Biological remediation treatments improve the health of a mixed contaminated soil before significantly reducing contaminant levels.

The remediation of mixed contaminated soil is challenging as it often requires actions to minimize metal-induced risks while degrading organic contaminants. Here, the effectiveness of different bioremediation strategies, namely, rhizoremediation with native plant species, mycoremediation with Pleurotus ostreatus spent mushroom substrate, and biostimulation with organic by-products (i.e., composted sewage sludge and spent mushroom substrate), for the recovery of a mixed contaminated soil from an abandoned gravel pit was studied. The combination of biostimulation and rhizoremediation led to the most significant increase in soil health, according to microbial indicator values. The application of composted sewage sludge led to the highest reduction in anthracene and polychlorinated biphenyls concentrations. None of the strategies managed to decrease contamination levels below regulatory limits, but they did enhance soil health. It was concluded that the biological remediation treatments improved soil functioning in a short time, before the concentration of soil contaminants was significantly reduced.

Induced development of oxytetracycline tolerance in bacterial communities from soil amended with well-aged cow manure.

The use of animal manure as organic fertilizer is a common agricultural practice that can improve soil health and crop yield. However, antibiotics and their metabolites are often present in animal manure and, hence, in manure-amended soil. The aim of this study was to assess the induced development of oxytetracycline (OTC) tolerance in soil bacterial communities as a result of the addition of OTC to soil amended with well-aged cow manure. To this purpose, soil amended with well-aged cow manure was repeatedly - three times - spiked with different OTC concentrations (0, 2, 20, 60, 150, and 500 mg OTC kg-1 dry weight soil, each time) according to a pollution-induced community tolerance (PICT) assay. The PICT detection phase was conducted in Biolog EcoPlatesTM in the presence of the following OTC concentration gradient in the wells: 0, 5, 20, 40, 60, and 100 mg L-1. For all treatments, the application of OTC in the PICT selection phase resulted in lower values of bacterial metabolic activity (i.e., lower values of average well color development) in the PICT detection phase. A significant increase in OTC tolerance was observed in soil bacterial communities that had been exposed three times to ≥ 20 mg OTC kg-1 DW soil during the PICT selection phase. In general, higher levels of OTC exposure during the PICT selection phase resulted in bacterial tolerance to higher OTC concentrations during the PICT detection phase, pointing to a dose-dependent induced tolerance. It is important to (i) rationalize the amount of antibiotics administered to livestock, and (ii) treat properly the antibiotic-containing manure before its application to agricultural soil as fertilizer.

Mycoremediation with Agaricus bisporus and Pleurotus ostreatus growth substrates versus phytoremediation with Festuca rubra and Brassica sp. for the recovery of a Pb and γ-HCH contaminated soil.

Mycoremediation with mushroom growth substrates can be used for the recovery of mixed contaminated soils due to the benefits derived from the physicochemical characteristics of the substrates, the activity of extracellular enzymes secreted by the fungi, and the presence of the fungal mycelia. The objective of this work was to assess the potential of Agaricus bisporus and Pleurotus ostreatus growth substrates (inoculated mushroom substrates vs. spent mushroom substrates) for the mycoremediation of soils co-contaminated with lead and lindane (γ-HCH). We compared the efficiency of these mycoremediation strategies with the phytoremediation with Brassica spp. Or Festuca rubra plants, in terms of both reduction in contaminant levels and enhancement of soil health. An enhanced soil health was achieved as a result of the application of mycoremediation treatments, compared to phytoremediation and control (untreated) treatments. The application of P. ostreatus inoculated substrate led to the most significant reduction in γ-HCH concentration (up to 88.9% compared to corresponding controls). In the presence of inoculated mushroom substrate, P. ostreatus fruiting bodies extracted more Pb than Brassica spp. Or F. rubra plants. Mycoremediation with P. ostreatus growth substrates appears a promising strategy for the recovery of the health of soils co-contaminated with Pb and γ-HCH.

Organic amendment treatments for antimicrobial resistance and mobile element genes risk reduction in soil-crop systems.

