Impacts of vegetable processing and cheese making effluent on soil microbial functional diversity, community structure, and denitrification potential of land treatment systems.

The cheese making and vegetable processing industries generate immense volumes of high-nitrogen wastewater that is often treated at rural facilities using land applications. Laboratory incubation results showed denitrification decreased with temperature in industry facility soils but remained high in soils from agricultural sites (75% at 2.1°C). 16S rRNA, phospholipid fatty acid (PLFA), and soil respiration analyses were conducted to investigate potential soil microbiome impacts. Biotic and abiotic system factor correlations showed no clear patterns explaining the divergent denitrification rates. In all three soil types at the phylum level, Actinobacteria, Proteobacteria, and Acidobacteria dominated, whereas at the class level, Nitrososphaeria and Alphaproteobacteria dominated, similar to denitrifying systems such as wetlands, wastewater resource recovery facilities, and wastewater-irrigated agricultural systems. Results show that potential denitrification drivers vary but lay the foundation to develop a better understanding of the key factors regulating denitrification in land application systems and protect local groundwater supplies. PRACTITIONER POINTS: Incubation study denitrification rates decreased as temperatures decreased, potentially leading to groundwater contamination issues during colder months. The three most dominant phyla for all systems are Actinobacteria, Proteobacteria, and Acidobacteria. The dominant class for all systems is Nitrosphaeria (phyla Crenarchaeota). No correlation patterns between denitrification rates and system biotic and abiotic factors were observed that explained system efficiency differences.

Shifts in bacterial traits under chronic nitrogen deposition align with soil processes in arbuscular, but not ectomycorrhizal-associated trees.

Nitrogen (N) deposition increases soil carbon (C) storage by reducing microbial activity. These effects vary in soil beneath trees that associate with arbuscular (AM) and ectomycorrhizal (ECM) fungi. Variation in carbon C and N uptake traits among microbes may explain differences in soil nutrient cycling between mycorrhizal associations in response to high N loads, a mechanism not previously examined due to methodological limitations. Here, we used quantitative Stable Isotope Probing (qSIP) to measure bacterial C and N assimilation rates from an added organic compound, which we conceptualize as functional traits. As such, we applied a trait-based approach to explore whether variation in assimilation rates of bacterial taxa can inform shifts in soil function under chronic N deposition. We show taxon-specific and community-wide declines of bacterial C and N uptake under chronic N deposition in both AM and ECM soils. N deposition-induced reductions in microbial activity were mirrored by declines in soil organic matter mineralization rates in AM but not ECM soils. Our findings suggest C and N uptake traits of bacterial communities can predict C cycling feedbacks to N deposition in AM soils, but additional data, for instance on the traits of fungi, may be needed to connect microbial traits with soil C and N cycling in ECM systems. Our study also highlights the potential of employing qSIP in conjunction with trait-based analytical approaches to inform how ecological processes of microbial communities influence soil functioning.

Evenness of soil organic carbon chemical components changes with tree species richness, composition and functional diversity across forests in China.

Higher tree species richness generally increases the storage of soil organic carbon (SOC). However, less attention is paid to the influence of varied tree species composition on SOC storage. Recently, the perspectives for the stronger persistence of SOC caused by the higher molecular diversity of organic compounds were proposed. Therefore, the influences of tree species richness and composition on the molecular diversity of SOC need to be explored. In this study, an index of the evenness of diverse SOC chemical components was proposed to represent the potential resistance of SOC to decomposition under disturbances. Six natural forest types were selected encompassing a diversity gradient, ranging from cold temperate to tropical forests. We examined the correlations of tree species richness, composition, and functional diversity, with the evenness of SOC chemical components at a molecular level by 13 C nuclear magnetic resonance. Across the range, tree species richness correlated to the evenness of SOC chemical components through tree species composition. The negative correlation of evenness of SOC chemical components with tree species composition, and the positive correlation of evenness of SOC chemical components with tree functional diversity were found. These indicate the larger difference in tree species composition and the lower community functional diversity resulted in the higher heterogeneity of SOC chemical components among the communities. The positive correlation of the evenness of SOC chemical components with the important value of indicator tree species, further revealed the specific tree species contributing to the higher evenness of SOC chemical components in each forest type. Soil fungal and bacterial α-diversity had effect on the evenness of SOC chemical components. These findings suggest that the indicator tree species conservation might be preferrable to simply increasing tree species richness, for enhancing the potential resistance of SOC to decomposition.

Fungivorous nematodes drive microbial diversity and carbon cycling in soil.

