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In vitro compartmentalization is a technique for generating water-in-oil microdroplets to establish the genotype (DNA information)-phenotype (biomolecule function) linkage required by many biological applications. Recently, fluorinated oils have become more widely used for making microdroplets due to their better biocompatibility. However, it is difficult to perform multi-step reactions requiring the addition of reagents in water-in-fluorinated-oil microdroplets. On-chip droplet manipulation is usually used for such purposes, but it may encounter some technical issues of low throughput or time delay of reagent delivery into different microdroplets. Hence, to address the above issues, we evaluated the feasibility of employing a nanodroplets-based approach for the delivery of copper ions and peptide molecules of middle-size (2 kDa).

The diverse microbial community of activated sludge used in biological treatment systems exhibits dynamic seasonal shifts in community composition and activity. Many wastewater treatment plants in temperate/continental climates experience seasonal cold temperature nitrification failure. “Seasonal nitrification failure” is the discharge of elevated concentrations of ammonia (greater than 4 mg/liter) with treated wastewater during the winter (influent wastewater temperatures below 13°C).

Prevailing global increases in population, urbanization, and agricultural production are causing increased pressures on water resources, especially as the use of chemicals in agriculture, industry, and medicine provide new challenges for water treatment and reuse. Organohalogen compounds are persistent contaminants that often evade current wastewater treatment technologies, resulting in their accumulation in the environment and posing a serious threat to ecosystem health. Recent advances in understanding pyrogenic carbons as electron shuttling and storing materials have exposed their potential for enhancing the dehalogenation and overall degradation of organohalide contaminants in soil, sediment, surface water, and wastewater systems. Biochar is a porous carbonaceous material produced during the thermochemical decomposition of biomass feedstock in the presence of little or no oxygen (pyrolysis). Interest in biochar for application towards environmental remediation is largely based on its three distinct benefits: I) carbon sequestration to offset greenhouse gas emissions, II) adsorption of (in-) organic contaminants and nutrients, and III) a strong electron exchange capacity. Due to the innate complexity of biochar materials, several electron transfer mechanisms exist by which biochar may mediate contaminant degradation. These electron transfer pathways include electron-accepting and donating cycles through redox-active functional groups and direct electron transfer via conductive carbon matrices. These mechanisms are responsible for biochar's participation in multiple redox-driven biogeochemical transformations with proven consequences for effective organohalogen remediation. This literature review summarizes the current knowledge on the mechanisms and processes through which biochar can directly or indirectly mediate the transformation of organohalogen compounds under various environmental conditions. Perspectives and research directions for future application of biochars for targeted remediation strategies are also discussed.

Understanding spatiotemporal patterns in microbial community composition is a central goal of microbial ecology. The objective of this study was to better understand the biogeography of activated sludge microbial communities, which are important for the protection of surface water quality. Monthly samples were collected from 20 facilities (25 bioreactors) within 442 km of each other for 1 year. Microbial community composition was characterized by sequencing of PCR-amplified 16S rRNA gene fragments. Statistically significant distance decay of community similarity was observed in these bioreactors independent of clustering method (operational taxonomic units [OTUs] at 97% similarity, genus-level phylotypes) and community dissimilarity metric (Sørensen, Bray-Curtis, and weighted Unifrac). Universal colonizers (i.e., detected in all samples) and ubiquitous genus-level phylotypes (i.e., detected in every facility at least once) also exhibited a significant distance decay relationship. Variation partitioning analysis of community composition showed that environmental characteristics (temperature, influent characteristics, etc.) explained more of the variance in community composition than geographic distance did, suggesting that environmental heterogeneity is more important than dispersal limitation as a mechanism for determining microbial community composition. Distance decay relationships also became stronger with increasing distance between facilities. Seasonal variation in community composition was also observed from selected bioreactors, but there was no clear seasonal pattern in the distance decay relationships. IMPORTANCE Understanding the spatiotemporal patterns of biodiversity is a central goal of ecology. The distance decay of community similarity is one of the spatial scaling patterns observed in many forms of life, including plants, animals, and microbial communities. Municipal wastewater treatment relies on microorganisms to prevent the release of excessive quantities of nutrients and other pollutants, but relatively few studies have explored distance decay relationships in wastewater treatment bioreactors. Our results demonstrate a strong distance decay pattern in wastewater treatment bioreactors, regardless of the sequence clustering method or the community dissimilarity metric. Our results suggest that microbial communities in wastewater treatment bioreactors are not randomly assembled but rather exhibit a statistically significant spatial pattern.

The goal of this research was to use and to validate different quantitative polymerase chain reaction (qPCR) assays to quantify the pertinent microbial populations in full-scale anaerobic d...

This study evaluated the efficacy of quantitative polymerase chain reaction (qPCR) to monitor several pertinent bacterial populations in 25 different full-scale wastewater treatment bioreac...

In this study, we investigated whether bacterial community composition in full-scale wastewater treatment bioreactors can be better explained by niche- or neutral-based theory (deterministic or stochastic) and whether bioreactor design (continuous flow versus fill and draw) affected community assembly. Four wastewater treatment facilities (one with quadruplicated continuous-flow bioreactors, two with one continuous-flow bioreactor each, and one with triplicate fill-and-draw bioreactors) were investigated. Bioreactor community composition was characterized by sequencing of PCR-amplified 16S rRNA gene fragments. Replicate bioreactors at the same wastewater treatment facility had largely reproducible (i.e., deterministic) bacterial community composition, although bacterial community composition in continuous-flow bioreactors was significantly more reproducible (P

Numerous wastewater treatment processes are designed by engineers to achieve specific treatment goals. However, the impact of these different process designs on bacterial community composition is poorly understood. In this study, 24 different municipal wastewater treatment facilities (37 bioreactors) with various system designs were analyzed by sequencing of PCR-amplified 16S rRNA gene fragments. Although a core microbiome was observed in all of the bioreactors, the overall microbial community composition (analysis of molecular variance; P = 0.001) as well as that of a specific population of Nitrosomonas spp. (P = 0.04) was significantly different between A/O (anaerobic/aerobic) systems and conventional activated sludge (CAS) systems. Community α-diversity (number of observed operational taxonomic units [OTUs] and Shannon diversity index) was also significantly higher in A/O systems than in CAS systems (Wilcoxon; P

Kinetic isotope effects have been used successfully to prove and characterize organic contaminant transformation on various scales including field and laboratory studies. For tetrachloroethene (PCE...

Wastewater treatment plants in temperate climate zones frequently undergo seasonal nitrification failure in the winter month yet maintain removal efficiency for other contaminants. We tested the hypothesis that nitrification failure can be correlated to shifts in the nitrifying microbial community. We monitored three parallel, full-scale sequencing batch reactors over the course of a year with respect to reactor performance, microbial community composition via 16S rRNA gene amplicon sequencing, and functional gene abundance using qPCR. All reactors demonstrated similar changes to their core microbiome, and only subtle variations among seasonal and transient taxa. We observed a decrease in species richness during the winter, with a slow recovery of the activated sludge community during spring. Despite the change in nitrification performance, ammonia monooxygenase gene abundances remained constant throughout the year, as did the relative sequence abundance of Nitrosomonadacae. This suggests that nitrification failure at colder temperatures might result from different reaction kinetics of nitrifying taxa, or that other organisms with strong seasonal shifts in population abundance, e.g. an uncultured lineage of Saprospiraceae, affect plant performance in the winter. This research is a comprehensive analysis of the seasonal microbial community dynamics in triplicate full-scale sequencing batch reactors and ultimately strengthens our basic understanding of the microbial ecology of activated sludge communities by revealing seasonal succession patterns of individual taxa that correlate with nutrient removal efficiency.

