types. • Aggregate-effects on protists were detectable regardless of the sieving method. [ABSTRACT FROM AUTHOR]

Soil stability and aggregates are important drivers of soil fertility and microbial diversity and are highly vulnerable to land degradation. However, the role of soil aggregates in driving the responses of microbial functional diversity and multiple ecosystem services and functions (multifunctionality) to further degradation (e.g., fertilization) remains largely unexplored and poorly understood. In this study, we used soils from long-term experiments involving inorganic and organic fertilization treatments to investigate the role soil aggregates (microscale) play in driving microbial functional gene diversity (via GeoChip) and the activity of multiple extracellular enzymes in an agricultural ecosystem. We found that microbial functional gene diversity has a significant and positive relationship with soil multifunctionality, which is enhanced in soil aggregates by organic fertilizer but is reduced by inorganic fertilizer. We also found that soil aggregate fractions indirectly controlled multiple ecosystem functions via changes in functional diversity. Smaller soil aggregates with higher resource availability (carbon and nitrogen) supported more ecological functions than larger aggregates under contrasting fertilizer management regimes. Soil multifunctionality is regulated by the differences in resource availability and not by microbial functional gene composition, which suggests that microbial functional diversity contributed more to multifunctionality than gene composition. Random forest analysis and structural equation modeling indicated that soil carbon and nitrogen and microbial functional diversity together determined the multifunctionality, whereas soil traits have more standardized total effects than functional diversity. Our study highlights that soil aggregation stratifies soil nutrition and microbial functional diversity, which leads to the differentiation of aggregate ecosystem multifunctionality. • Microbial functional diversity positively regulates soil multifunctionality. • Microbial diversity contributes more to multifunctionality than taxonomic traits. • Soil aggregate size may indirectly and negatively determine multifunctionality. • Organic and inorganic fertilizers have opposite effects on multifunctionality. • Soil nutrition and functional diversity together control multifunctionality. [ABSTRACT FROM AUTHOR]

Soil amendments such as lime, biochar and straw are expected to be effective for in situ remediation of cadmium (Cd)-contaminated soil, while increasing the taxonomic diversity of soil microbial community and their functions. The objective of this three-year field experiment was to evaluate the effects of five commonly used soil amendments on Cd stabilization, as well as bacterial phylogenetic and functional attributes. The taxonomic structure of the soil bacterial community did not change after soil amendments were applied. Most of bacterial functional genes involved in carbon, nitrogen and phosphorus cycling were more abundant in soil amended with CaCO 3 and its combination with biochar/straw than soil without amendments. A greater number of functional genes were associated with the growth of rare bacterial species, which were 1.3 times more abundant in the CaCO 3 -amended soil than the unamended. Repeated application of CaCO 3 and CaCO 3 together with biochar/straw increased the number and abundance of functional genes, and the complexity and connectivity of the soil bacterial network, probably because the increased soil pH altered keystone taxa which were highly associated with functional genes. This study demonstrates that soil amendments applied to Cd-contaminated soils can have unexpected consequences on soil bacterial communities and their functions. Image 1 • Soil pH and Cd speciation affect soil bacterial taxonomy and functions. • Bacterial functional genes are sensitive to lime, biochar and straw amendments. • Repeated application of amendments increased gene abundance of C/N/P cycling. • Soil pH altered keystone taxa which are highly associated with functional genes. [ABSTRACT FROM AUTHOR]

Differently sized soil aggregates may provide spatially heterogeneous microenvironments for microorganisms, but little is known about the impacts of soil spatial structure on the co-occurrence patterns of bacterial and fungal taxa. Here, we investigated the compositions and networks of bacterial and fungal communities within different aggregate-size fractions (macroaggregates: 2000–250 μm; microaggregates: 53–250 μm; and silt + clay: <53 μm) of an agricultural Inceptisol to examine the micro-scale location and interaction of microbial populations. Our results showed that aggregate size was positively correlated with soil C, N and P, while negatively correlated with soil C/N ratio. Phospholipid fatty acid and high-throughput sequencing indicated that bacterial and fungal community structures were affected more strongly by aggregate size than by fertilization. Soil microbial biomass and community compositions were significantly correlated with environmental factors. The influences of fertilization treatments on soil microorganisms varied with aggregate size and the greatest were found for macroaggregates. Bacterial-fungal networks across different aggregate-size fractions were analyzed by MENA, CoNet and R software. Total links, average degree and average clustering coefficient increased with aggregate size, whereas average path distance followed the opposite trend. More bacterial-bacterial correlations were detected in macroaggregate network. Our results suggest that bacterial-fungal network was much more complex in macroaggregates than in microaggregates and silt + clay, indicating greater cooperation and competition among microbes in larger aggregates. Overall, our study demonstrates the significant effects of soil structure on microbial community structure and network. [ABSTRACT FROM AUTHOR]

