Direct Evidence for Deterministic Assembly of Bacterial Communities in Full-Scale Municipal Wastewater Treatment Facilities.

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 < 0.001) than in fill-and-draw bioreactors (Bray-Curtis dissimilarity, µ = 0.48 ± 0.06 versus 0.58 ± 0.08). Next, we compared our results to previously used indirect methods for distinguishing between deterministic and stochastic community assembly mechanisms. Synchronicity was observed in the bacterial community composition among bioreactors within the same metropolitan region, consistent with deterministic community assembly. Similarly, a null model-based analysis also indicated that all wastewater bioreactor communities were controlled by deterministic factors and that continuous-flow bioreactors were significantly more deterministic (P < 0.001) than fill-and-draw bioreactors (nearest-taxon index, µ = 3.8 ± 0.6 versus 2.7 ± 0.8). Our results indicate that bacterial community composition in wastewater treatment bioreactors is better explained by deterministic community assembly theory; simultaneously, our results validate previously used but indirect methods to quantify whether microbial communities were assembled via deterministic or stochastic mechanisms. IMPORTANCE Understanding the mechanisms of bacterial community assembly is one of the grand challenges of microbial ecology. In environmental systems, this challenge is exacerbated because replicate experiments are typically impossible; that is, microbial ecologists cannot fabricate multiple field-scale experiments of identical, natural ecosystems. Our results directly demonstrate that deterministic mechanisms are more prominent than stochastic mechanisms in the assembly of wastewater treatment bioreactor communities. Our results also suggest that wastewater treatment bioreactor design is pertinent, such that the imposition of feast-famine conditions (i.e., fill-and-draw bioreactors) nudge bacterial community assembly more toward stochastic mechanisms than the imposition of stringent nutrient limitation (i.e., continuous-flow bioreactors). Our research also validates the previously used indirect methods (synchronous community dynamics and an application of a null model) for characterizing the relative importance of deterministic versus stochastic mechanisms of community assembly.

Different Engineering Designs Have Profoundly Different Impacts on the Microbiome and Nitrifying Bacterial Populations in Municipal Wastewater Treatment Bioreactors.

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 < 2 × 10-16). In addition, wastewater bioreactors with short mean cell residence time (<2 days) had very low community α-diversity and fewer nitrifying bacteria compared to those of other system designs. Nitrospira spp. (0.71%) and Nitrotoga spp. (0.41%) were the most prominent nitrite-oxidizing bacteria (NOB); because these two genera were rarely prominent at the same time, these populations appeared to be functionally redundant. Weak evidence (AOB:NOB « 2; substantial quantities of Nitrospira sublineage II) was also obtained suggesting that complete ammonia oxidation by a single organism was occurring in system designs known to impose stringent nutrient limitation. This research demonstrates that design decisions made by wastewater treatment engineers significantly affect the microbiome of wastewater treatment bioreactors. IMPORTANCE Municipal wastewater treatment facilities rely on the application of numerous "activated sludge" process designs to achieve site-specific treatment goals. A plethora of microbiome studies on municipal wastewater treatment bioreactors have been performed previously; however, the role of process design on the municipal wastewater treatment microbiome is poorly understood. In fact, wastewater treatment engineers have attempted to control the microbiome of wastewater bioreactors for decades without sufficient empirical evidence to support their design paradigms. Our research demonstrates that engineering decisions with respect to system design have a significant impact on the microbiome of wastewater treatment bioreactors.

Seasonal Dynamics of the Activated Sludge Microbiome in Sequencing Batch Reactors, Assessed Using 16S rRNA Transcript Amplicon Sequencing.

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.

Deciphering the Variability of Stable Isotope (C, Cl) Fractionation of Tetrachloroethene Biotransformation by Desulfitobacterium strains Carrying Different Reductive Dehalogenases Enzymes.