Dehalogenation is one of the most important reactions for eliminating trace organic pollutants in natural and engineering systems. This study investigated the dehalogenation of a model organohalogen compound, triclosan (TCS), by aqueous biochars (a-BCs) ( 0.76) and suggest the first-step dechlorination as the rate-limiting step among the possible pathways. These results showcased that the reduced a-BCs can reductively degrade organohalogens with potential applications for wastewater treatment and groundwater remediation. While TCS was used as a model compound in this study, a-BC-based degradation can be likely applied to a range of redox-sensitive trace organic compounds.

Activated sludge is comprised of diverse microorganisms which remediate wastewater. Previous research has characterized activated sludge using 16S rRNA gene amplicon sequencing, which can help to address questions on the relative abundance of microorganisms. In this study, we used 16S rRNA transcript sequencing in order to characterize “active” populations (via protein synthesis potential) and gain a deeper understanding of microbial activity patterns within activated sludge. Seasonal abundances of individual populations in activated sludge change over time, yet a persistent group of core microorganisms remains throughout the year which are traditionally classified on presence or absence without monitoring of their activity or growth. The goal of this study was to further our understanding of how the activated sludge microbiome changes between seasons with respect to population abundance, activity, and growth. Triplicate sequencing batch reactors were sampled at 10-min intervals throughout reaction cycles during all four seasons. We quantified the gene and transcript copy numbers of 16S rRNA amplicons using real-time PCR and sequenced the products to reveal community abundance and activity changes. We identified 108 operational taxonomic units (OTUs) with stable abundance, activity, and growth throughout the year. Nonproliferating OTUs were commonly human health related, while OTUs that showed seasonal abundance changes have previously been identified as being associated with floc formation and bulking. We observed significant differences in 16S rRNA transcript copy numbers, particularly at lower temperatures in winter and spring. The study provides an analysis of the seasonal dynamics of microbial activity variations in activated sludge based on quantifying and sequencing 16S rRNA transcripts. IMPORTANCE Sequencing batch reactors are a common design for wastewater treatment plants, particularly in smaller municipalities, due to their low footprint and ease of operations. However, like for most treatment plants in temperate/continental climates, the microbial community involved in water treatment is highly seasonal and its biological processes can be sensitive to cold temperatures. The seasonality of these microbial communities has been explored primarily in conventional treatment plants and not in sequencing batch reactors. Furthermore, most studies often only address which organisms are present. However, the activated sludge microbial community is very diverse, and it is often hard to discern which organisms are active and which organisms are simply present. In this study, we applied additional sequencing techniques to also address the issues of which organisms are active and which organisms are growing. By addressing these issues, we gained new insights into seasonal microbial populations dynamics and activity patterns affecting wastewater treatment.

N2O is a major greenhouse gas and the majority of anthropogenic N2O emissions originate from agriculturally managed soils. Therefore, developing N2O mitigation strategies is a key challenge for the agricultural sector and biochar soil treatment is one reported option. Biochar's capacity to increase soil pH and to foster activity of specialized N2O reducers has been proposed as possible mechanisms for N2O mitigation. An experiment was undertaken to investigate whether changes in the community composition of N2O reducers was observed under field conditions after biochar application. The study objective was to assess the abundance and taxonomic composition of the functional marker genes nosZ and nosZ –II across a vegetation period of Zea mays L. after biochar or lime addition compared to an untreated control. After fertilization, biochar amendment resulted in a significant increase of nosZ gene copy numbers compared to the control and the lime treatment. Simultaneously a shift in community composition of nosZ-II bearing bacteria was observed in the biochar treatment that went beyond the sole liming effect. This study broadens our understanding of the functional impact of biochar on N2O emissions and emphasizes the possibility to shape the functioning of the N2O reducing microbial community through the addition of biochar at a field scale.

In order to ensure stable performance of engineered biotechnologies that rely on mixed microbial community systems, it is important to identify process-specific microbial traits and study their in-situ activity and responses to changing environmental conditions and system operational parameters. We used BioOrthogonal Non-Canonical Amino acid Tagging (BONCAT) in combination with Fluorescence-Activated Cell Sorting (FACS) and 16S rRNA gene amplicon sequencing to identify translationally active cells in activated sludge. We found that only a subset of the activated sludge microbiome is translationally active during the aerobic treatment phase of a full-scale sequencing batch reactor designed to enhance biological phosphorus removal from municipal wastewater. Relative abundance of amplicon sequence variants was not a reliable predictor of species activity. BONCAT-positive and -negative cells revealed a broad range of population-wide and taxa-specific translational heterogeneity. BONCAT-FACS in combination with amplicon sequencing can provide new insights into the ecophysiology of highly dynamic microbiomes in activated sludge systems.

Biochars have been recognized as an important material to improve soil properties. In a number of studies their beneficial properties have been found to increase with residence time in soil and during the composting process. The beneficial properties have been correlated with surface functional groups resulting from the interactions between char particles, inorganic and organic matter in the soil and soil biota. These interactions result in the formation of organo-mineral phases on the internal and external surfaces of the biochar. A paucity of information exists, particularly from longer-term field trials, on organo-mineral phases present on both the internal and external surfaces of the biochar. To characterize the structure of, and interface between, the carbon and mineral phases, we examined biochars recovered from two field trials and after composting from different countries using high resolution scanning electron microscopy (SEM), atomic resolution transmission electron microscopy (TEM) and scanning TEM (STEM), energy electron loss spectroscopy (EELS) and energy dispersive X-ray spectroscopy (EDS) at resolutions of 1–20 nm. The work revealed the formation of porous agglomerates of different minerals/inorganic compounds bound together with organic compounds on the surfaces of the biochar. In some cases, these agglomerates were bound together to form organo-mineral associations. The analyses also showed that the organic compounds containing both N and C functional groups and mixed valence iron oxide nanoparticles are possibly interacting with the organic compounds. The analysis also showed the formation of pores at the interface of the carbon matrix and organo-mineral aggregates.

Kinetic isotope effects have been used successfully to prove and characterize organic contaminant transformation on various scales including field and laboratory studies. For tetrachloroethene (PCE) biotransformation, however, causes for the substantial variability of reported isotope enrichment factors (ε) are still not deciphered (ε

The application of biochar as a soil amendment to improve soil fertility has been suggested as a tool to reduce soil-borne CO2 and non-CO2 greenhouse gas emissions, especially nitrous oxide (N2O). Both laboratory and field trials have demonstrated N2O emission reduction by biochar amendment, but the long-term effect (>1 year) has been questioned. Here, we present results of a combined microcosm and field study using a powdered beech wood biochar from slow pyrolysis. The field experiment showed that both CO2 and N2O emissions were still effectively reduced by biochar in the third year after application. However, biochar did not influence the biomass yield of sunflower for biogas production (Helianthus annuus L.). Biochar reduced bulk density and increased soil aeration and thus reduced the water-filled pore space (WFPS) in the field, but was also able to suppress N2O emission in the microcosms experiment conducted at constant WFPS. For both experiments, biochar had limited impact on soil mineral nitrogen speciation, but it reduced the accumulation of nitrite in the microcosms. Extraction of soil DNA and quantification of functional marker genes by quantitative polymerase chain reaction showed that biochar did not alter the abundance of nitrogen-transforming bacteria and archaea in both field and microcosm experiments. In contradiction to previous experiments, this study demonstrates the long-term N2O emission suppression potential of a wood biochar and thus highlights its overall climate change mitigation potential. While a detailed understanding of the underlying mechanisms requires further research, we provide evidence for a range of biochar-induced changes to the soil environment and their change with time that might explain the often observed N2O emission suppression.