Soil bacteria and fungi mediate terrestrial biogeochemical cycling, but we know relatively little about how trophic interactions influence their community composition, diversity, and function. Specifically, it is unclear how consumer populations affect the activity of microbial taxa they consume, and therefore the interaction of those taxa with other members of the microbial community. Due to its extreme diversity, studying trophic dynamics in soil is a complex feat. Seeking to address these challenges, we performed a microcosm-based consumer manipulation experiment to determine the impact of a common fungal-feeding nematode (Aphelenchus avenae) on soil microbial community composition, diversity, and activity (e.g., C cycling parameters). Fungivory decreased fungal and bacterial α-diversity and stimulated C and N cycling, possibly via cascading impacts of fungivory on bacterial communities. Our results present experimental evidence that soil trophic dynamics are intimately linked with microbial diversity and function, factors that are key in understanding global patterns in biogeochemical cycling.

Exploring Microbiome Functional Dynamics through Space and Time with Trait-Based Theory.

Microbiomes play essential roles in the health and function of animal and plant hosts and drive nutrient cycling across ecosystems. Integrating novel trait-based approaches with ecological theory can facilitate the prediction of microbial functional traits important for ecosystem functioning and health. In particular, the yield-acquisition-stress (Y-A-S) framework considers dominant microbial life history strategies across gradients of resource availability and stress. However, microbiomes are dynamic, and spatial and temporal shifts in taxonomic and trait composition can affect ecosystem functions. We posit that extending the Y-A-S framework to microbiomes during succession and across biogeographic gradients can lead to generalizable rules for how microbiomes and their functions respond to resources and stress across space, time, and diverse ecosystems. We demonstrate the potential of this framework by applying it to the microbiomes hosted by the carnivorous pitcher plant Sarracenia purpurea, which have clear successional trajectories and are distributed across a broad climatic gradient.

Land use intensification destabilizes stream microbial biodiversity and decreases metabolic efficiency.

Urbanization and agricultural intensification can transform landscapes. Changes in land-use can lead to increases in storm runoff and nutrient loadings which can impair the health and function of stream ecosystems. Microorganisms are an integral component of stream ecosystems. Due to the sensitivity of microorganisms to perturbations, changes in hydrology and water chemistry may alter microbial activity and structure. These shifts in microbial community dynamics may alter stream metabolism and water quality, potentially impacting higher trophic levels. Here we examine the effects of land-use and associated changes in water chemistry on sediment microbial communities by studying the West Run Watershed (WRW) a mixed-land-use system in West Virginia, USA. Streams were sampled throughout the growing season at six sites within the WRW spanning different levels of land use intensification. The proportion of land impacted by agricultural and urban development was positively correlated with temporal variation in stream sediment microbial community composition (adj R2 = 0.65), suggesting development can destabilize microbial communities. Moreover, streams in developed watersheds had an increased metabolic quotient (20-50% higher), this indicates that microorganisms have greater respiration per unit biomass and signifies reduced metabolic efficiency. Further, our results suggest that land use associated changes in water chemistry alter microbial function both directly and indirectly via changes in microbial community composition and biomass. Taken together our results suggest that highly developed watersheds with elevated conductivity, metal ion concentration, and pH impose stress on microbial communities resulting in reduced microbial efficiency and elevated respiration.

Environment-host-microbial interactions shape the Sarracenia purpurea microbiome at the continental scale.

The importance of climate, habitat structure, and higher trophic levels on microbial diversity is only beginning to be understood. Here, we examined the influence of climate variables, plant morphology, and the abundance of aquatic invertebrates on the microbial biodiversity of the northern pitcher plant Sarracenia purpurea. The plant's cup-shaped leaves fill with rainwater and support a miniature, yet full-fledged, ecosystem with a diverse microbiome that decomposes captured prey and a small network of shredding and filter-feeding aquatic invertebrates that feed on microbes. We characterized pitcher microbiomes of 108 plants sampled at 36 sites from Florida to Quebec. Structural equation models revealed that annual precipitation and temperature, plant size, and midge abundance had direct effects on microbiome taxonomic and phylogenetic diversity. Climate variables also exerted indirect effects through plant size and midge abundance. Further, spatial structure and climate influenced taxonomic composition, but not phylogenetic composition. Our results suggest that direct effects of midge abundance and climate and indirect effects of climate through its effect on plant-associated factors lead to greater richness of microbial phylotypes in warmer, wetter sites.

Differences in microbial community response to nitrogen fertilization result in unique enzyme shifts between arbuscular and ectomycorrhizal-dominated soils.