Agricultural fertilization with organic amendments of animal origin often leads to antibiotic resistance dissemination. In this study, we evaluated the effect of different treatments (anaerobic digestion, biochar application, ozonation, zerovalent iron nanoparticle application, and spent mushroom substrate addition) on the resistome in dairy cow manure-derived amendments (slurry, manure, and compost). Anaerobic digestion and biochar application resulted in the highest reduction in antibiotic resistance gene (ARG) and mobile genetic element (MGE) gene abundance. These two treatments were applied to cow manure compost, which was then used to fertilize the soil for lettuce growth. After crop harvest, ARG and MGE gene absolute and relative abundances in the soil and lettuce samples were determined by droplet digital PCR and high-throughput qPCR, respectively. Prokaryotic diversity in cow manure-amended soils was determined using 16S rRNA metabarcoding. Compared to untreated compost, anaerobic digestion led to a 38% and 83% reduction in sul2 and intl1 absolute abundances in the soil, respectively, while biochar led to a 60% reduction in intl1 absolute abundance. No differences in lettuce gene abundances were observed among treatments. We conclude that amendment treatments can minimize the risk of antibiotic resistance in agroecosystems.

Reduction of the resistome risk from cow slurry and manure microbiomes to soil and vegetable microbiomes.

In cow farms, the interaction between animal and environmental microbiomes creates hotspots for antibiotic resistance dissemination. A shotgun metagenomic approach was used to survey the resistome risk in five dairy cow farms. To this purpose, 10 environmental compartments were sampled: 3 of them linked to productive cows (fresh slurry, stored slurry, slurry-amended pasture soil); 6 of them to non-productive heifers and dry cows (faeces, fresh manure, aged manure, aged manure-amended orchard soil, vegetables-lettuces and grazed soil); and, finally, unamended control soil. The resistome risk was assessed using MetaCompare, a computational pipeline which scores the resistome risk according to possible links between antibiotic resistance genes (ARGs), mobile genetic elements (MGEs) and human pathogens. The resistome risk decreased from slurry and manure microbiomes to soil and vegetable microbiomes. In total (sum of all the compartments), 18,157 ARGs were detected: 24% related to ansamycins, 21% to multidrugs, 14% to aminoglycosides, 12% to tetracyclines, 9% to ß-lactams, and 9% to macrolide-lincosamide-streptogramin B. All but two of the MGE-associated ARGs were only found in the animal dejections (not in soil or vegetable samples). Several ARGs with potential as resistome risk markers (based on their presence in hubs of co-occurrence networks and high dissemination potential) were identified. As a precautionary principle, improved management of livestock dejections is necessary to minimize the risk of antibiotic resistance.

Agricultural Soils Amended With Thermally-Dried Anaerobically-Digested Sewage Sludge Showed Increased Risk of Antibiotic Resistance Dissemination.

The application of sewage sludge (SS) to agricultural soil can help meet crop nutrient requirements and enhance soil properties, while reusing an organic by-product. However, SS can be a source of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs), resulting in an increased risk of antibiotic resistance dissemination. We studied the effect of the application of thermally-dried anaerobically-digested SS on (i) soil physicochemical and microbial properties, and (ii) the relative abundance of 85 ARGs and 10 MGE-genes in soil. Soil samples were taken from a variety of SS-amended agricultural fields differing in three factors: dose of application, dosage of application, and elapsed time after the last application. The relative abundance of both ARGs and MGE-genes was higher in SS-amended soils, compared to non-amended soils, particularly in those with a more recent SS application. Some physicochemical parameters (i.e., cation exchange capacity, copper concentration, phosphorus content) were positively correlated with the relative abundance of ARGs and MGE-genes. Sewage sludge application was the key factor to explain the distribution pattern of ARGs and MGE-genes. The 30 most abundant families within the soil prokaryotic community accounted for 66% of the total variation of ARG and MGE-gene relative abundances. Soil prokaryotic α-diversity was negatively correlated with the relative abundance of ARGs and MGE-genes. We concluded that agricultural soils amended with thermally-dried anaerobically-digested sewage sludge showed increased risk of antibiotic resistance dissemination.

Antibiotic Resistance in Agricultural Soil and Crops Associated to the Application of Cow Manure-Derived Amendments From Conventional and Organic Livestock Farms.