Soil biochar amendment has been described as a promising tool to improve soil quality, sequester carbon, and mitigate nitrous oxide (N2O) emissions. N2O is a potent greenhouse gas. The main sources of N2O in soils are microbially-mediated nitrogen transformation processes such as nitrification and denitrification. While previous studies have focused on the link between N2O emission mitigation and the abundance and activity of N2O-reducing microorganisms in biochar-amended soils, the impact of biochar on the taxonomic composition of the nosZ gene carrying soil microbial community has not been subject of systematic study to date. We used 454 pyrosequencing in order to study the microbial diversity in biochar-amended and biochar-free soil microcosms. We sequenced bacterial 16S rRNA gene amplicons as well as fragments of common (typical) nosZ genes and the recently described ‘atypical’ nosZ genes. The aim was to describe biochar-induced shifts in general bacterial community diversity and taxonomic variations among the nosZ gene containing N2O-reducing microbial communities. While soil biochar amendment significantly altered the 16S rRNA gene-based community composition and structure, it also led to the development of distinct functional traits capable of N2O reduction containing typical and atypical nosZ genes related to nosZ genes found in Pseudomonas stutzeri and Pedobacter saltans, respectively. Our results showed that biochar amendment can affect the relative abundance and taxonomic composition of N2O-reducing functional microbial traits in soil. Thus these findings broaden our knowledge on the impact of biochar on soil microbial community composition and nitrogen cycling.

Nitrate-dependent ferrous iron [Fe(II)] oxidation (NDFO) is a well-recognized chemolithotrophic pathway in anoxic sediments. The neutrophilic chemolithoautotrophic enrichment culture KS originally obtained from a freshwater sediment (K. L. Straub, M. Benz, B. Schink, and F. Widdel, Appl Environ Microbiol 62:1458–1460, 1996) has been used as a model system to study NDFO. However, the primary Fe(II) oxidizer in this culture has not been isolated, despite extensive efforts to do so. Here, we present a metagenomic analysis of this enrichment culture in order to gain insight into electron transfer pathways and the roles of different bacteria in the culture. We obtained a near-complete genome of the primary Fe(II) oxidizer, a species in the family Gallionellaceae , and draft genomes from its flanking community members. A search of the putative extracellular electron transfer pathways in these genomes led to the identification of a homolog of the MtoAB complex [a porin-multiheme cytochrome c system identified in neutrophilic microaerobic Fe(II)-oxidizing Sideroxydans lithotrophicus ES-1] in a Gallionellaceae sp., and findings of other putative genes involving cytochrome c and multicopper oxidases, such as Cyc2 and OmpB. Genome-enabled metabolic reconstruction revealed that this Gallionellaceae sp. lacks nitric oxide and nitrous oxide reductase genes and may partner with flanking populations capable of complete denitrification to avoid toxic metabolite accumulation, which may explain its resistance to growth in pure culture. This and other revealed interspecies interactions and metabolic interdependencies in nitrogen and carbon metabolisms may allow these organisms to cooperate effectively to achieve robust chemolithoautotrophic NDFO. Overall, the results significantly expand our knowledge of NDFO and suggest a range of genetic targets for further exploration.

Iron is abundant in sediments, where it can be biogeochemically cycled between its divalent and trivalent redox states. The neutrophilic microbiological Fe cycle involves Fe(III)-reducing and three different physiological groups of Fe(II)-oxidizing microorganisms, i.e., microaerophilic, anoxygenic phototrophic, and nitrate-reducing Fe(II) oxidizers. However, it is unknown whether all three groups coexist in one habitat and how they are spatially distributed in relation to gradients of O 2 , light, nitrate, and Fe(II). We examined two coastal marine sediments in Aarhus Bay, Denmark, by cultivation and most probable number (MPN) studies for Fe(II) oxidizers and Fe(III) reducers and by quantitative-PCR (qPCR) assays for microaerophilic Fe(II) oxidizers. Our results demonstrate the coexistence of all three metabolic types of Fe(II) oxidizers and Fe(III) reducers. In qPCR, microaerophilic Fe(II) oxidizers ( Zetaproteobacteria ) were present with up to 3.2 × 10 6 cells g dry sediment −1 . In MPNs, nitrate-reducing Fe(II) oxidizers, anoxygenic phototrophic Fe(II) oxidizers, and Fe(III) reducers reached cell numbers of up to 3.5 × 10 4 , 3.1 × 10 2 , and 4.4 × 10 4 g dry sediment −1 , respectively. O 2 and light penetrated only a few millimeters, but the depth distribution of the different iron metabolizers did not correlate with the profile of O 2 , Fe(II), or light. Instead, abundances were homogeneous within the upper 3 cm of the sediment, probably due to wave-induced sediment reworking and bioturbation. In microaerophilic Fe(II)-oxidizing enrichment cultures, strains belonging to the Zetaproteobacteria were identified. Photoferrotrophic enrichments contained strains related to Chlorobium and Rhodobacter ; the nitrate-reducing Fe(II) enrichments contained strains related to Hoeflea and Denitromonas . This study shows the coexistence of all three types of Fe(II) oxidizers in two near-shore marine environments and the potential for competition and interrelationships between them.

The accumulation of the widely-used antibacterial and antifungal compound triclosan (TCS) in freshwaters raises concerns about the impact of this harmful chemical on the biofilms that are the dominant life style of microorganisms in aquatic systems. However, investigations to-date rarely go beyond effects at the cellular, physiological or morphological level. The present paper focuses on bacterial biofilms addressing the possible chemical impairment of their functionality, while also examining their substratum stabilization potential as one example of an important ecosystem service. The development of a bacterial assemblage of natural composition – isolated from sediments of the Eden Estuary (Scotland, UK) – on non-cohesive glass beads (

