Wood ash decreases cadmium toxicity to the soil nematode Caenorhabditis elegans.

Wood ash is a beneficial fertilizer and liming agent in nutrient depleted soils, but it also contains considerable amounts of cadmium (Cd), which can be toxic to organisms in the environment. Therefore, risk assessments regarding utilization of wood ash is required. Here, we studied how wood ash (applied in doses equivalent to 0, 3 and 6 t ha-1) and Cd (applied in doses of 0, 10, 150, 300, 600, 1200 and 2000 mg kg-1) affected growth of the soil nematode Caenorhabditis elegans. The treatments were combined in a full factorial design. Wood ash alone greatly stimulated both soil respiration and growth of C. elegans, whereas Cd alone had a toxic effect. However, unrealistically high Cd levels were needed to severely affect growth of C. elegans and soil respiration, especially soil respiration was very resilient to Cd amendment. Ash addition decreased Cd toxicity to C. elegans, with an EC50 value of 390 mg Cd kg-1 in the 3 t ash ha-1 treatment, and an increase of EC50 to 1894 mg Cd kg-1 in the 6 t ash ha-1 treatment. This is probably because ash increases the Cd sorption capacity of the soil, and thereby decreases the bio-availability of Cd. The results suggest that there is no acute toxic effect of Cd to nematodes associated with wood ash recycling; in fact, our results suggest that ash actually decrease Cd toxicity.

Groundwater chemistry determines the prokaryotic community structure of waterworks sand filters.

Rapid sand filtration is essential at most waterworks that treat anaerobic groundwater. Often the filtration depends on microbiological processes, but the microbial communities of the filters are largely unknown. We determined the prokaryotic community structures of 11 waterworks receiving groundwater from different geological settings by 16S rRNA gene-based 454 pyrosequencing and explored their relationships to filtration technology and raw water chemistry. Most of the variation in microbial diversity observed between different waterworks sand filters could be explained by the geochemistry of the inlet water. In addition, our findings suggested four features of particular interest: (1) Nitrospira dominated over Nitrobacter at all waterworks, suggesting that Nitrospira is a key nitrifying bacterium in groundwater-treating sand filters. (2) Hyphomicrobiaceae species were abundant at all waterworks, where they may be involved in manganese oxidation. (3) Six of 11 waterworks had significant concentrations of methane in their raw water and very high abundance of the methanotrophic Methylococcaceae. (4) The iron-oxidizing bacteria Gallionella was present at all waterworks suggesting that biological iron oxidation is occurring in addition to abiotic iron oxidation. Elucidation of key members of the microbial community in groundwater-treating sand filters has practical potential, for example, when methods are needed to improve filter function.

Steam-treatment-based soil remediation promotes heat-tolerant, potentially pathogenic microbiota.

We investigated microbiota in surface and subsurface soil from a site, above steam-treated deep sub-soil originally contaminated with chlorinated solvents. During the steam treatment, the surface soil reached temperatures c. 30 degrees C higher than the temperature in untreated soil; whereas the subsurface soil, at a depth of about 40 cm, reached a temperature c. 45 degrees C higher than untreated soil. The soil was examined prior to, during, and 6, 12, 14, 20 and 31 months after treatment. Numbers of bacteria cultivable at 42 degrees C increased significantly in subsurface soil. Similarly, substrate utilization in ECOLOG plates, incubated at 42 degrees C, increased from less than 10% of available carbon sources in the untreated soil to more than 60% of the available carbon sources in the steam-treated soil. Aspergillus fumigatus was quantified as an example ofheat-tolerant fungi normally found in compost. These organisms are rarely detected in Danish soils but high numbers (c. 10(5) hyphal forming units g(-1)) occurred in the treated soil up to 31 months after the steam-treatment. We conclude that steam-treatment leads to changes of the microbial communities. Some changes are temporary while others can last for years after termination of the steam-treatment; reflecting different strategies that soil microorganisms follow.

DNA metabarcoding reveals the impact of Cu2+ on soil cercozoan diversity.