While the effect of nitrogen (N) deposition on belowground carbon (C) cycling varies, emerging evidence shows that forest soils dominated by trees that associate with ectomycorrhizal fungi (ECM) store more C than soils dominated by trees that associate with arbuscular mycorrhizae (AM) with increasing N deposition. We hypothesized that this is due to unique nutrient cycling responses to N between AM and ECM-dominated soils. ECM trees primarily obtain N through fungal mining of soil organic matter subsidized by root-C. As such, we expected the largest N-induced responses of C and N cycling to occur in ECM rhizospheres and be driven by fungi. Conversely, as AM trees rely on bacterial scavengers in bulk soils to cycle N, we predicted the largest AM responses to be driven by shifts in bacteria and occur in bulk soils. To test this hypothesis, we measured microbial community composition, metatranscriptome profiles, and extracellular enzyme activity in bulk, rhizosphere, and organic horizon (OH) soils in AM and ECM-dominated soils at Bear Brook Watershed in Maine, USA. After 27 years of N fertilization, fungal community composition shifted across ECM soils, but bacterial communities shifted across AM soils. These shifts were mirrored by enhanced C relative to N mining enzyme activities in both mycorrhizal types, but this occurred in different soil fractions. In ECM stands these shifts occurred in rhizosphere soils, but in AM stands they occurred in bulk soils. Additionally, ECM OH soils exhibited the opposite response with declines in C relative to N mining. As rhizosphere soils account for only a small portion of total soil volume relative to bulk soils, coupled with declines in C to N enzyme activity in ECM OH soils, we posit that this may partly explain why ECM soils store more C than AM soils as N inputs increase.

Soil microbial succession following surface mining is governed primarily by deterministic factors.

Understanding the successional dynamics governing soil microbial community assembly following disturbance can aid in developing remediation strategies for disturbed land. However, the influences shaping microbial communities during succession following soil disturbance remain only partially understood. One example of a severe disturbance to soil is surface mining for natural resources, which displaces communities and changes the physical and chemical soil environment. These changes may alter community composition through selective pressure on microbial taxa (i.e. deterministic processes). Dispersal and ecological drift may also shape communities following disturbance (i.e. stochastic processes). Here, the relative influence of stochastic and deterministic processes on microbial community succession was investigated using a chronosequence of reclaimed surface mines ranging from 2-32 years post-reclamation. Sequencing of bacterial and fungal ribosomal gene amplicons coupled with a linear modeling approach revealed that following mine reclamation, while bacterial communities are modestly influenced by stochastic factors, the influence of deterministic factors was ∼7 × greater. Fungal communities were influenced only by deterministic factors. Soil organic matter, texture, and pH emerged as the most influential environmental factors on both bacterial and fungal communities. Our results suggest that management of deterministic soil characteristics over a sufficient time period could increase the microbial diversity and productivity of mine soils.

Anthropogenic N deposition, fungal gene expression, and an increasing soil carbon sink in the Northern Hemisphere.

Terrestrial ecosystems in the Northern Hemisphere are a globally important sink for anthropogenic CO2 in the Earth's atmosphere, slowing its accumulation as well as the pace of climate warming. With the use of a long-term field experiment (ca. 20 yr), we show that the expression of fungal class II peroxidase genes, which encode enzymes mediating the rate-limiting step of organic matter decay, are significantly downregulated (-60 to -80%) because of increases in anthropogenic N deposition; this response was consistent with a decline in extracellular peroxidase enzyme activity in soil, the slowing of organic-matter decay, and greater soil C storage. The reduction in peroxidase expression we document here occurred in the absence of a compositional shift in metabolically active fungi, indicating that an overall reduction in peroxidase expression underlies the slowing of decay and increases in soil C storage. This molecular mechanism has global implications for soil C storage and should be represented in coupled climate-biogeochemical models simulating the influence of enhanced terrestrial C storage on atmospheric CO2 and the future climate of an N-enriched Earth.

Seasonal, sub-seasonal and diurnal variation of soil bacterial community composition in a temperate deciduous forest.

The temporal dynamics of soil bacterial communities are understudied, but such understanding is critical to elucidating the drivers of community variation. The goal of this study was to characterize how soil bacterial communities vary across diurnal, sub-seasonal and seasonal time-scales in a 5.8 m2 plot and test the hypothesis that bacterial diversity varies on each of these scales. We used 16S rDNA gene amplicon sequencing to quantify the alpha and beta diversity of soil bacteria as well as the Net Relatedness Index and Nearest Taxon Indices to assess the degree of phylogenetic clustering, and the extent to which community shifts were driven by stochastic vs. deterministic limitation. We found that species richness was highest in winter, lowest in fall and that communities were compositionally distinct across seasons. There was no evidence of diurnal-scale shifts; the finest temporal scale over which community shifts were detected using our DNA-based analysis was between sampling dates separated by 6 weeks. Phylogenetic analyses suggested that seasonal-scale differences in community composition were the result of environmental filtering and homogeneous selection. Our findings provide insight into temporal variation of soil bacterial communities across the hourly to seasonal scales while minimizing the potential confounding effect of spatial variation.