The enrichment culture KS is one of the few existing autotrophic, nitrate-reducing, Fe(II)-oxidizing cultures that can be continuously transferred without an organic carbon source. We used a combination of catalyzed amplification reporter deposition fluorescence in situ hybridization (CARD-FISH) and nanoscale secondary ion mass spectrometry (NanoSIMS) to analyze community dynamics, single-cell activities, and interactions among the two most abundant microbial community members (i.e., Gallionellaceae sp. and Bradyrhizobium spp.) under autotrophic and heterotrophic growth conditions. CARD-FISH cell counts showed the dominance of the Fe(II) oxidizer Gallionellaceae sp. under autotrophic conditions as well as of Bradyrhizobium spp. under heterotrophic conditions. We used NanoSIMS to monitor the fate of 13 C-labeled bicarbonate and acetate as well as 15 N-labeled ammonium at the single-cell level for both taxa. Under autotrophic conditions, only the Gallionellaceae sp. was actively incorporating 13 C-labeled bicarbonate and 15 N-labeled ammonium. Interestingly, both Bradyrhizobium spp. and Gallionellaceae sp. became enriched in [ 13 C]acetate and [ 15 N]ammonium under heterotrophic conditions. Our experiments demonstrated that Gallionellaceae sp. was capable of assimilating [ 13 C]acetate while Bradyrhizobium spp. were not able to fix CO 2 , although a metagenomics survey of culture KS recently revealed that Gallionellaceae sp. lacks genes for acetate uptake and that the Bradyrhizobium sp. carries the genetic potential to fix CO 2 . The study furthermore extends our understanding of the microbial reactions that interlink the nitrogen and Fe cycles in the environment. IMPORTANCE Microbial mechanisms by which Fe(II) is oxidized with nitrate as the terminal electron acceptor are generally referred to as “nitrate-dependent Fe(II) oxidation” (NDFO). NDFO has been demonstrated in laboratory cultures (such as the one studied in this work) and in a variety of marine and freshwater sediments. Recently, the importance of NDFO for the transport of sediment-derived Fe in aquatic ecosystems has been emphasized in a series of studies discussing the impact of NDFO for sedimentary nutrient cycling and redox dynamics in marine and freshwater environments. In this article, we report results from an isotope labeling study performed with the autotrophic, nitrate-reducing, Fe(II)-oxidizing enrichment culture KS, which was first described by Straub et al. (1) about 20 years ago. Our current study builds on the recently published metagenome of culture KS (2).

Most isolated nitrate-reducing Fe(II)-oxidizing microorganisms are mixotrophic, meaning that Fe(II) is chemically oxidized by nitrite that forms during heterotrophic denitrification, and it is debated to which extent Fe(II) is enzymatically oxidized. One exception is the chemolithoautotrophic enrichment culture KS, a consortium consisting of a dominant Fe(II) oxidizer, Gallionellaceae sp., and less abundant heterotrophic strains (e.g., Bradyrhizobium sp., Nocardioides sp.). Currently, this is the only nitrate-reducing Fe(II)-oxidizing culture for which autotrophic growth has been demonstrated convincingly for many transfers over more than 2 decades. We used 16S rRNA gene amplicon sequencing and physiological growth experiments to analyze the community composition and dynamics of culture KS with various electron donors and acceptors. Under autotrophic conditions, an operational taxonomic unit (OTU) related to known microaerophilic Fe(II) oxidizers within the family Gallionellaceae dominated culture KS. With acetate as an electron donor, most 16S rRNA gene sequences were affiliated with Bradyrhizobium sp. Gallionellaceae sp. not only was able to oxidize Fe(II) under autotrophic and mixotrophic conditions but also survived over several transfers of the culture on only acetate, although it then lost the ability to oxidize Fe(II). Bradyrhizobium spp. became and remained dominant when culture KS was cultivated for only one transfer under heterotrophic conditions, even when conditions were reverted back to autotrophic in the next transfer. This study showed a dynamic microbial community in culture KS that responded to changing substrate conditions, opening up questions regarding carbon cross-feeding, metabolic flexibility of the individual strains in KS, and the mechanism of Fe(II) oxidation by a microaerophile in the absence of O 2 . IMPORTANCE Nitrate-reducing Fe(II)-oxidizing microorganisms are present in aquifers, soils, and marine and freshwater sediments. Most nitrate-reducing Fe(II) oxidizers known are mixotrophic, meaning that they need organic carbon to continuously oxidize Fe(II) and grow. In these microbes, Fe(II) was suggested to be chemically oxidized by nitrite that forms during heterotrophic denitrification, and it remains unclear whether or to what extent Fe(II) is enzymatically oxidized. In contrast, the enrichment culture KS was shown to oxidize Fe(II) autotrophically coupled to nitrate reduction. This culture contains the designated Fe(II) oxidizer Gallionellaceae sp. and several heterotrophic strains (e.g., Bradyrhizobium sp.). We showed that culture KS is able to metabolize Fe(II) and a variety of organic substrates and is able to adapt to dynamic environmental conditions. When the community composition changed and Bradyrhizobium became the dominant community member, Fe(II) was still oxidized by Gallionellaceae sp., even when culture KS was cultivated with acetate/nitrate [Fe(II) free] before being switched back to Fe(II)/nitrate.

Little is known about the potential activity of microbial communities in hypersaline sediment ecosystems. Ribosomal tag libraries of DNA and RNA extracted from the sediment of Lake Strawbridge (Western Australia) revealed bacterial and archaeal operational taxonomic units (OTUs) with high RNA/DNA ratios providing evidence for the presence of ‘rare’ but potentially “active” taxa. Among the ‘rare’ bacterial taxa Halomonas, Salinivibrio and Idiomarina showed the highest protein synthesis potential. Rare but ‘active’ archaeal OTUs were related to the KTK 4A cluster and the Marine-Benthic-Groups B and D. We present the first molecular analysis of the microbial diversity and protein synthesis potential of rare microbial taxa in a hypersaline sediment ecosystem.

In previous studies on microbial ferric iron (Fe(III)) reduction varying results regarding reduction rates and secondary mineral formation have been reported for almost identical conditions regarding temperature, pH, medium composition, Fe(III) mineral identity and bulk iron concentration. Here we show that in addition to physico-chemical parameters also geometric aspects, i.e., incubation orientation and dimension of cultivation vessels, influence the reduction rates and mineralogy. We incubated the Fe(III)-reducer Shewanella oneidensis MR-1 in test tubes at ferrihydrite (FH) concentrations of 1.3–50 mM either in vertical or horizontal orientation. Cells and minerals formed a pellet at the bottom of the tubes with different thicknesses at the same initial FH concentration depending on the incubation orientation. In vertically incubated tubes thick FH pellets were present at the bottom of the tubes and magnetite was formed in all setups with ≥2.5 mM initial FH. In tubes that were incubated horizontally no...

The remediation of metal-contaminated soils by phytoextraction depends on plant growth and plant metal accessibility. Soil microorganisms can affect the accumulation of metals by plants either by directly or indirectly stimulating plant growth and activity or by (im)mobilizing and/or complexing metals. Understanding the intricate interplay of metal-accumulating plants with their rhizosphere microbiome is an important step toward the application and optimization of phytoremediation. We compared the effects of a “native” and a strongly disturbed (gamma-irradiated) soil microbial communities on cadmium and zinc accumulation by the plant Arabidopsis halleri in soil microcosm experiments. A. halleri accumulated 100% more cadmium and 15% more zinc when grown on the untreated than on the gamma-irradiated soil. Gamma irradiation affected neither plant growth nor the 1 M HCl-extractable metal content of the soil. However, it strongly altered the soil microbial community composition and overall cell numbers. Pyrosequencing of 16S rRNA gene amplicons of DNA extracted from rhizosphere samples of A. halleri identified microbial taxa ( Lysobacter , Streptomyces , Agromyces , Nitrospira , “ Candidatus Chloracidobacterium”) of higher relative sequence abundance in the rhizospheres of A. halleri plants grown on untreated than on gamma-irradiated soil, leading to hypotheses on their potential effect on plant metal uptake. However, further experimental evidence is required, and wherefore we discuss different mechanisms of interaction of A. halleri with its rhizosphere microbiome that might have directly or indirectly affected plant metal accumulation. Deciphering the complex interactions between A. halleri and individual microbial taxa will help to further develop soil metal phytoextraction as an efficient and sustainable remediation strategy.