Although copper (Cu2+) is a micronutrient, the metal may be toxic if present in high concentrations in soil ecosystems and subsequently affect various organisms, ranging from microorganisms to earthworms. We performed a microcosm study with an array of Cu2+ concentrations, with a specific focus on Cercozoa, an important protozoan group in most soil food webs. Research on Cercozoa is still scarce in terms of both diversity and ecology; hence, to explore this group in more depth, we used high-throughput sequencing to detect Cu2+ induced community changes. Increased levels of Cu2+ caused a shift in the cercozoan community, and we observed decreased cercozoan relative abundance across the majority of orders, families and genera. Due to their key role in soil food webs, especially as bacterial predators and providers of nutrients to plants, the reduction of cercozoan abundance and diversity may seriously affect soil functionality. Our results indicate that the increase of Cu2+ concentrations in the soil could potentially have this effect and the consequences need exploration.

Activity of root-knot nematodes associated with composition of a nematode-attached microbiome and the surrounding soil microbiota.

We investigated if activity of the pre-infective juveniles (J2s) of root-knot nematodes is linked to the recruitment of a specific microbiome on the nematode surface and/or to the composition of the surrounding microbiota. For this, we determined the J2 activity (active vs. non-motile, which referred to dead and immobile J2s) upon a 3-day incubation in soil suspensions and studied the composition of bacteria, protists, and fungi present on the nematode surface and in the suspensions using amplicon sequencing of the 16S/18S rRNA genes, and ITS region. We also amended suspensions with Pseudomonas protegens strain CHA0 to study its effects on J2 activity and microbial composition. The J2 activity was suppressed in soil suspensions, but increased when suspensions were amended with P. protegens CHA0. The active and non-motile J2s differed in the composition of surface-attached bacteria, which was altered by the presence of P. protegens CHA0 in the soil suspensions. The bacterial genera Algoriphagus, Pedobacter, and Bdellovibrio were enriched on active J2s and may have protected the J2s against antagonists. The incubation time appeared short for attachment of fungi and protists. Altogether, our study is a step forward in disentangling the complex nematode-microbe interactions in soil for more successful nematode control.

Abrupt permafrost thaw triggers activity of copiotrophs and microbiome predators.

Permafrost soils store a substantial part of the global soil carbon and nitrogen. However, global warming causes abrupt erosion and gradual thaw, which make these stocks vulnerable to microbial decomposition into greenhouse gases. Here, we investigated the microbial response to abrupt in situ permafrost thaw. We sequenced the total RNA of a 1 m deep soil core consisting of up to 26 500-year-old permafrost material from an active abrupt erosion site. We analysed the microbial community in the active layer soil, the recently thawed, and the intact permafrost, and found maximum RNA:DNA ratios in recently thawed permafrost indicating a high microbial activity. In thawed permafrost, potentially copiotrophic Burkholderiales and Sphingobacteriales, but also microbiome predators dominated the community. Overall, both thaw-dependent and long-term soil properties significantly correlated with changes in community composition, as did microbiome predator abundance. Bacterial predators were dominated in shallower depths by Myxococcota, while protozoa, especially Cercozoa and Ciliophora, almost tripled in relative abundance in thawed layers. Our findings highlight the ecological importance of a diverse interkingdom and active microbial community highly abundant in abruptly thawing permafrost, as well as predation as potential biological control mechanism.

Green waste and sewage sludge feeding ratio alters co-composting performance: Emphasis on the role of bacterial community during humification.

Composting with five levels of green waste and sewage sludge was compared to examine how feeding ratios affected composting performance with special focus on humification, and the underlying mechanisms. The results showed that the raw material ratio persistently affected compost nutrients and stability. Humification and mineralization were promoted by higher proportion of sewage sludge. Bacterial community composition and within-community relationships were also significantly affected by the raw material feeding ratio. Network analysis indicated that clusters 1 and 4 which dominated by Bacteroidetes, Proteobacteria, and Acidobacteria shown significantly positive correlation with humic acid concentration. Notably, the structural equational model and variance partitioning analysis demonstrated that bacterial community structure (explained 47.82% of the variation) mediated the effect of raw material feeding ratio on humification, and exceeded the effect of environmental factors (explained 19.30% of the variation) on humic acid formation. Accordingly, optimizing the composting raw material improves the composting performance.

Phylogenomic analysis of kinetoplastids supports that trypanosomatids arose from within bodonids.