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 (εC = -0.4 to -19.0‰). Factors such as different reaction mechanisms and masking of isotope fractionation by either limited intracellular mass transfer or rate-limitations within the enzymatic multistep reaction are under discussion. This study evaluated the contribution of these factors to the magnitude of carbon and chlorine isotope fractionation of Desulfitobacterium strains harboring three different PCE-transforming enzymes (PCE-RdhA). Despite variable single element isotope fractionation (εC = -5.0 to -19.7‰; εCl = -1.9 to -6.3‰), similar slopes of dual element isotope plots (ΛC/Cl values of 2.4 ± 0.1 to 3.6 ± 0.1) suggest a common reaction mechanism for different PCE-RdhAs. Cell envelope properties of the Desulfitobacterium strains allowed to exclude masking effects due to PCE mass transfer limitation. Our results thus revealed that different rate-limiting steps (e.g., substrate channel diffusion) in the enzymatic multistep reactions of individual PCE-RdhAs rather than different reaction mechanisms determine the extent of PCE isotope fractionation in the Desulfitobacterium genus.

Anaerobic Dehalogenation by Reduced Aqueous Biochars.

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) (<450 nm). We found that TCS can be anaerobically degraded by reduced a-BCs with a pseudo first-order degradation rate constant of 0.0011-0.011 h-1. The 288 h degradation fraction of TCS correlated significantly with the amount of a-BC-bound electrons (0.055 ± 0.00024 to 0.11 ± 0.0016 mol e-/mol C) available for donation after 24 h of pre-reduction by Shewanella putrefaciens CN32. Within the reduction period, the recovery of chlorine based on residual TCS and generated Cl- ranged from 73.6 to 85.2%, implying that a major fraction of TCS was fully dechlorinated, together with mass spectroscopic analysis of possible degradation byproducts. Least-squares numerical fitting, accounting for the reactions of hydroquinones/semiquinones in a-BCs with TCS and byproducts, can simulate the reaction kinetics well (R2 > 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.

Growth and Population Dynamics of the Anaerobic Fe(II)-Oxidizing and Nitrate-Reducing Enrichment Culture KS.

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 O2IMPORTANCE 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.

Insights into Carbon Metabolism Provided by Fluorescence In Situ Hybridization-Secondary Ion Mass Spectrometry Imaging of an Autotrophic, Nitrate-Reducing, Fe(II)-Oxidizing Enrichment Culture.

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.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).

Metagenomic Analyses of the Autotrophic Fe(II)-Oxidizing, Nitrate-Reducing Enrichment Culture KS.

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 cytochromec system identified in neutrophilic microaerobic Fe(II)-oxidizing Sideroxydans lithotrophicus ES-1] in a Gallionellaceae sp., and findings of other putative genes involving cytochromecand 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.

Coexistence of Microaerophilic, Nitrate-Reducing, and Phototrophic Fe(II) Oxidizers and Fe(III) Reducers in Coastal Marine Sediment.

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 O2, 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. O2 and light penetrated only a few millimeters, but the depth distribution of the different iron metabolizers did not correlate with the profile of O2, 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.

Rhizosphere microbial community composition affects cadmium and zinc uptake by the metal-hyperaccumulating plant Arabidopsis halleri.

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.

Resiliency of Stable Isotope Fractionation (δ(13)C and δ(37)Cl) of Trichloroethene to Bacterial Growth Physiology and Expression of Key Enzymes.

Quantification of in situ (bio)degradation using compound-specific isotope analysis requires a known and constant isotope enrichment factor (ε). 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 (<1 pceA transcript per cell). Cultures initially transferred or subcultivated on TCE showed very similar isotope fractionation, both for carbon (εcarbon: -8.6‰ ± 0.3‰ or -8.8‰ ± 0.2‰) and chlorine (εchlorine: -2.7‰ ± 0.3‰) with little variation (0.7‰) for the different experimental conditions. Thus, TCE isotope fractionation by D. hafniense strain Y51 was affected by neither growth phase, pceA transcription, or translation, nor by PceA content per cell, suggesting that transport limitations did not affect isotope fractionation. Previously reported variable ε values for other organohalide-respiring bacteria might thus be attributed to different expression levels of their multiple reductive dehalogenases.

High spatial resolution of distribution and interconnections between Fe- and N-redox processes in profundal lake sediments.

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 as ammonia exchange biofilm carrier for enhanced aerobic nitrification in activated sludge.