Aggregates are the basic structural units of soils. Fine soil aggregates are crucial pools for the retention of heavy metals. In this study, we evaluated the accumulation characteristics of exogenous Cu in the <2 μm aggregate fractions from a Histosol for an aging of 28 months by nano-scale secondary ion mass spectrometry (NanoSIMS) and synchrotron micro-X-ray fluorescence (micro-XRF). Results showed that the correlations between Cu and Fe/Al/Mn increased from 0.10–0.17 to 0.55–0.63, while those between Cu and C decreased sharply from 0.61 to 0.10 and then increased to 0.36, indicating that exogenous Cu tended to accumulate on inorganic mineral components. The carbon NEXAFS data suggested that the relative content of aromatic and carboxyl carbon decreased from 8.1% and 30.8% to 3.6% and 17.8% at month 12, and increased to 5.9% and 26.0% at month 28, respectively. However, an opposite trend was found for alkyl and carbonyl carbon which showed an increase at month 12 followed by a decrease afterwards. The consistency for the correlations between Cu and C with the changes of aromatic and carboxyl carbon indicated their key roles in the binding of Cu on the organic components in the <2 μm aggregates of Histosol. These direct observations offer a better understanding on the interactions of heavy metals with various soil components which is critical for the risk assessment and fate evaluation of exogenous Cu in soil ecosystems. Image 1 • Fine soil aggregates play vital roles in retention of heavy metals. • Aging promotes binding of Cu onto inorganic components in fine aggregates. • Cu tends to be accumulated with amorphous metal oxides during aging. • Cu is governed mainly by aromatic and carboxyl C within organic fractions. [ABSTRACT FROM AUTHOR]

• Fertilization treatments affect soil aggregate fractionation. • Fertilization regimes influence phosphorus fraction. • Fertilization treatments affect P-cycling-related bacterial abundance. • Soil aggregate showed greater impact on P-cycling-related bacterial community than fertilization. • P components accounted for P-cycling-related bacterial community composition. Fertilization could promote phosphorus (P) cycling in soil; however, it is unclear how long-term fertilization regimes affect P-cycling-related microbial communities in different soil aggregates. In this study, we compiled promising strategies to evaluate the effect of 40-year long-term fertilization treatments on the P-cycling-related bacterial communities associated with different soil aggregates, of which P fraction and physicochemical properties were also measured. We found that both organic and mineral fertilization treatments increased the proportion of silt + clay (< 53 μm) and decreased the proportion of macroaggregate (250–2000 μm). Organic fertilization increased microaggregate (53–250 μm) proportion while mineral fertilization resulted in the opposite. The abundances of phoC- , phoD- , phnX- , gcd- , and pstS- harboring bacteria increased in silt + clay in five fertilization treatments, while bpp -harboring bacterial abundance increased in microaggregate applied with nitrogen chemical fertilizer and nitrogen with combined manure fertilizer. Phytase activity was higher in macroaggregate in no fertilizer and mineral fertilization treatments, while phosphatase activity was higher in silt + clay in five fertilization treatments. Besides, fertilization significantly affected P fraction, with higher proportions of inorganic P, organic P, non-apatite inorganic P, and apatite inorganic P in silt + clay. For P-cycling-related bacterial community composition, 43.09% of the variation explained by fertilization treatment and aggregate fractionation, 73.92% of the variation explained by soil physicochemical properties and P fraction were found. Besides, P components especially organic P, non-apatite inorganic P, and labile P presented great effects on P-cycling-related bacterial community composition. To our knowledge, our findings first suggest that silt + clay is beneficial to enrich P-cycling-related bacteria, nutrient-driven aggregate fractionation and P fraction via fertilization treatments can shape P-cycling-related bacterial community composition. [ABSTRACT FROM AUTHOR]