Kinetoplastids are a large group of free-living and parasitic eukaryotic flagellates, including the medically important trypanosomatids (e.g., Trypanosoma and Leishmania) and the widespread free-living and parasitic bodonids. Small subunit rRNA- and conserved protein-based phylogenies support the division of kinetoplastids into five orders (Prokinetoplastida, Neobodonida, Parabodonida, Eubodonida, and Trypanosomatida), but they produce incongruent results regarding their relative branching order, in particular for the position of the Trypanosomatida. In general, small subunit rRNA tends to support their early emergence, whereas protein phylogenies most often support a more recent origin from within bodonids. In order to resolve this question through a phylogenomic approach, we carried out massive parallel sequencing of cDNA from representatives of three bodonid orders (Bodo saltans -Eubodonida-, Procryptobia sorokini -Parabodonida-, and Rhynchomonas nasuta -Neobodonida-). We identified 64 well-conserved proteins shared by these species, four trypanosomatids, and two closely related outgroup species (Euglena gracilis and Diplonema papillatum). Phylogenetic analysis of a concatenated data set yielded a strongly supported tree showing the late emergence of trypanosomatids as a sister group of the Eubodonida. In addition, we identified homologues of proteins involved in trypanosomatid mitochondrial mRNA editing in the three bodonid species, suggesting that editing may be widespread in kinetoplastids. Comparison of expressed sequences from mitochondrial genes showed variability at U positions, in agreement with the existence of editing activity in the three bodonid orders most closely related to trypanosomatids (Neobodonida, Parabodonida, and Eubodonida). Mitochondrial mRNA editing appears to be an ancient phenomenon in kinetoplastids.

Is wood ash amendment a suitable mitigation strategy for N2O emissions from soil?

Wood ash, the by-product of biomass combustion to energy, can return important nutrients back to the soil and counteract acidification. However, the application of wood ash may affect the emission of greenhouse gases. Here, the effect of wood ash application on nitrous oxide (N2O) emissions from different soil environments were investigated in a 40 days incubation experiment comprising ten different soil types amended with five different wood ash concentrations (0, 3, 9, 20, and 54 t ash ha-1). The emitted N2O was measured continuously, and initial soil properties without ash application (carbon (C), nitrogen (N), ammonium (NH4+), nitrate (NO3-), and pH) and resulting soil properties (pH, NH4+, and NO3-) were measured prior and after the incubation period, respectively. The Random Forests (RF) model was used to identify which factors (initial and resulting soil properties, vegetation, management, wood ash doze, and respiration rate) were the most important to predict the development of emitted N2O after ash application. Wood ash either increased, decreased, or had no effect on the amount of emitted N2O depending on soil type and ash dose. The RF model identified the final resulting pH as the most important factor for the prediction of emitted N2O. The results suggest that wood ash can mitigate N2O emissions from soil, however, this effect depends on soil type where a mitigating effect of wood ash application was observed mainly in low pH soils with high soil organic matter whereas an increase in N2O emissions was observed in mineral soils that had previously received N fertilization. This study emphasises the importance of pH manipulation in regards to N2O emissions from soil.

Total RNA sequencing reveals multilevel microbial community changes and functional responses to wood ash application in agricultural and forest soil.

Recycling of wood ash from energy production may counteract soil acidification and return essential nutrients to soils. However, wood ash amendment affects soil physicochemical parameters that control composition and functional expression of the soil microbial community. Here, we applied total RNA sequencing to simultaneously assess the impact of wood ash amendment on the active soil microbial communities and the expression of functional genes from all microbial taxa. Wood ash significantly affected the taxonomic (rRNA) as well as functional (mRNA) profiles of both agricultural and forest soil. Increase in pH, electrical conductivity, dissolved organic carbon and phosphate were the most important physicochemical drivers for the observed changes. Wood ash amendment increased the relative abundance of the copiotrophic groups Chitinonophagaceae (Bacteroidetes) and Rhizobiales (Alphaproteobacteria) and resulted in higher expression of genes involved in metabolism and cell growth. Finally, total RNA sequencing allowed us to show that some groups of bacterial feeding protozoa increased concomitantly to the enhanced bacterial growth, which shows their pivotal role in the regulation of bacterial abundance in soil.