Amending soil with biochar (pyrolized biomass) is suggested as a globally applicable approach to address climate change and soil degradation by carbon sequestration, reducing soil-borne greenhouse-gas emissions and increasing soil nutrient retention. Biochar was shown to promote plant growth, especially when combined with nutrient-rich organic matter, e.g., co-composted biochar. Plant growth promotion was explained by slow release of nutrients, although a mechanistic understanding of nutrient storage in biochar is missing. Here we identify a complex, nutrient-rich organic coating on co-composted biochar that covers the outer and inner (pore) surfaces of biochar particles using high-resolution spectro(micro)scopy and mass spectrometry. Fast field cycling nuclear magnetic resonance, electrochemical analysis and gas adsorption demonstrated that this coating adds hydrophilicity, redox-active moieties, and additional mesoporosity, which strengthens biochar-water interactions and thus enhances nutrient retention. This implies that the functioning of biochar in soil is determined by the formation of an organic coating, rather than biochar surface oxidation, as previously suggested., Biochar promotes plant growth via a slow release of nutrients; however, a mechanistic understanding of nutrient storage in biochar is lacking. Here, using high-resolution spectromicroscopy and mass spectrometry, the authors identify an organic coating on co-composted particles that enhances nutrient retention.

The enrichment culture KS is one of the few existing autotrophic, nitrate-reducing, Fe(II)-oxidizing cultures that can be continuously transferred without an organic carbon source. We used a combination of catalyzed amplification reporter deposition fluorescence in situ hybridization (CARD-FISH) and nanoscale secondary ion mass spectrometry (NanoSIMS) to analyze community dynamics, single-cell activities, and interactions among the two most abundant microbial community members (i.e., Gallionellaceae sp. and Bradyrhizobium spp.) under autotrophic and heterotrophic growth conditions. CARD-FISH cell counts showed the dominance of the Fe(II) oxidizer Gallionellaceae sp. under autotrophic conditions as well as of Bradyrhizobium spp. under heterotrophic conditions. We used NanoSIMS to monitor the fate of 13C-labeled bicarbonate and acetate as well as 15N-labeled ammonium at the single-cell level for both taxa. Under autotrophic conditions, only the Gallionellaceae sp. was actively incorporating 13C-labeled bicarbonate and 15N-labeled ammonium. Interestingly, both Bradyrhizobium spp. and Gallionellaceae sp. became enriched in [13C]acetate and [15N]ammonium under heterotrophic conditions. Our experiments demonstrated that Gallionellaceae sp. was capable of assimilating [13C]acetate while Bradyrhizobium spp. were not able to fix CO2, although a metagenomics survey of culture KS recently revealed that Gallionellaceae sp. lacks genes for acetate uptake and that the Bradyrhizobium sp. carries the genetic potential to fix CO2. The study furthermore extends our understanding of the microbial reactions that interlink the nitrogen and Fe cycles in the environment.

Most described nitrate-reducing Fe(II)-oxidizing bacteria (NRFeOB) are mixotrophic and depend on organic cosubstrates for growth. Encrustation of cells in Fe(III) minerals has been observed for mixotrophic NRFeOB but not for autotrophic phototrophic and microaerophilic Fe(II) oxidizers. So far, little is known about cell-mineral associations in the few existing autotrophic NRFeOB. Here, we investigate whether the designated autotrophic Fe(II)-oxidizing strain (closely related to Gallionella and Sideroxydans ) or the heterotrophic nitrate reducers that are present in the autotrophic nitrate-reducing Fe(II)-oxidizing enrichment culture KS form mineral crusts during Fe(II) oxidation under autotrophic and mixotrophic conditions. In the mixed culture, we found no significant encrustation of any of the cells both during autotrophic oxidation of 8 to 10 mM Fe(II) coupled to nitrate reduction and during cultivation under mixotrophic conditions with 8 to 10 mM Fe(II), 5 mM acetate, and 4 mM nitrate, where higher numbers of heterotrophic nitrate reducers were present. Two pure cultures of heterotrophic nitrate reducers ( Nocardioides and Rhodanobacter ) isolated from culture KS were analyzed under mixotrophic growth conditions. We found green rust formation, no cell encrustation, and only a few mineral particles on some cell surfaces with 5 mM Fe(II) and some encrustation with 10 mM Fe(II). Our findings suggest that enzymatic, autotrophic Fe(II) oxidation coupled to nitrate reduction forms poorly crystalline Fe(III) oxyhydroxides and proceeds without cellular encrustation while indirect Fe(II) oxidation via heterotrophic nitrate-reduction-derived nitrite can lead to green rust as an intermediate mineral and significant cell encrustation. The extent of encrustation caused by indirect Fe(II) oxidation by reactive nitrogen species depends on Fe(II) concentrations and is probably negligible under environmental conditions in most habitats. IMPORTANCE Most described nitrate-reducing Fe(II)-oxidizing bacteria (NRFeOB) are mixotrophic (their growth depends on organic cosubstrates) and can become encrusted in Fe(III) minerals. Encrustation is expected to be harmful and poses a threat to cells if it also occurs under environmentally relevant conditions. Nitrite produced during heterotrophic denitrification reacts with Fe(II) abiotically and is probably the reason for encrustation in mixotrophic NRFeOB. Little is known about cell-mineral associations in autotrophic NRFeOB such as the enrichment culture KS. Here, we show that no encrustation occurs in culture KS under autotrophic and mixotrophic conditions while heterotrophic nitrate-reducing isolates from culture KS become encrusted. These findings support the hypothesis that encrustation in mixotrophic cultures is caused by the abiotic reaction of Fe(II) with nitrite and provide evidence that Fe(II) oxidation in culture KS is enzymatic. Furthermore, we show that the extent of encrustation caused by indirect Fe(II) oxidation by reactive nitrogen species depends on Fe(II) concentrations and is probably negligible in most environmental habitats.

Microbial nitrogen transformation processes such as denitrification represent major sources of the potent greenhouse gas nitrous oxide (N2O). Soil biochar amendment has been shown to significantly decrease N2O emissions in various soils. However, the effect of biochar on the structure and function of microbial communities that actively perform nitrogen redox transformations has not been studied in detail yet. To analyse the community composition of actively denitrifying and N2O-reducing microbial communities, we collected RNA samples at different time points from a soil microcosm experiment conducted under denitrifying conditions and performed Illumina amplicon sequencing targeting nirK, typical nosZ and atypical nosZ mRNA transcripts. Within 10 days, biochar significantly increased the diversity of nirK and typical nosZ transcripts and resulted in taxonomic shifts among the typical nosZ-expressing microbial community. Furthermore, biochar addition led to a significant increase in transcript production among microbial species that are specialized on direct N2O reduction from the environment. Our results point towards a potential coupling of biochar-induced N2O emission reduction and an increase in microbial N2O reduction activity among specific groups of typical and atypical N2O reducers. However, experiments with other soils and biochars will be required to verify the transferability of these findings to other soil-biochar systems.

Recent studies have reported the formation of Cu0 nanoparticles (CuNP) by suspended bacteria in pore water of periodically flooded soils, but the bacteria have not yet been identified. The aim of this study was to identify the CuNP-forming bacteria and to determine the location of CuNP formation relative to the bacterial cell surface. Electron microscopy revealed that the bacteria were rod-shaped spore formers and suggested that CuNP were formed in the periplasm. Combined results from denaturing gradient gel electrophoresis, 16S rRNA gene clone libraries, and classic microbiological cultivation techniques provided strong evidence for a Clostridium sp. strain as the CuNP-forming bacteria. Clostridia are well-adapted to frequent flooding and drying due to their ability to form spores and may play an important role in Cu cycling and metallic Cu formation in redox-dynamic environments.