The effects of biochar on aerobic nitrification in activated sludge were investigated in sequencing batch reactors. Biochar amended reactors exhibited 87-94 % lower ammonia in effluent and 16-71 % greater removal of total Kjeldahl nitrogen compared to control reactors. Quantitative qPCR analyses revealed that the relative abundance of ammonia oxidizing bacteria (AOB, amoA/16S rRNA genes) was greater in biochar than in control reactors. AOB were enriched on biochar surfaces, with biochar particles having up to 12.1 times greater relative abundance of AOB compared to suspended biomass. Biochar's maximum ammonia sorption capacity of 4.4 mg N/g at pH 7 decreased with decreasing pH, however a pH-sensitive fluorescent probe was used to show that biofilms growing on biochar surfaces maintain a median pH of > 6.7 despite reactor acidification by nitrification. Microbial colonization of biochar in activated sludge creates a pH-sheltered environment that sustains biochar's ammonia sorption capacity, resulting in enrichment of AOB on biochar particles and improved nitrification.

Effect of target gene sequence evenness and dominance on real-time PCR quantification of artificial sulfate-reducing microbial communities.

Quantitative real-time PCR of phylogenetic and functional marker genes is among the most commonly used techniques to quantify the abundance of microbial taxa in environmental samples. However, in most environmental applications, the approach is a rough assessment of population abundance rather than an exact absolute quantification method because of PCR-based estimation biases caused by multiple factors. Previous studies on these technical issues have focused on primer or template sequence features or PCR reaction conditions. However, how target gene sequence characteristics (e.g., evenness and dominance) in environmental samples affect qPCR quantifications has not been well studied. Here, we compared three primer sets targeting the beta subunit of the dissimilatory sulfite reductase (dsrB) to investigate qPCR quantification performance under different target gene sequence evenness and dominance conditions using artificial gBlock template mixtures designed accordingly. Our results suggested that the qPCR quantification performance of all tested primer sets was determined by the comprehensive effect of the target gene sequence evenness and dominance in environmental samples. Generally, highly degenerate primer sets have equivalent or better qPCR quantification results than a more target-specific primer set. Low template concentration in this study (~105 copies/L) will exaggerate the qPCR quantification results difference among tested primer sets. Improvements to the accuracy and reproducibility of qPCR assays for gene copy number quantification in environmental microbiology and microbial ecology studies should be based on prior knowledge of target gene sequence information acquired by metagenomic analysis or other approaches, careful selection of primer sets, and proper reaction conditions optimization.

Fate of Cd during microbial Fe(III) mineral reduction by a novel and Cd-tolerant Geobacter species.

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.

Organic carbon and reducing conditions lead to cadmium immobilization by secondary Fe mineral formation in a pH-neutral soil.

Cadmium (Cd) is of environmental relevance as it enters soils via Cd-containing phosphate fertilizers and endangers human health when taken up by crops. Cd is known to associate with Fe(III) (oxyhydr)oxides in pH-neutral to slightly acidic soils, though it is not well understood how the interrelation of Fe and Cd changes under Fe(III)-reducing conditions. Therefore, we investigated how the mobility of Cd changes when a Cd-bearing soil is faced with organic carbon input and reducing conditions. Using fatty acid profiles and quantitative PCR, we found that both fermenting and Fe(III)-reducing bacteria were stimulated by organic carbon-rich conditions, leading to significant Fe(III) reduction. The reduction of Fe(III) minerals was accompanied by increasing soil pH, increasing dissolved inorganic carbon, and decreasing Cd mobility. SEM-EDX mapping of soil particles showed that a minor fraction of Cd was transferred to Ca- and S-bearing minerals, probably carbonates and sulfides. Most of the Cd, however, correlated with a secondary iron mineral phase that was formed during microbial Fe(III) mineral reduction and contained mostly Fe, suggesting an iron oxide mineral such as magnetite (Fe3O4). Our data thus provide evidence that secondary Fe(II) and Fe(II)/Fe(III) mixed minerals could be a sink for Cd in soils under reducing conditions, thus decreasing the mobility of Cd in the soil.

Ammonia-Oxidizing Bacteria Maintain Abundance but Lower amoA-Gene Expression during Cold Temperature Nitrification Failure in a Full-Scale Municipal Wastewater Treatment Plant.