The application of organic amendments to agricultural soil can enhance crop yield, while improving the physicochemical and biological properties of the recipient soils. However, the use of manure-derived amendments as fertilizers entails environmental risks, such as the contamination of soil and crops with antibiotic residues, antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). In order to delve into these risks, we applied dairy cow manure-derived amendments (slurry, fresh manure, aged manure), obtained from a conventional and an organic farm, to soil. Subsequently, lettuce and wheat plants were grown in the amended soils. After harvest, the abundance of 95 ARGs and MGE-genes from the amended soils and plants were determined by high-throughput qPCR. The structure of soil prokaryotic communities was determined by 16S rRNA amplicon sequencing and qPCR. The absolute abundance of ARGs and MGE-genes differed between treatments (amended vs. unamended), origins of amendment (conventional vs. organic), and types of amendment (slurry vs. fresh manure vs. aged manure). Regarding ARG-absolute abundances in the amendments themselves, higher values were usually found in slurry vs. fresh or aged manure. These abundances were generally higher in soil than in plant samples, and higher in wheat grain than in lettuce plants. Lettuce plants fertilized with conventional amendments showed higher absolute abundances of tetracycline resistance genes, compared to those amended with organic amendments. No single treatment could be identified as the best or worst treatment regarding the risk of antibiotic resistance in soil and plant samples. Within the same treatment, the resistome risk differed between the amendment, the amended soil and, finally, the crop. In other words, according to our data, the resistome risk in manure-amended crops cannot be directly inferred from the analysis of the amendments themselves. We concluded that, depending on the specific question under study, the analysis of the resistome risk should specifically focus on the amendment, the amended soil or the crop.

Keep and promote biodiversity at polluted sites under phytomanagement.

The phytomanagement concept combines a sustainable reduction of pollutant linkages at risk-assessed contaminated sites with the generation of both valuable biomass for the (bio)economy and ecosystem services. One of the potential benefits of phytomanagement is the possibility to increase biodiversity in polluted sites. However, the unique biodiversity present in some polluted sites can be severely impacted by the implementation of phytomanagement practices, even resulting in the local extinction of endemic ecotypes or species of great conservation value. Here, we highlight the importance of promoting measures to minimise the potential adverse impact of phytomanagement on biodiversity at polluted sites, as well as recommend practices to increase biodiversity at phytomanaged sites without compromising its effectiveness in terms of reduction of pollutant linkages and the generation of valuable biomass and ecosystem services.

Long-term phytomanagement with compost and a sunflower - Tobacco rotation influences the structural microbial diversity of a Cu-contaminated soil.

At a former wood preservation site contaminated with Cu, various phytomanagement options have been assessed in the last decade through physicochemical, ecotoxicological and biological assays. In a field trial at this site, phytomanagement with a crop rotation based on tobacco and sunflower, combined with the incorporation of compost and dolomitic limestone, has proved to be efficient in Cu-associated risk mitigation, ecological soil functions recovery and net gain of economic and social benefits. To demonstrate the long-term effectiveness and sustainability of phytomanagement, we assessed here the influence of this remediation option on the diversity, composition and structure of microbial communities over time, through a metabarcoding approach. After 9 years of phytomanagement, no overall effect was identified on microbial diversity; the soil amendments, notably the repeated compost application, led to shifts in soil microbial populations. This phytomanagement option induced changes in the composition of soil microbial communities, promoting the growth of microbial groups belonging to Alphaproteobacteria, many being involved in N cycling. Populations of Nitrososphaeria, which are crucial in nitrification, as well as taxa from phyla Planctomycetacia, Chloroflexi and Gemmatimonadetes, which are tolerant to metal contamination and adapted to oligotrophic soil conditions, decreased in amended phytomanaged plots. Our study provides an insight into population dynamics within soil microbial communities under long-term phytomanagement, in line with the assessment of soil ecological functions and their recovery.

The microbiota of technosols resembles that of a nearby forest soil three years after their establishment.

Technosols can be used to rehabilitate degraded land and reuse wastes. Ideally, these newly formed soils should also fulfil the main soil functions. In this study, initially, we characterized the physicochemical and microbial properties of different formulations and their ingredients (i.e., dirt from a waste recovery plant, recycled bentonite, sewage sludge). When these technosols were then used for the rehabilitation of a quarry, the evolution of such properties was monitored for three consecutive years. Physicochemical and microbial properties were compared to those of a reference soil from a nearby forest. Diversity and composition of prokaryotes and eukaryotes were determined using 16S and 18S rRNA amplicon sequencing. Three years after establishment, as much as 78.8% and 63.9% of the prokaryotic and eukaryotic orders, respectively, were shared between the technosols and the reference forest soil. Although technosols initially showed lower values of CO2 emission, compaction and functional diversity (Biolog EcoPlates™), at the end of the study these values were similar to those observed in the reference forest soil. It was concluded that the microbiota of the studied technosols resembles that of the nearby forest soil after just three years of establishment.