The application of culture-dependent studies to quantify Fe-metabolizing microorganisms from the environment is a necessity, as there are so far no universal functional marker genes for application in culture-independent studies. Media composition can vary between studies, therefore, we determined the effects of three different growth media on the quantification (MPNs) and identity (via cloning and sequencing of dominant DGGE bands) of nitrate-reducing Fe(II)-oxidizers and lactate- or acetate-oxidizing Fe(III)-reducers from a lacustrine sediment: low sulphate freshwater medium (FWM), sterile filtered bicarbonate-buffered lake water (BLW) and a mixture of both (MIX). We consistently found fewer cells in the BLW than in the FWM and the MIX. The DGGE banding patterns of the microbial communities enriched in different media types clustered together according to the e− donor and acceptor couples and not according to the medium used. Thus, although the medium composition significantly influenced the quantificat...

The Fe and N biogeochemical cycles play key roles in freshwater environments. We aimed to determine the spatial positioning and interconnections of the N and Fe cycles in profundal lake sediments. The gradients of O2, NO3(-), NH4(+), pH, Eh, Fe(II) and Fe(III) were determined and the distribution of microorganisms was assessed by most probable numbers and quantitative polymerase chain reaction. The redox zones could be divided into an oxic zone (0-8 mm), where microaerophiles (Gallionellaceae) were most abundant at a depth of 7 mm. This was followed by a denitrification zone (6-12 mm), where NO3(-)-dependent Fe(II) oxidizers and organoheterotrophic denitrifiers both reduce nitrate. Lastly, an iron redox transition zone was identified at 12.5-22.5 mm. Fe(III) was most abundant above this zone while Fe(II) was most abundant beneath. The high abundance of poorly crystalline iron suggested iron cycling. The Fe and N cycles are biologically connected through nitrate-reducing Fe(II) oxidizers and chemically by NOx(-) species formed during denitrification, which can chemically oxidize Fe(II). This study combines high resolution chemical, molecular and microbiological data to pinpoint sedimentary redox zones in which Fe is cycled between Fe(II) and Fe(III) and where Fe and N-redox processes interact.

Biochar influences soil fertility, N2O emissions, and atmospheric CO2 budgets, and because of its quinone and aromatic structures, it is redox-active. Here we demonstrate that biochar concentrations of 5 and 10 g L–1 stimulate both the rate and the extent of microbial reduction of the Fe(III) oxyhydroxide mineral ferrihydrite (15 mM) by Shewanella oneidensis MR-1, while lower biochar concentrations (0.5 and 1 g L–1) have a negative effect on ferrihydrite reduction. Control experiments showed that biochar particles and not biochar-derived water-soluble organic compounds are responsible for the stimulating and inhibiting effect. We also found that biochar changed the mineral product of ferrihydrite reduction from magnetite (Fe3O4) to siderite (FeCO3). Our study suggests that biochar can influence soil biogeochemistry not only indirectly by changing the soil structure and chemistry but also by directly mediating electron transfer processes, i.e., by functioning as an electron shuttle.

Volatile halogenated organic compounds (VOX) contribute to ozone depletion and global warming. There is evidence of natural VOX formation in many environments ranging from forest soils to salt lakes. Laboratory studies have suggested that VOX formation can be chemically stimulated by reactive Fe species while field studies have provided evidence for direct biological (enzymatic) VOX formation. However, the relative contribution of abiotic and biotic processes to global VOX budgets is still unclear. The goals of this study were to quantify VOX release from sediments from a hypersaline lake in Western Australia (Lake Strawbridge) and to distinguish between the relative contributions of biotic and abiotic VOX formation in microbially active and sterilized microcosms. Our experiments demonstrated that the release of organochlorines from Lake Strawbridge sediments was mainly biotic. Among the organochlorines detected were monochlorinated, e.g., chloromethane (CH3Cl), and higher chlorinated VOX compounds such as trichloromethane (CHCl3). Amendment of sediments with either Fe(III) oxyhydroxide (ferrihydrite) or a mixture of lactate/acetate or both ferrihydrite and lactate/acetate did not stimulate VOX formation. This suggests that although microbial Fe(III) reduction took place, there was no stimulation of VOX formation via Fe redox transformations or the formation of reactive Fe species under our experimental conditions.

Microorganisms are known to couple the degradation of hydrocarbons to Fe(III) reduction leading to the dissolution and (trans)formation of Fe minerals including ferro(i)magnetic Fe minerals such as magnetite. The screening of soil magnetic properties, in particular magnetic susceptibility (MS), has the potential to assist in locating and assessing hydrocarbon (e.g. gasoline) contamination in the environment. In order to evaluate this, it must be understood how changes in soil geochemistry and hydrocarbon input impact MS. To this end, we incubated microcosms with soils from six different field sites anoxically and followed the changes in soil MS. In parallel we simulated hydrocarbon (i.e., gasoline) contamination in the same soils under anoxic conditions. We found that in microbially active microcosms both with or without added gasoline, average changes in MS of 6.9 ± 2.6% occurred, whereas in sterile controls the changes were less than 2.5% demonstrating that microbial metabolism played a major role in the (trans)formation of ferro(i)magnetic minerals. The microcosms reached stable MS values after a few weeks to months in four out of the six soils showing an increase in MS while in two soils the MS decreased over time. After stable MS values were reached, further addition of labile organic carbon (i.e., lactate/acetate) did not lead to further changes in MS, but the addition of Fe(III) oxyhydroxides (ferrihydrite) led to increases in MS suggesting that the changes in MS were limited by bioavailable Fe and not by bioavailable organic carbon. In the control experiments without carbon amendment, we observed that natural organic matter was mobilized from the soil matrix by water or microbial growth medium (0.33-0.47 mL/g field moist soil) added to the microcosms, and that this mobilized organic matter also stimulated microbial Fe metabolism and thus also led to a microbially driven change in MS. This study shows that changes in MS after an increase of the amount of bioavailable organic carbon can occur in a variety of soils. It also suggests that whether MS increases or decreases depends on the initial MS of the soil and the extent of the MS change seems to depend upon the amount of bioavailable Fe(III).

Fe(III) (oxyhydr)oxides affect the mobility of contaminants in the environment by providing reactive surfaces for sorption. This includes the toxic metal cadmium (Cd), which prevails in agricultural soils and is taken up by crops. Fe(III)-reducing bacteria can mobilize such contaminants by Fe(III) mineral dissolution or immobilize them by sorption to or coprecipitation with secondary Fe minerals. To date, not much is known about the fate of Fe(III) mineral-associated Cd during microbial Fe(III) reduction. Here, we describe the isolation of a new Geobacter sp. strain Cd1 from a Cd-contaminated field site, where the strain accounts for 10(4) cells g(-1) dry soil. Strain Cd1 reduces the poorly crystalline Fe(III) oxyhydroxide ferrihydrite in the presence of at least up to 112 mg Cd L(-1). During initial microbial reduction of Cd-loaded ferrihydrite, sorbed Cd was mobilized. However, during continuous microbial Fe(III) reduction, Cd was immobilized by sorption to and/or coprecipitation within newly formed secondary minerals that contained Ca, Fe, and carbonate, implying the formation of an otavite-siderite-calcite (CdCO3-FeCO3-CaCO3) mixed mineral phase. Our data shows that microbially mediated turnover of Fe minerals affects the mobility of Cd in soils, potentially altering the dynamics of Cd uptake into food or phyto-remediating plants.