AgNO3 Sterilizes Grains of Barley (Hordeum vulgare) without Inhibiting Germination-A Necessary Tool for Plant-Microbiome Research.

To understand and manipulate the interactions between plants and microorganisms, sterile seeds are a necessity. The seed microbiome (inside and surface microorganisms) is unknown for most plant species and seed-borne microorganisms can persist and transfer to the seedling and rhizosphere, thereby obscuring the effects that purposely introduced microorganisms have on plants. This necessitates that these unidentified, seed-borne microorganisms are removed before seeds are used for studies on plant-microbiome interactions. Unfortunately, there is no single, standardized protocol for seed sterilization, hampering progress in experimental plant growth promotion and our study shows that commonly applied sterilization protocols for barley grains using H2O2, NaClO, and AgNO3 yielded insufficient sterilization. We therefore developed a sterilization protocol with AgNO3 by testing several concentrations of AgNO3 and added two additional steps: Soaking the grains in water before the sterilization and rinsing with salt water (1% (w/w) NaCl) after the sterilization. The most efficient sterilization protocol was to soak the grains, sterilize with 10% (w/w) AgNO3, and to rinse with salt water. By following those three steps, 97% of the grains had no culturable, viable microorganism after 21 days based on microscopic inspection. The protocol left small quantities of AgNO3 residue on the grain, maintained germination percentage similar to unsterilized grains, and plant biomass was unaltered. Hence, our protocol using AgNO3 can be used successfully for experiments on plant-microbiome interactions.

Bacterial feeders, the nematode Caenorhabditis elegans and the flagellate Cercomonas longicauda, have different effects on outcome of competition among the Pseudomonas biocontrol strains CHA0 and DSS73.

How bacterial feeding fauna affects colonization and survival of bacteria in soil is not well understood, which constrains the applicability of bacterial inoculants in agriculture. This study aimed to unravel how food quality of bacteria and bacterial feeders with different feeding habits (the selective feeding flagellate Cercomonas longicauda versus the non-selective feeding nematode Caenorhabditis elegans) influence the abundance of two bacteria that compete for resources in simple model communities. Microcosms consisted of either one gfp-tagged bacterial strain (Pseudomonas fluorescens DSM50090 or one of two biocontrol strains P. fluorescens CHA0 or Pseudomonas sp. DSS73) or combinations of two bacterial strains. DSM50090 is a suitable food bacterium, DSS73 is of intermediate food quality, and CHA0 is inedible to the bacterial feeders. Bacterial and protozoan cell numbers were measured by flow cytometry. In the presence of flagellates, CHA0 increased its abundance as compared to the other biocontrol strain DSS73 or to DSM50090, which were both eaten by the flagellates. In contrast, the number of CHA0 declined as compared to DSS73 when the model community was subjected to nematode predation pressure. Hence, the results suggested that the outcome of competition among bacteria depended on their ability to cope with the prevailing bacterial predator.

Species-specific effects of live roots and shoot litter on soil decomposer abundances do not forecast plant litter-nitrogen uptake.

Plant species produce litter of varying quality and differ in the quality and quantity of compounds they release from live roots, which both can induce different decomposer growth in the soil. To test whether differences in decomposer growth can forecast the amount of N species acquire from plant litter, as suggested by theory, we grew individuals of three grassland plants-Holcus lanatus, Plantago lanceolata and Lotus corniculatus-in soils into which (15)N-labelled litter of either Holcus, Plantago or Lotus was added. We measured the effects of live roots and litter of each species on soil microbes and their protozoan and nematode feeders, and to link decomposer growth and plant nutrient uptake, we measured the amount of N taken up by plants from the added litter. We hypothesised that those species that induce the highest growth of microbes, and especially that of microbial feeders, will also take up the highest amount of N from the litter. We found, however, that although numbers of bacterial-feeding Protozoa and nematodes were on average lower after addition of Holcus than Plantago or Lotus litter, N uptake was higher from Holcus litter. Further, although the effects on Protozoa and bacterial- and fungal-feeding nematodes did not differ between the live plants, litter-N uptake differed, with Holcus being the most efficient compared to Plantago and Lotus. Hence, although microbes and their feeders unquestionably control N mineralization in the soil, and their growth differs among plant species, these differences cannot predict differences in litter-N uptake among plant species. A likely reason is that for nutrient uptake, other species-specific plant traits, such as litter chemistry, root proliferation ability and competitiveness for soil N, override in significance the species-specific ability of plants to induce decomposer growth.

