Proterozoic Acquisition of Archaeal Genes for Extracellular Electron Transfer: A Metabolic Adaptation of Aerobic Ammonia-Oxidizing Bacteria to Oxygen Limitation.

Many aerobic microbes can utilize alternative electron acceptors under oxygen-limited conditions. In some cases, this is mediated by extracellular electron transfer (or EET), wherein electrons are transferred to extracellular oxidants such as iron oxide and manganese oxide minerals. Here, we show that an ammonia-oxidizer previously known to be strictly aerobic, Nitrosomonas communis, may have been able to utilize a poised electrode to maintain metabolic activity in anoxic conditions. The presence and activity of multiheme cytochromes in N. communis further suggest a capacity for EET. Molecular clock analysis shows that the ancestors of ß-proteobacterial ammonia oxidizers appeared after Earth's atmospheric oxygenation when the oxygen levels were >10-4pO2 (present atmospheric level [PAL]), consistent with aerobic origins. Equally important, phylogenetic reconciliations of gene and species trees show that the multiheme c-type EET proteins in Nitrosomonas and Nitrosospira lineages were likely acquired by gene transfer from γ-proteobacteria when the oxygen levels were between 0.1 and 1 pO2 (PAL). These results suggest that ß-proteobacterial EET evolved during the Proterozoic when oxygen limitation was widespread, but oxidized minerals were abundant.

DNA- and RNA-SIP Reveal Nitrospira spp. as Key Drivers of Nitrification in Groundwater-Fed Biofilters.

Nitrification, the oxidative process converting ammonia to nitrite and nitrate, is driven by microbes and plays a central role in the global nitrogen cycle. Our earlier investigations based on 16S rRNA and amoA amplicon analysis, amoA quantitative PCR and metagenomics of groundwater-fed biofilters indicated a consistently high abundance of comammox Nitrospira Here, we hypothesized that these nonclassical nitrifiers drive ammonia-N oxidation. Hence, we used DNA and RNA stable isotope probing (SIP) coupled with 16S rRNA amplicon sequencing to identify the active members in the biofilter community when subjected to a continuous supply of NH4+ or NO2- in the presence of 13C-HCO3- (labeled) or 12C-HCO3- (unlabeled). Allylthiourea (ATU) and sodium chlorate were added to inhibit autotrophic ammonia- and nitrite-oxidizing bacteria, respectively. Our results confirmed that lineage II Nitrospira dominated ammonia oxidation in the biofilter community. A total of 78 (8 by RNA-SIP and 70 by DNA-SIP) and 96 (25 by RNA-SIP and 71 by DNA-SIP) Nitrospira phylotypes (at 99% 16S rRNA sequence similarity) were identified as complete ammonia- and nitrite-oxidizing, respectively. We also detected significant HCO3- uptake by Acidobacteria subgroup10, Pedomicrobium, Rhizobacter, and Acidovorax under conditions that favored ammonia oxidation. Canonical Nitrospira alone drove nitrite oxidation in the biofilter community, and activity of archaeal ammonia-oxidizing taxa was not detected in the SIP fractions. This study provides the first in situ evidence of ammonia oxidation by comammox Nitrospira in an ecologically relevant complex microbiome.IMPORTANCE With this study we provide the first in situ evidence of ecologically relevant ammonia oxidation by comammox Nitrospira in a complex microbiome and document an unexpectedly high H13CO3- uptake and growth of proteobacterial and acidobacterial taxa under ammonia selectivity. This finding raises the question of whether comammox Nitrospira is an equally important ammonia oxidizer in other environments.

Diversity of Iron Oxidizers in Groundwater-Fed Rapid Sand Filters: Evidence of Fe(II)-Dependent Growth by Curvibacter and Undibacterium spp.

Although earlier circumstantial observations have suggested the presence of iron oxidizing bacteria (IOB) in groundwater-fed rapid sand filters (RSF), ferrous iron (Fe(II)) oxidation in this environment is often considered a chemical process due to the highly oxic and circumneutral pH conditions. The low water temperature (5-10°C), typical of groundwaters, on the other hand, may reduce the rates of chemical Fe(II) oxidation, which may allow IOB to grow and compete with chemical Fe(II) oxidation. Hence, we hypothesized that IOB are active and abundant in groundwater-fed RSFs. Here, we applied a combination of cultivation and molecular approaches to isolate, quantify, and confirm the growth of IOB from groundwater-fed RSFs, operated at different influent Fe(II) concentrations. Isolates related to Undibacterium and Curvibacter were identified as novel IOB lineages. Gallionella spp. were dominant in all waterworks, whereas Ferriphaselus and Undibacterium were dominant at pre-filters of waterworks receiving groundwaters with high (>2 mg/l) Fe(II) concentrations. The high density and diversity of IOB in groundwater-fed RSFs suggest that neutrophilic IOB may not be limited to oxic/anoxic interfaces.