Surface sediments at the former military base Hradcany, Czech Republic, heavily contaminated with hydrocarbons, were remediated over years by air-sparging. The sediments show a strong magnetic enhancement at the groundwater fluctuation zone. Here we describe the isolation of a new Fe(III)-reducing and magnetite-producing bacterial strain Geothrix fermentans HradG1from this magnetic and redox-dynamic layer. This isolation underlines that the genus Geothrix is a relevant group of bacteria in hydrocarbon-contaminated environments that undergo dynamic oxic-anoxic redox fluctuations. The Fe(III)-reducing metabolic activity of these organisms potentially leads to changing magnetic soil properties that can potentially be used to identify biogeochemical hotspots.

10 páginas.-- 7 figuras.-- 4 tablas.-- 56 referencias.-- Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.scitotenv.2017.08.161, Biochar, a material defined as charred organic matter applied in agriculture, is suggested as a beneficial additive and bulking agent in composting. Biochar addition to the composting feedstock was shown to reduce greenhouse gas emissions and nutrient leaching during the composting process, and to result in a fertilizer and plant growth medium that is superior to non-amended composts. However, the impact of biochar on the quality and carbon speciation of the organic matter in bulk compost has so far not been the focus of systematic analyses, although these parameters are key to determine the long-term stability and carbon sequestration potential of biochar-amended composts in soil. In this study, we used different spectroscopic techniques to compare the organic carbon speciation of manure compost amended with three different biochars. A non-biochar-amended compost served as control. Based on Fourier-transformed infrared (FTIR) and 13C nuclear magnetic resonance (NMR) spectroscopy we did not observe any differences in carbon speciation of the bulk compost independent of biochar type, despite a change in the FTIR absorbance ratio 2925 cm− 1/1034 cm− 1, that is suggested as an indicator for compost maturity. Specific UV absorbance (SUVA) and emission-excitation matrixes (EEM) revealed minor differences in the extractable carbon fractions, which only accounted for ~ 2–3% of total organic carbon. Increased total organic carbon content of biochar-amended composts was only due to the addition of biochar-C and not enhanced preservation of compost feedstock-C. Our results suggest that biochars do not alter the carbon speciation in compost organic matter under conditions optimized for aerobic decomposition of compost feedstock. Considering the effects of biochar on compost nutrient retention, mitigation of greenhouse gas emissions and carbon sequestration, biochar addition during aerobic composting of manure might be an attractive strategy to produce a sustainable, slow release fertilizer., The Spanish Ministry of Economy and Competitiveness (MINECO) for his “Ramón y Cajal” post-doctoral contract (RYC-2014-16338). MINECO is also thanked for the financial support of the projects CGL2015-64811-P and CGL2016-76498-R, which funded the NMR analyses. NH was financially supported by a BMBF PhD scholarship provided by the Rosa Luxemburg Foundation, Berlin, Germany. The composts were produced during a “Short Term Scientific Mission” (STSM) granted to NH by the EU COST Action TD1107 “Biochar as option for sustainable resource management”.

In soils halogens (fluorine, chlorine, bromine, iodine) are cycled through the transformation of inorganic halides into organohalogen compounds and vice versa. There is evidence that these reactions are microbially driven but the key enzymes and groups of microorganisms involved are largely unknown. Our aim was to uncover the diversity, abundance and distribution of genes encoding for halogenating and dehalogenating enzymes in a German forest soil by shotgun metagenomic sequencing. Metagenomic libraries of three soil horizons revealed the presence of genera known to be involved in halogenation and dehalogenation processes such as Bradyrhizobium or Pseudomonas. We detected a so far unknown diversity of genes encoding for (de)halogenating enzymes in the soil metagenome including specific and unspecific halogenases as well as metabolic and cometabolic dehalogenases. Genes for non-heme, no-metal chloroperoxidases and haloalkane dehalogenases were the most abundant halogenase and dehalogenase genes, respectively. The high diversity and abundance of (de)halogenating enzymes suggests a strong microbial contribution to natural halogen cycling. This was also confirmed in microcosm experiments in which we quantified the biotic formation of chloroform and bromoform. Knowledge on microorganisms and genes that catalyze (de)halogenation reactions is critical because they are highly relevant to industrial biotechnologies and bioremediation applications.

Hypersaline lakes are characteristic for Western Australia and display a rare combination of geochemical and mineralogical properties that make these lakes potential analogues for past conditions on Mars. In our study, we focused on the geochemistry and mineralogy of Lake Orr and Lake Whurr. While both lakes are poor in organic carbon (1%), the sediments' pH values differ and range from 3.8 to 4.8 in Lake Orr and from 5.4 to 6.3 in Lake Whurr sediments. Lake Whurr sediments were dominated by orange and red sediment zones in which the main Fe minerals were identified as hematite, goethite, and tentatively jarosite and pyrite. Lake Orr was dominated by brownish and blackish sediments where the main Fe minerals were goethite and another paramagnetic Fe(III)-phase that could not be identified. Furthermore, a likely secondary Fe(II)-phase was observed in Lake Orr sediments. The mineralogy of these two salt lakes in the sampling area is strongly influenced by events such as flooding, evaporation, and desiccation, processes that explain at least to some extent the observed differences between Lake Orr and Lake Whurr. The iron mineralogy of Lake Whurr sediments and the high salinity make this lake a suitable analogue for Meridiani Planum on Mars, and in particular the tentative identification of pyrite in Lake Whurr sediments has implications for the interpretation of the Fe mineralogy of Meridiani Planum sediments.Western Australia-Salt lakes-Jarosite-Hematite-Pyrite-Mars analogue. Astrobiology 16, 525-538.

The extreme osmotic conditions prevailing in hypersaline environments result in decreasing metabolic diversity with increasing salinity. Various microbial metabolisms have been shown to occur even at high salinity, including photosynthesis as well as sulfate and nitrate reduction. However, information about anaerobic microbial iron metabolism in hypersaline environments is scarce. We studied the phylogenetic diversity, distribution, and metabolic activity of iron(II)-oxidizing and iron(III)-reducing Bacteria and Archaea in pH-neutral, iron-rich salt lake sediments (Lake Kasin, southern Russia; salinity, 348.6 g liter −1 ) using a combination of culture-dependent and -independent techniques. 16S rRNA gene clone libraries for Bacteria and Archaea revealed a microbial community composition typical for hypersaline sediments. Most-probable-number counts confirmed the presence of 4.26 × 10 2 to 8.32 × 10 3 iron(II)-oxidizing Bacteria and 4.16 × 10 2 to 2.13 × 10 3 iron(III)-reducing microorganisms per gram dry sediment. Microbial iron(III) reduction was detected in the presence of 5 M NaCl, extending the natural habitat boundaries for this important microbial process. Quantitative real-time PCR showed that 16S rRNA gene copy numbers of total Bacteria , total Archaea , and species dominating the iron(III)-reducing enrichment cultures (relatives of Halobaculum gomorrense , Desulfosporosinus lacus , and members of the Bacilli ) were highest in an iron oxide-rich sediment layer. Combined with the presented geochemical and mineralogical data, our findings suggest the presence of an active microbial iron cycle at salt concentrations close to the solubility limit of NaCl.