Application of sewage sludge to agricultural soil increases the abundance of antibiotic resistance genes without altering the composition of prokaryotic communities.

The application of sewage sludge as soil amendment is a common agricultural practice. However, wastewater treatment plants, sewage sludge and sewage sludge-amended soils have been reported as hotspots for the appearance and dissemination of antibiotic resistance, driven, among other factors, by selection pressure exerted by co-exposure to antibiotics and heavy metals. To address this threat to environmental and human health, soil samples from a long-term (24 years) field experiment, carried out to study the impact of thermally dried and anaerobically digested sewage sludge (at different doses and frequencies of application) on agricultural soil quality, were investigated for the presence of genes encoding antibiotic resistance (ARGs) and mobile genetic elements (MGEs). Sewage sludge-induced changes in specific soil physicochemical and microbial properties, as indicators of soil quality, were also investigated. The application of sewage sludge increased the total concentration of copper and zinc in amended soils, but without affecting the bioavailability of these metals, possibly due to the high values of soil pH and organic matter content. Soil microbal quality, as reflected by the value of the Soil Quality Index, was higher in sewage sludge-amended soils. Similarly, the application of sewage sludge increased soil microbial activity and biomass, as well as the abundance of ARGs and MGE genes, posing a risk of dissemination of antibiotic resistance. In contrast, the composition of soil prokaryotic communities was not significantly altered by the application of sewage sludge. We found correlation between soil Cu and Zn concentrations and the abundance of ARGs and MGE genes. It was concluded that sewage sludge-derived amendments must be properly treated and managed if they are to be applied to agricultural soil.

The impact of nanoscale zero-valent iron particles on soil microbial communities is soil dependent.

The application of nanoscale zero-valent iron particles (nZVI) for the remediation of contaminated sites is very promising. However, information concerning the ecotoxicity of nZVI on soil microbial communities and, hence, soil quality, is still scarce. We carried out a three-month experiment to evaluate the impact of the application of different concentrations of nZVI (from 1 to 20 mg g DW soil-1) on soil microbial properties in a clay-loam versus a sandy-loam soil. Data on microbial biomass (total bacteria and fungi by qPCR, microbial biomass carbon), activity (ß-glucosidase, arylsulphatase and urease activities), and functional (Biolog Ecoplates™) and structural (ARISA, 16S rRNA amplicon sequencing) diversity evidenced that the sandy-loam soil was more vulnerable to the presence of nZVI than the clay-loam soil. In the sandy-loam soil, arylsulphatase activity and bacterial abundance, richness and diversity were susceptible to the presence of nZVI. The high content of clay and organic matter present in the clay-loam soil may explain the observed negligible effects of nZVI on soil microbial properties. It was concluded that the impact of nZVI on soil microbial communities and, hence, soil quality, is soil dependent.

Data on the selection of biostimulating agents for the bioremediation of soil simultaneously contaminated with lindane and zinc.

The bioremediation of contaminated soil often involves the addition of organic/inorganic amendments and mobilizing agents (e.g. surfactants, detergents), in order to stimulate the growth and degrading activity of soil microbial populations and increase contaminant bioavailability. For this data article we carried out an experiment to select biostimulating agents for the bioremediation of soil simultaneously contaminated with lindane (HCH, 10 mg kg-1 DW soil) and Zinc (Zn, 1500 mg kg-1 DW soil). To this purpose, a factorial design was used to test the effect of three organic amendments (i.e. hen manure, composted horse manure, cow slurry) and three mobilizing agents (i.e. sodium dodecylbenzenesulfonate (SDS), rhamnolipids and Tween-80) on the reduction of total HCH and bioavailable Zn concentration in soil. Similarly, the effect of the addition of cyclohexane, as chemical inducer of HCH degradation, was also studied. The addition of SDS, rhamnolipids and Tween-80 significantly reduced HCH concentration in soil, regardless of the presence of other biostimulating agents. When added individually, the three organic amendments (hen manure, composted horse manure, cow slurry) significantly reduced bioavailable Zn concentration in soil. These data provide useful information for the bioremediation, through biostimulation, of soils simultaneously contaminated with HCH and Zn.