Reactor staging influences microbial community composition and diversity of denitrifying MBBRs- Implications on pharmaceutical removal.

The subdivision of biofilm reactor in two or more stages (i.e., reactor staging) represents an option for process optimisation of biological treatment. In our previous work, we showed that the gradient of influent organic substrate availability (induced by the staging) can influence the microbial activity (i.e., denitrification and pharmaceutical biotransformation kinetics) of a denitrifying three-stage Moving Bed Biofilm Reactor (MBBR) system. However, it is unclear whether staging and thus the long-term exposure to varying organic carbon type and loading influences the microbial community structure and diversity. In this study, we investigated biofilm structure and diversity in the three-stage MBBR system (S) compared to a single-stage configuration (U) and their relationship with microbial functions. Results from 16S rRNA amplicon libraries revealed a significantly higher microbial richness in the staged MBBR (at 99% sequence similarity) compared to single-stage MBBR. A more even and diverse microbial community was selected in the last stage of S (S3), likely due to exposure to carbon limitation during continuous-flow operation. A core of OTUs was shared in both systems, consisting of Burkholderiales, Xanthomonadales, Flavobacteriales and Sphingobacteriales, while MBBR staging selected for specific taxa (i.e., Candidate division WS6 and Deinococcales). Results from quantitative PCR (qPCR) showed that S3 exhibited the lowest abundance of 16S rRNA but the highest abundance of atypical nosZ, suggesting a selection of microbes with more diverse N-metabolism (i.e., incomplete denitrifiers) in the stage exposed to the lowest carbon availability. A positive correlation (p < 0.05) was observed between removal rate constants of several pharmaceuticals with abundance of relevant denitrifying genes, but not with biodiversity. Despite the previously suggested positive relationship between microbial diversity and functionality in macrobial and microbial ecosystems, this was not observed in the current study, indicating a need to further investigate structure-function relationships for denitrifying systems.

Density and distribution of nitrifying guilds in rapid sand filters for drinking water production: Dominance of Nitrospira spp.

We investigated the density and distribution of total bacteria, canonical Ammonia Oxidizing Bacteria (AOB) (Nitrosomonas plus Nitrosospira), Ammonia Oxidizing Archaea (AOA), as well as Nitrobacter and Nitrospira in rapid sand filters used for groundwater treatment. To investigate the spatial distribution of these guilds, filter material was sampled at four drinking water treatment plants (DWTPs) in parallel filters of the pre- and after-filtration stages at different locations and depths. The target guilds were quantified by qPCR targeting 16S rRNA and amoA genes. Total bacterial densities (ignoring 16S rRNA gene copy number variation) were high and ranged from 109 to 1010 per gram (1015 to 1016 per m3) of filter material. All examined guilds, except AOA, were stratified at only one of the four DWTPs. Densities varied spatially within filter (intra-filter variation) at two of the DWTPs and in parallel filters (inter-filter variation) at one of the DWTPs. Variation analysis revealed random sampling as the most efficient strategy to yield accurate mean density estimates, with collection of at least 7 samples suggested to obtain an acceptable (below half order of magnitude) density precision. Nitrospira was consistently the most dominant guild (5-10% of total community), and was generally up to 4 orders of magnitude more abundant than Nitrobacter and up to 2 orders of magnitude more abundant than canonical AOBs. These results, supplemented with further analysis of the previously reported diversity of Nitrospira in the studied DWTPs based on 16S rRNA and nxrB gene phylogeny (Gülay et al., 2016; Palomo et al., 2016), indicate that the high Nitrospira abundance is due to their comammox (complete ammonia oxidation) physiology. AOA densities were lower than AOB densities, except in the highly stratified filters, where they were of similar abundance. In conclusion, rapid sand filters are microbially dense, with varying degrees of spatial heterogeneity, which requires replicate sampling for a sufficiently precise determination of total microbial community and specific population densities. A consistently high Nitrospira to bacterial and archaeal AOB density ratio suggests that non-canonical pathways for nitrification may dominate the examined RSFs.

Nitrotoga is selected over Nitrospira in newly assembled biofilm communities from a tap water source community at increased nitrite loading.