Summary Environmental microbiology research increasingly focuses on the single microbial cell as the defining entity that drives environmental processes. The interactions of individual microbial cells with each other, the environment and with higher organisms shape microbial communities and control the functioning of whole ecosystems. A single-cell view of microorganisms in their natural environment requires analytical tools that measure both cell function and chemical speciation at the submicrometre scale. Here we review the technical capabilities and limitations of high-resolution secondary ion mass spectrometry (NanoSIMS) and scanning transmission (soft) X-ray microscopy (STXM) and give examples of their applications. Whereas NanoSIMS can be combined with isotope-labelling, thereby localizing the distribution of cellular activities (e.g. carbon/nitrogen fixation/turnover), STXM provides information on the location and chemical speciation of metabolites and products of redox reactions. We propose the combined use of both techniques and discuss the technical challenges of their joint application. Both techniques have the potential to enhance our understanding of cellular mechanisms and activities that contribute to microbially mediated processes, such as the biogeochemical cycling of elements, the transformation of contaminants and the precipitation of mineral phases.

We investigated the distribution and activity of chloroethene-degrading microorganisms and associated functional genes during reductive dehalogenation of tetrachloroethene to ethene in a laboratory continuous-flow column. Using real-time PCR, we quantified “ Dehalococcoides ” species 16S rRNA and chloroethene-reductive dehalogenase (RDase) genes ( pceA, tceA, vcrA , and bvcA ) in nucleic acid extracts from different sections of the column. Dehalococcoides 16S rRNA gene copies were highest at the inflow port [(3.6 ± 0.6) × 10 6 (mean ± standard deviation) per gram soil] where the electron donor and acceptor were introduced into the column. The highest transcript numbers for tceA, vcrA , and bvcA were detected 5 to 10 cm from the column inflow. bvcA was the most highly expressed of all RDase genes and the only vinyl chloride reductase-encoding transcript detectable close to the column outflow. Interestingly, no expression of pceA was detected in the column, despite the presence of the genes in the microbial community throughout the column. By comparing the 16S rRNA gene copy numbers to the sum of all four RDase genes, we found that 50% of the Dehalococcoides population in the first part of the column did not contain either one of the known chloroethene RDase genes. Analysis of 16S rRNA gene clone libraries from both ends of the flow column revealed a microbial community dominated by members of Firmicutes and Actinobacteria . Higher clone sequence diversity was observed near the column outflow. The results presented have implications for our understanding of the ecophysiology of reductively dehalogenating Dehalococcoides spp. and their role in bioremediation of chloroethenes.

A continuous-flow anaerobic column experiment was conducted to evaluate the reductive dechlorination of tetrachloroethene (PCE) in Hanford aquifer material after bioaugmentation with the Evanite (EV) culture. An influent PCE concentration of 0.09 mM was transformed to vinyl chloride (VC) and ethene (ETH) within a hydraulic residence time of 1.3 days. The experimental breakthrough curves were described by the one-dimensional two-site-nonequilibrium transport model. PCE dechlorination was observed after bioaugmentation and after the lactate concentration was increased from 0.35 to 0.67 mM. At the onset of reductive dehalogenation, cis-dichloroethene (c-DCE) concentrations in the column effluent exceeded the influent PCE concentration indicating enhanced PCE desorption and transformation. When the lactate concentration was increased to 1.34 mM, c-DCE reduction to vinyl chloride (VC) and ethene (ETH) occurred. Spatial rates of PCE and VC transformation were determined in batch-incubated microcosms constructed with aquifer samples obtained from the column. PCE transformation rates were highest in the first 5 cm from the column inlet and decreased towards the column effluent. Dehalococcoides cell numbers dropped from approximately 73.5% of the total Bacterial population in the original inocula, to about 0.5% to 4% throughout the column. The results were consistent with estimates of electron donor utilization, with 4% going towards dehalogenation reactions.

Since the development of effective N2O mitigation options is a key challenge for future agricultural practice, we studied the interactive effect of tillage systems on fertilizer-derived N2O emissions and the abundance of microbial communities involved in N2O production and reduction. Soil samples from 0–10 cm and 10–20 cm depth of reduced tillage and ploughed plots were incubated with dairy slurry (SL) and manure compost (MC) in comparison with calcium ammonium nitrate (CAN) and an unfertilized control (ZERO) for 42 days. N2O and CO2 fluxes, ammonium, nitrate, dissolved organic C, and functional gene abundances (16S rRNA gene, nirK, nirS, nosZ, bacterial and archaeal amoA) were regularly monitored. Averaged across all soil samples, N2O emissions decreased in the order CAN and SL (CAN = 748.8 ± 206.3, SL = 489.4 ± 107.2 μg kg−1) followed by MC (284.2 ± 67.3 μg kg−1) and ZERO (29.1 ± 5.9 μg kg−1). Highest cumulative N2O emissions were found in 10–20 cm of the reduced tilled soil in CAN and SL. N2O fluxes were assigned to ammonium as source in CAN and SL and correlated positively to bacterial amoA abundances. Additionally, nosZ abundances correlated negatively to N2O fluxes in the organic fertilizer treatments. Soils showed a gradient in soil organic C, 16S rRNA, nirK, and nosZ with greater amounts in the 0–10 than 10–20 cm layer. Abundances of bacterial and archaeal amoA were higher in reduced tilled soil compared to ploughed soils. The study highlights that tillage system induced biophysicochemical stratification impacts net N2O emissions within the soil profile according to N and C species added during fertilization.

Biochar has been shown to improve soil function through increased cation and anion exchange capacity, soil water retention, increased soil buffer capacity, and enhanced microbial growth. Proposed mechanisms by which biochar will increase microbial activity include provision of labile carbon, increased nutrient retention, facilitated electron transfer (shuttling) to microorganisms, and increased microbial habitat space given the high specific surface area and micropore volume of biochar. Furthermore, some studies have shown that it reduces N2O emissions during denitrification in soils. Here we discuss the impact of biochar amendment on soil microbial nitrogen cycling.

Vinyl chloride reductases (VC-RDase) are the key enzymes for complete microbial reductive dehalogenation of chloroethenes, including the groundwater pollutants tetrachloroethene and trichloroethene. Analysis of the codon usage of the VC-RDase genes vcrA and bvcA showed that these genes are highly unusual and are characterized by a low G+C fraction at the third position. The third position of codons in VC-RDase genes is biased toward the nucleotide T, even though available Dehalococcoides genome sequences indicate the absence of any tRNAs matching codons that end in T. The comparatively high level of abnormality in the codon usage of VC-RDase genes suggests an evolutionary history that is different from that of most other Dehalococcoides genes.

Quantification of in situ (bio)degradation using compound-specific isotope analysis requires a known and constant isotope enrichment factor (e). Because reported isotope enrichment factors for microbial dehalogenation of chlorinated ethenes vary considerably we studied the potential effects of metabolic adaptation to TCE respiration on isotope fractionation (δ(13)C and δ(37)Cl) using a model organism (Desulfitobacterium hafniesne Y51), which only has one reductive dehalogenase (PceA). Cells grown on TCE for the first time showed exponential growth until 10(9) cells/mL. During exponential growth, the cell-normalized amount of PceA enzyme increased steadily in the presence of TCE (up to 21 pceA transcripts per cell) but not with alternative substrates (