From phytoremediation of soil contaminants to phytomanagement of ecosystem services in metal contaminated sites.

Since the emergence of phytoremediation, much research has focused on its development for (i) the removal of metals from soil and/or (ii) the reduction of metal bioavailability, mobility, and ecotoxicity in soil. Here, we review the lights and shades of the two main strategies (i.e., phytoextraction and phytostabilization) currently used for the phytoremediation of metal contaminated soils, irrespective of the level of such contamination. Both strategies face limitations to become successful at commercial scale and, then, often generate skepticism regarding their usefulness. Recent innovative approaches and paradigms are gradually establishing these phytoremediation strategies as suitable options for the management of metal contaminated soils. The combination of these phytotechnologies with a sustainable and profitable site use (a strategy called phytomanagement) grants value to the many benefits that can be obtained during the phytoremediation of metal contaminated sites, such as, for instance, the restoration of important ecosystem services, e.g. nutrient cycling, carbon storage, water flow regulation, erosion control, water purification, fertility maintenance, etc.

Short-term effects of non-grazing on plants, soil biota and aboveground-belowground links in Atlantic mountain grasslands.

Mountain grasslands in the Iberian Peninsula are the result of extensive grazing. However, a progressive abandonment of grazing activity is currently observed in the study region. The objective of this work was to evaluate the short-term (2 years) effects of non-grazing on the diversity and composition of plants, soil microorganisms (prokaryotes, fungi, arbuscular mycorrhiza), mesofauna, macrofauna and aboveground-belowground links, through the study of 16 grazed vs. non-grazed areas in Atlantic grasslands located in the Basque Country (Spain). Sites were divided between 4 habitat types with different elevation, pasture productivity, vegetation type and parent material. Herbivores appeared to influence plant community composition, contributing to increase aboveground diversity, while having unequal effects on belowground communities depending on the organisms analysed. This may be explained by the different habitat and trophic level of each soil organism, which may be more or less affected by the predominating negative effects of grazing, such as soil compaction, and only partially compensated by other positive effects. Finally, habitat type appeared to be the strongest influence on both above- and belowground communities, also influencing the effect of the absence of grazing.

Plasmid-Mediated Bioaugmentation for the Bioremediation of Contaminated Soils.

Bioaugmentation, or the inoculation of microorganisms (e.g., bacteria harboring the required catabolic genes) into soil to enhance the rate of contaminant degradation, has great potential for the bioremediation of soils contaminated with organic compounds. Regrettably, cell bioaugmentation frequently turns into an unsuccessful initiative, owing to the rapid decrease of bacterial viability and abundance after inoculation, as well as the limited dispersal of the inoculated bacteria in the soil matrix. Genes that encode the degradation of organic compounds are often located on plasmids and, consequently, they can be spread by horizontal gene transfer into well-established, ecologically competitive, indigenous bacterial populations. Plasmid-mediated bioaugmentation aims to stimulate the spread of contaminant degradation genes among indigenous soil bacteria by the introduction of plasmids, located in donor cells, harboring such genes. But the acquisition of plasmids by recipient cells can affect the host's fitness, a crucial aspect for the success of plasmid-mediated bioaugmentation. Besides, environmental factors (e.g., soil moisture, temperature, organic matter content) can play important roles for the transfer efficiency of catabolic plasmids, the expression of horizontally acquired genes and, finally, the contaminant degradation activity. For plasmid-mediated bioaugmentation to be reproducible, much more research is needed for a better selection of donor bacterial strains and accompanying plasmids, together with an in-depth understanding of indigenous soil bacterial populations and the environmental conditions that affect plasmid acquisition and the expression and functioning of the catabolic genes of interest.

Environmental parameters altered by climate change affect the activity of soil microorganisms involved in bioremediation.

Bioremediation, based on the use of microorganisms to break down pollutants, can be very effective at reducing soil pollution. But the climate change we are now experiencing is bound to have an impact on bioremediation performance, since the activity and degrading abilities of soil microorganisms are dependent on a series of environmental parameters that are themselves being altered by climate change, such as soil temperature, moisture, amount of root exudates, etc. Many climate-induced effects on soil microorganisms occur indirectly through changes in plant growth and physiology derived from increased atmospheric CO2 concentrations and temperatures, the alteration of precipitation patterns, etc., with a concomitant effect on rhizoremediation performance (i.e. the plant-assisted microbial degradation of pollutants in the rhizosphere). But these effects are extremely complex and mediated by processes such as acclimation and adaptation. Besides, soil microorganisms form complex networks of interactions with a myriad of organisms from many taxonomic groups that will also be affected by climate change, further complicating data interpretation.