Anthropogenic N Deposition Alters the Composition of Expressed Class II Fungal Peroxidases.

Here, we present evidence that ca. 20 years of experimental N deposition altered the composition of lignin-decaying class II peroxidases expressed by forest floor fungi, a response which has occurred concurrently with reductions in plant litter decomposition and a rapid accumulation of soil organic matter. This finding suggests that anthropogenic N deposition has induced changes in the biological mediation of lignin decay, the rate limiting step in plant litter decomposition. Thus, an altered composition of transcripts for a critical gene that is associated with terrestrial C cycling may explain the increased soil C storage under long-term increases in anthropogenic N deposition.IMPORTANCE Fungal class II peroxidases are enzymes that mediate the rate-limiting step in the decomposition of plant material, which involves the oxidation of lignin and other polyphenols. In field experiments, anthropogenic N deposition has increased soil C storage in forests, a result which could potentially arise from anthropogenic N-induced changes in the composition of class II peroxidases expressed by the fungal community. In this study, we have gained unique insight into how anthropogenic N deposition, a widespread agent of global change, affects the expression of a functional gene encoding an enzyme that plays a critical role in a biologically mediated ecosystem process.

Soil microbial communities and elk foraging intensity: implications for soil biogeochemical cycling in the sagebrush steppe.

Foraging intensity of large herbivores may exert an indirect top-down ecological force on soil microbial communities via changes in plant litter inputs. We investigated the responses of the soil microbial community to elk (Cervus elaphus) winter range occupancy across a long-term foraging exclusion experiment in the sagebrush steppe of the North American Rocky Mountains, combining phylogenetic analysis of fungi and bacteria with shotgun metagenomics and extracellular enzyme assays. Winter foraging intensity was associated with reduced bacterial richness and increasingly distinct bacterial communities. Although fungal communities did not respond linearly to foraging intensity, a greater ß-diversity response to winter foraging exclusion was observed. Furthermore, winter foraging exclusion increased soil cellulolytic and hemicellulolytic enzyme potential and higher foraging intensity reduced chitinolytic gene abundance. Thus, future changes in winter range occupancy may shape biogeochemical processes via shifts in microbial communities and subsequent changes to their physiological capacities to cycle soil C and N.

Anthropogenic N deposition increases soil organic matter accumulation without altering its biochemical composition.

Accumulating evidence indicates that future rates of atmospheric N deposition have the potential to increase soil C storage by reducing the decay of plant litter and soil organic matter (SOM). Although the microbial mechanism underlying this response is not well understood, a decline in decay could alter the amount, as well as biochemical composition of SOM. Here, we used size-density fractionation and solid-state 13 C-NMR spectroscopy to explore the extent to which declines in microbial decay in a long-term (ca. 20 yrs.) N deposition experiment have altered the biochemical composition of forest floor, bulk mineral soil, as well as free and occluded particulate organic matter. Significant amounts of organic matter have accumulated in occluded particulate organic matter (~20%; oPOM); however, experimental N deposition had not altered the abundance of carboxyl, aryl, alkyl, or O/N-alkyl C in forest floor, bulk mineral soil, or any soil fraction. These observations suggest that biochemically equivalent organic matter has accumulated in oPOM at a greater rate under experimental N deposition, relative to the ambient treatment. Although we do not understand the process by which experimental N deposition has fostered the occlusion of organic matter by mineral soil particles, our results highlight the importance of interactions among the products of microbial decay and the chemical and physical properties of silt and clay particles that occlude organic matter from microbial attack. Because oPOM can reside in soils for decades to centuries, organic matter accumulating under future rates of anthropogenic N deposition could remain in soil for long periods of time. If temperate forest soils in the Northern Hemisphere respond like those in our experiment, then unabated deposition of anthropogenic N from the atmosphere has the potential to foster greater soil C storage, especially in fine-texture forest soils.

Microbial Potential for Ecosystem N Loss Is Increased by Experimental N Deposition.