Bacterial and protozoan dynamics upon thawing and freezing of an active layer permafrost soil.

The active layer of soil overlaying permafrost in the Arctic is subjected to annual changes in temperature and soil chemistry, which we hypothesize to affect the overall soil microbial community. We investigated changes in soil microorganisms at different temperatures during warming and freezing of the active layer soil from Svalbard, Norway. Soil community data were obtained by direct shotgun sequencing of total extracted RNA. No changes in soil microbial communities were detected when warming from -10 to -2 °C or when freezing from -2 to -10 °C. In contrast, within a few days we observed changes when warming from -2 to +2 °C with a decrease in fungal rRNA and an increase in several OTUs belonging to Gemmatimonadetes, Bacteroidetes and Betaproteobacteria. Even more substantial changes occurred when incubating at 2 °C for 16 days, with declines in total fungal potential activity and decreases in oligotrophic members from Actinobacteria and Acidobacteria. Additionally, we detected an increase in transcriptome sequences of bacterial phyla Bacteriodetes, Firmicutes, Betaproteobacteria and Gammaproteobacteria-collectively presumed to be copiotrophic. Furthermore, we detected an increase in putative bacterivorous heterotrophic flagellates, likely due to predation upon the bacterial community via grazing. Although this grazing activity may explain relatively large changes in the bacterial community composition, no changes in total 16S rRNA gene copy number were observed and the total RNA level remained stable during the incubation. Together, these results are showing the first comprehensive ecological evaluation across prokaryotic and eukaryotic microbial communities on thawing and freezing of soil by application of the TotalRNA technique.

Successional trophic complexity and biogeographical structure of eukaryotic communities in waterworks' rapid sand filters.

As groundwater-fed waterworks clean their raw inlet water with sand filters, a variety of pro- and eukaryotic microbial communities develop on these filters. While several studies have targeted the prokaryotic sand filter communities, little is known about the eukaryotic communities, despite the obvious need for knowledge of microorganisms that get in contact with human drinking water. With a new general eukaryotic primer set (18S, V1-V3 region), we performed FLX-454 sequencing of material from 21 waterworks' sand filters varying in age (3-40 years) and geographical location on a 250 km east-west axis in Denmark, and put the data in context of their previously published prokaryotic communities. We find that filters vary highly in trophic complexity depending on age, from simple systems with bacteria and protozoa (3-6 years) to complex, mature systems with nematodes, rotifers and turbellarians as apex predators (40 years). Unlike the bacterial communities, the eukaryotic communities display a clear distance-decay relationship that predominates over environmental variations, indicating that the underlying aquifers feeding the filters harbor distinct eukaryotic communities with limited dispersal in between. Our findings have implications for waterworks' filter management, and offer a window down to the largely unexplored eukaryotic microbiology of groundwater aquifers.

Wood ash effects on growth and cadmium uptake in Deschampsia flexuosa (Wavy hair-grass).

Wood ash recycling to forests is beneficial because it regains nutrients and prevents acidification, but wood ash application is restricted due to its cadmium (Cd) content. We question if Cd in wood ash represents a problem, since decreases in Cd bioavailability due to ash-induced pH changes may counteract increased total Cd concentration. We studied effects of wood ash (0, 3, 9 and 30 t ha-1) and lime (pH increase equivalent to the wood ash treatments) on growth and Cd uptake in Deschampsia flexuosa. After four months, we measured plant biomass and Cd accumulation, and extracted Cd from the soil using three different methods; HNO3 (total), EDTA (chelator-based) and NH4NO3 (salt-based). Wood ash and lime strongly stimulated plant growth. Cd concentration in the plant tissue decreased with wood ash and lime addition, and correlated positively with the NH4NO3 extractable fraction of Cd in the soil. In contrast, HNO3 and EDTA extracted more Cd with increased wood ash application. We conclude that wood ash amendment increases soil pH, total Cd concentration, nutrient levels and stimulates plant growth. However, it does not increase Cd accumulation in D. flexuosa, as pH-driven decreases in Cd bioavailability leads to reduced plant Cd uptake. Finally, soil bioavailable Cd is best determined using NH4NO3-extraction.