Ecosystem services and plant physiological status during endophyte-assisted phytoremediation of metal contaminated soil.

Mining sites shelter a characteristic biodiversity with large potential for the phytoremediation of metal contaminated soils. Endophytic plant growth-promoting bacteria were isolated from two metal-(hyper)accumulator plant species growing in a metal contaminated mine soil. After characterizing their plant growth-promoting traits, consortia of putative endophytes were used to carry out an endophyte-assisted phytoextraction experiment using Noccaea caerulescens and Rumex acetosa (singly and in combination) under controlled conditions. We evaluated the influence of endophyte-inoculated plants on soil physicochemical and microbial properties, as well as plant physiological parameters and metal concentrations. Data interpretation through the grouping of soil properties within a set of ecosystem services was also carried out. When grown together, we observed a 41 and 16% increase in the growth of N. caerulescens and R. acetosa plants, respectively, as well as higher values of Zn phytoextraction and soil microbial biomass and functional diversity. Inoculation of the consortia of putative endophytes did not lead to higher values of plant metal uptake, but it improved the plants' physiological status, by increasing the content of chlorophylls and carotenoids by up to 28 and 36%, respectively, indicating a reduction in the stress level of plants. Endophyte-inoculation also stimulated soil microbial communities: higher values of acid phosphatase activity (related to the phosphate solubilising traits of the endophytes), bacterial and fungal abundance, and structural diversity. The positive effects of plant growth and endophyte inoculation on soil properties were reflected in an enhancement of some ecosystem services (biodiversity, nutrient cycling, water flow regulation, water purification and contamination control).

Long-term effects of aided phytostabilisation on microbial communities of metal-contaminated mine soil.

Aided phytostabilisation uses metal-tolerant plants, together with organic or inorganic amendments, to reduce metal bioavailability in soil while improving soil quality. The long-term effects of the following organic amendments were examined as part of an aided phytostabilisation field study in an abandoned Pb/Zn mining area: cow slurry, sheep manure and paper mill sludge mixed with poultry manure. In the mining area, two heavily contaminated vegetated sites, showing different levels of soil metal contamination (LESS and MORE contaminated site), were selected for this study. Five years after amendment application, metal bioavailability (CaCl2 extractability) along with a variety of indicators of soil microbial activity, biomass and diversity were analysed. Paper mill sludge mixed with poultry manure treatment resulted in the highest reduction of Cd, Pb and Zn bioavailability, as well as in stimulation of soil microbial activity and diversity, especially at the LESS contaminated site. In contrast, cow slurry was the least successful treatment. Our results emphasise the importance of the (i) long-term monitoring of soil quality at sites subjected to aided phytostabilisation and (ii) selection of the most efficient amendments and plants in terms of both reduction of metal bioavailability and improvement of soil quality.

Multi-targeted metagenetic analysis of the influence of climate and environmental parameters on soil microbial communities along an elevational gradient.

Mountain elevation gradients are invaluable sites for understanding the effects of climate change on ecosystem function, community structure and distribution. However, relatively little is known about the impact on soil microbial communities, in spite of their importance for the functioning of the soil ecosystem. Previous studies of microbial diversity along elevational gradients were often limited by confounding variables such as vegetation, pH, and nutrients. Here, we utilised a transect in the Pyrenees established to minimise variation in such parameters, to examine prokaryotic, fungal, protist and metazoan communities throughout three consecutive years. We aimed to determine the influences of climate and environmental parameters on soil microbial community structure; as well as on the relationships between those microbial communities. Further, functional diversity of heterotrophic bacteria was determined using Biolog. Prokaryotic and fungal community structure, but not alpha-diversity, correlated significantly with elevation. However, carbon-to-nitrogen ratio and pH appeared to affect prokaryotic and protist communities more strongly. Both community structure and physicochemical parameters varied considerably between years, illustrating the value of long-term monitoring of the dynamic processes controlling the soil ecosystem. Our study also illustrates both the challenges and strengths of using microbial communities as indicators of potential impacts of climate change.