Fossil fuel combustion and fertilizer use has increased the amount of biologically available N entering terrestrial ecosystems. Nonetheless, our understanding of how anthropogenic N may alter the physiological mechanisms by which soil microorganisms cycle N in soil is still developing. Here, we applied shotgun metagenomics to a replicated long-term field experiment to determine how two decades of experimental N deposition, at a rate expected by mid-century, has affected the genetic potential of the soil microbial community to cycle N in soils. Experimental N deposition lead to a significant and persistent increase in functional assemblages mediating N cycle transformations associated with ecosystem N loss (i.e., denitrification and nitrification), whereas functional assemblages associated with N input and retention (i.e., N fixation and microbial N assimilation) were less positively affected. Furthermore, the abundance and composition of microbial taxa, as well as functional assemblages involved in housekeeping functions (i.e., DNA replication) were unaffected by experimental N deposition. Taken together, our results suggest that functional genes and gene pathways associated with ecosystem N loss have been favored by experimental N deposition, which may represent a genetic mechanism fostering increased N loss as anthropogenic N deposition increases in the future.

Active microorganisms in forest soils differ from the total community yet are shaped by the same environmental factors: the influence of pH and soil moisture.

Predicting the impact of environmental change on soil microbial functions requires an understanding of how environmental factors shape microbial composition. Here, we investigated the influence of environmental factors on bacterial and fungal communities across an expanse of northern hardwood forest in Michigan, USA, which spans a 500-km regional climate gradient. We quantified soil microbial community composition using high-throughput DNA sequencing on coextracted rDNA (i.e. total community) and rRNA (i.e. active community). Within both bacteria and fungi, total and active communities were compositionally distinct from one another across the regional gradient (bacteria P = 0.01; fungi P < 0.01). Taxonomically, the active community was a subset of the total community. Compositional differences between total and active communities reflected changes in the relative abundance of dominant taxa. The composition of both the total and active microbial communities varied by site across the gradient (P < 0.01) and was shaped by differences in soil moisture, pH, SOM carboxyl content, as well as C and N concentration. Our study highlights the importance of distinguishing between metabolically active microorganisms and the total community, and emphasizes that the same environmental factors shape the total and active communities of bacteria and fungi in this ecosystem.

Anthropogenic N Deposition Slows Decay by Favoring Bacterial Metabolism: Insights from Metagenomic Analyses.

Litter decomposition is an enzymatically-complex process that is mediated by a diverse assemblage of saprophytic microorganisms. It is a globally important biogeochemical process that can be suppressed by anthropogenic N deposition. In a northern hardwood forest ecosystem located in Michigan, USA, 20 years of experimentally increased atmospheric N deposition has reduced forest floor decay and increased soil C storage. Here, we paired extracellular enzyme assays with shotgun metagenomics to assess if anthropogenic N deposition has altered the functional potential of microbial communities inhabiting decaying forest floor. Experimental N deposition significantly reduced the activity of extracellular enzymes mediating plant cell wall decay, which occurred concurrently with changes in the relative abundance of metagenomic functional gene pathways mediating the metabolism of carbohydrates, aromatic compounds, as well as microbial respiration. Moreover, experimental N deposition increased the relative abundance of 50 of the 60 gene pathways, the majority of which were associated with saprotrophic bacteria. Conversely, the relative abundance and composition of fungal genes mediating the metabolism of plant litter was not affected by experimental N deposition. Future rates of atmospheric N deposition have favored saprotrophic soil bacteria, whereas the metabolic potential of saprotrophic fungi appears resilient to this agent of environmental change. Results presented here provide evidence that changes in the functional capacity of saprotrophic soil microorganisms mediate how anthropogenic N deposition increases C storage in soil.

Atmospheric N deposition alters connectance, but not functional potential among saprotrophic bacterial communities.

The use of co-occurrence patterns to investigate interactions between micro-organisms has provided novel insight into organismal interactions within microbial communities. However, anthropogenic impacts on microbial co-occurrence patterns and ecosystem function remain an important gap in our ecological knowledge. In a northern hardwood forest ecosystem located in Michigan, USA, 20 years of experimentally increased atmospheric N deposition has reduced forest floor decay and increased soil C storage. This ecosystem-level response occurred concomitantly with compositional changes in saprophytic fungi and bacteria. Here, we investigated the influence of experimental N deposition on biotic interactions among forest floor bacterial assemblages by employing phylogenetic and molecular ecological network analysis. When compared to the ambient treatment, the forest floor bacterial community under experimental N deposition was less rich, more phylogenetically dispersed and exhibited a more clustered co-occurrence network topology. Together, our observations reveal the presence of increased biotic interactions among saprotrophic bacterial assemblages under future rates of N deposition. Moreover, they support the hypothesis that nearly two decades of experimental N deposition can modify the organization of microbial communities and provide further insight into why anthropogenic N deposition has reduced decomposition, increased soil C storage and accelerated phenolic DOC production in our field experiment.