Ribosomal RNA genes challenge the monophyly of the Hyalospheniidae (Amoebozoa: Arcellinida).

To date only five partial and two complete SSU rRNA gene sequences are available for the lobose testate amoebae (Arcellinida). Consequently, the phylogenetic relationships among taxa and the definition of species are still largely dependant on morphological characters of uncertain value, which causes confusion in the phylogeny, taxonomy and the debate on cosmopolitanism of free-living protists. Here we present a SSU rRNA-based phylogeny of the Hyalospheniidae including the most common species. Similar to the filose testate amoebae of the order Euglyphida the most basal clades have a terminal aperture; the ventral position of the pseudostome appears to be a derived character. Family Hyalospheniidae appears paraphyletic and is separated into three clades: (1) Heleopera sphagni, (2) Heleopera rosea and Argynnia dentistoma and (3) the rest of the species from genera Apodera, Hyalosphenia, Porosia and Nebela. Our data support the validity of morphological characters used to define species among the Hyalospheniidae and even suggest that taxa described as varieties may deserve the rank of species (e.g. N. penardiana var. minor). Finally our results suggest that the genera Hyalosphenia and Nebela are paraphyletic, and that Porosia bigibbosa branches inside the main Nebela clade.

Toxicity of cadmium and zinc to small soil protists.

Small heterotrophic protists (flagellates and naked amoebae) are very abundant in soil and play a key role in maintaining soil services. Hence, knowledge on how xenobiotics affect these organisms is essential in ecosystem management. Cadmium (Cd) is an increasing environmental issue as both industrial deposition and recycling of heavy metal rich waste products have led to Cd enrichment of soils. Evaluation of toxicity of Cd to micro-organisms is often performed using a solution of pure Cd (e.g. CdCl) in liquid culture. This approach may be highly misleading as interactions between Cd and other substances, e.g. various ions or inherent soil components often strongly modify Cd toxicity. Hence, we compared the toxic effect of Cd to small heterotrophic protists in soil microcosms and liquid culture. We also evaluated how zinc (Zn) affects Cd toxicity, as Zn usually accompanies Cd in a ratio of c. 100:1, and is known to impede Cd toxicity. In the soil microcosms, we also monitored the primary food source of the protists, i.e. culturable bacteria, and used soil respiration as a proxy of soil functioning. Finally, we examined to what extent Cd actually sorbs to soil. We found 1) that c. 103 times more Cd was required to obtain the same effect in the soil microcosms compared to the liquid culture, 2) that soil sorption explains why Cd, even though highly toxic in aqueous solutions, has very limited effect when applied to soil, and 3) (very surprisingly) that in our experimental systems Zn was as toxic as Cd. Our study suggests that Cd toxicity to soil protists will be small because most Cd in soil will be sorbed to the soil matrix and because the Zn:Cd ratio of 100:1 in most substances, incl. pollutants, will mean that lethal Zn effects will occur before Cd reaches toxic levels.

Soil protists: a fertile frontier in soil biology research.

Protists include all eukaryotes except plants, fungi and animals. They are an essential, yet often forgotten, component of the soil microbiome. Method developments have now furthered our understanding of the real taxonomic and functional diversity of soil protists. They occupy key roles in microbial foodwebs as consumers of bacteria, fungi and other small eukaryotes. As parasites of plants, animals and even of larger protists, they regulate populations and shape communities. Pathogenic forms play a major role in public health issues as human parasites, or act as agricultural pests. Predatory soil protists release nutrients enhancing plant growth. Soil protists are of key importance for our understanding of eukaryotic evolution and microbial biogeography. Soil protists are also useful in applied research as bioindicators of soil quality, as models in ecotoxicology and as potential biofertilizers and biocontrol agents. In this review, we provide an overview of the enormous morphological, taxonomical and functional diversity of soil protists, and discuss current challenges and opportunities in soil protistology. Research in soil biology would clearly benefit from incorporating more protistology alongside the study of bacteria, fungi and animals.