In this study, we explore the relationship between community structure and transcriptional activity of ammonia-oxidizing bacteria during cold temperature nitrification failure in three parallel full-scale sequencing batch reactors (SBRs) treating municipal wastewater. In the three reactors, ammonia concentrations increased with declines in wastewater temperature below 15°C. We quantified and sequenced 16S rRNA and ammonia monooxygenase (amoA) gene fragments in DNA and RNA extracts from activated sludge samples collected from the SBRs during the warmer seasons (summer and fall) and when water temperatures were below 15°C (winter and spring). Taxonomic community composition of amoA genes and transcripts did not vary much between the warmer and colder seasons. However, we observed significant differences in amoA transcript copy numbers between fall (highest) and spring (lowest). Ammonia-oxidizing bacteria of the genus Nitrosomonas sp. could maintain their population abundance despite lowering their amoA gene expression during winter and spring. In spite of relatively low population abundance, an amoA amplicon sequence variant (ASV) cluster identified as most similar to the amoA gene of Nitrosospira briensis showed the highest amoA transcript-to-gene ratio throughout all four seasons, indicating that some nitrifiers remain active at wastewater temperatures below 15°C. Our results show that 16S rRNA and amoA gene copy numbers are limited predictors of cell activity. To optimize function and performance of mixed community bioprocesses, we need to collect high-resolution quantitative transcriptomic and potentially proteomic data to resolve the response of individual species to changes in environmental parameters in engineered systems. IMPORTANCE 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). This study aims at expanding our understanding of how ammonia-oxidizing bacteria in activated sludge change in activity and growth across seasons. We quantified the ammonia monooxygenase (amoA) gene and transcript copy numbers using real-time PCR and sequenced the amoA amplicons to reveal community structure and activity changes of nitrifying microbial populations during seasonal nitrification failure in three full-scale sequencing batch reactors (SRBs) treating municipal wastewater. Relevant findings presented in this study contribute to explain seasonal nitrification performance variability in SRBs.

Nanodroplet-Based Reagent Delivery into Water-in-Fluorinated-Oil Droplets.

In vitro compartmentalization (IVC) 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 such as low throughput or time delay of reagent delivery into different microdroplets. Hence, to overcome the above issues, we demonstrated a nanodroplet-based approach for the delivery of copper ions and middle-sized peptide molecules (human p53 peptide, 2 kDa). We confirmed the ion delivery by microscopic inspection of crystal formation inside the microdroplet, and confirmed the peptide delivery using a fluorescent immunosensor. We believe that this nanodroplet-based delivery method is a promising approach to achieving precise control for a broad range of fluorocarbon IVC-based biological applications, including molecular evolution, cell factory engineering, digital nucleic acid detection, or drug screening.

Linking environmental processes to the in situ functioning of microorganisms by high-resolution secondary ion mass spectrometry (NanoSIMS) and scanning transmission X-ray microscopy (STXM).

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.

Influence of seasonal and geochemical changes on the geomicrobiology of an iron carbonate mineral water spring.

Fuschna Spring in the Swiss Alps (Engadin region) is a bicarbonate iron(II)-rich, pH-neutral mineral water spring that is dominated visually by dark green microbial mats at the side of the flow channel and orange iron(III) (oxyhydr)oxides in the flow channel. Gradients of O(2), dissolved iron(II), and bicarbonate establish in the water. Our goals were to identify the dominating biogeochemical processes and to determine to which extent changing geochemical conditions along the flow path and seasonal changes influence mineral identity, crystallinity, and microbial diversity. Geochemical analysis showed microoxic water at the spring outlet which became fully oxygenated within 2.3 m downstream. X-ray diffraction and Mössbauer spectroscopy revealed calcite (CaCO(3)) and ferrihydrite [Fe(OH)(3)] to be the dominant minerals which increased in crystallinity with increasing distance from the spring outlet. Denaturing gradient gel electrophoresis banding pattern cluster analysis revealed that the microbial community composition shifted mainly with seasons and to a lesser extent along the flow path. 16S rRNA gene sequence analysis showed that microbial communities differ between the flow channel and the flanking microbial mat. Microbial community analysis in combination with most-probable-number analyses and quantitative PCR (qPCR) showed that the mat was dominated by cyanobacteria and the channel was dominated by microaerophilic Fe(II) oxidizers (1.97 × 10(7) ± 4.36 × 10(6) 16S rRNA gene copies g(-1) using Gallionella-specific qPCR primers), while high numbers of Fe(III) reducers (10(9) cells/g) were identified in both the mat and the flow channel. Phototrophic and nitrate-reducing Fe(II) oxidizers were present as well, although in lower numbers (10(3) to 10(4) cells/g). In summary, our data suggest that mainly seasonal changes caused microbial community shifts, while geochemical gradients along the flow path influenced mineral crystallinity.