Community assembly is a central topic in microbial ecology: how do assembly processes interact and what is the relative contribution of stochasticity and determinism? Here, we exposed replicate flow-through biofilm systems, fed with nitrite-supplemented tap water, to continuous immigration from a source community, present in the tap water, to determine the extent of selection and neutral processes in newly assembled biofilm communities at both the community and the functional guild (of nitrite-oxidizing bacteria, NOB) levels. The community composition of biofilms assembled under low and high nitrite loading was described after 40 days of complete nitrite removal. The total community assembly, as well as the NOB guild assembly were largely governed by a combination of deterministic and stochastic processes. Furthermore, we observed deterministic enrichment of certain types of NOB in the biofilms. Specifically, elevated nitrite loading selected for a single Nitrotoga representative, while lower nitrite conditions selected for a number of Nitrospira. Therefore, even when focusing on ecologically coherent ensembles, assembly is the result of complex stochastic and deterministic processes that can only be interrogated by observing multiple assemblies under controlled conditions.

Metal stressors consistently modulate bacterial conjugal plasmid uptake potential in a phylogenetically conserved manner.

The environmental stimulants and inhibitors of conjugal plasmid transfer in microbial communities are poorly understood. Specifically, it is not known whether exposure to stressors may cause a community to alter its plasmid uptake ability. We assessed whether metals (Cu, Cd, Ni, Zn) and one metalloid (As), at concentrations causing partial growth inhibition, modulate community permissiveness (that is, uptake ability) against a broad-host-range IncP-type plasmid (pKJK5). Cells were extracted from an agricultural soil as recipient community and a cultivation-minimal filter mating assay was conducted with an exogenous E. coli donor strain. The donor hosted a gfp-tagged pKJK5 derivative from which conjugation events could be microscopically quantified and transconjugants isolated and phylogenetically described at high resolution via FACS and 16S rRNA amplicon sequencing. Metal stress consistently decreased plasmid transfer frequencies to the community, while the transconjugal pool richness remained unaffected with OTUs belonging to 12 bacterial phyla. The taxonomic composition of the transconjugal pools was distinct from their respective recipient communities and clustered dependent on the stress type and dose. However, for certain OTUs, stress increased or decreased permissiveness by more than 1000-fold and this response was typically correlated across different metals and doses. The response to some stresses was, in addition, phylogenetically conserved. This is the first demonstration that community permissiveness is sensitive to metal(loid) stress in a manner that is both partially consistent across stressors and phylogenetically conserved.

Short-sludge age EBPR process - Microbial and biochemical process characterisation during reactor start-up and operation.

The new paradigm for used water treatment suggests the use of short solid retention times (SRT) to minimize organic substrate mineralization and to maximize resource recovery. However, little is known about the microbes and the underlying biogeochemical mechanisms driving these short-SRT systems. In this paper, we report the start-up and operation of a short-SRT enhanced biological phosphorus removal (EBPR) system operated as a sequencing batch reactor (SBR) fed with preclarified municipal wastewater, which is supplemented with propionate. The microbial community was analysed via 16S rRNA amplicon sequencing. During start-up (SRT = 8 d), the EBPR was removing up to 99% of the influent phosphate and completely oxidized the incoming ammonia. Furthermore, the sludge showed excellent settling properties. However, once the SRT was shifted to 3.5 days nitrification was inhibited and bacteria of the Thiothrix taxon proliferated in the reactor, thereby leading to filamentous bulking (sludge volume index up to SVI = 1100 mL/g). Phosphorus removal deteriorated during this period, likely due to the out-competition of polyphosphate accumulating organisms (PAO) by sulphate reducing bacteria (SRB). Subsequently, SRB activity was suppressed by reducing the anaerobic SRT from 1.2 day to 0.68 day, with a consequent rapid SVI decrease to ∼200 ml/g. The short-SRT EBPR effectively removed phosphate and nitrification was mitigated at SRT = 3 days and oxygen levels ranging from 2 to 3 mg/L.

Metagenomic analysis of rapid gravity sand filter microbial communities suggests novel physiology of Nitrospira spp.

Rapid gravity sand filtration is a drinking water production technology widely used around the world. Microbially catalyzed processes dominate the oxidative transformation of ammonia, reduced manganese and iron, methane and hydrogen sulfide, which may all be present at millimolar concentrations when groundwater is the source water. In this study, six metagenomes from various locations within a groundwater-fed rapid sand filter (RSF) were analyzed. The community gene catalog contained most genes of the nitrogen cycle, with particular abundance in genes of the nitrification pathway. Genes involved in different carbon fixation pathways were also abundant, with the reverse tricarboxylic acid cycle pathway most abundant, consistent with an observed Nitrospira dominance. From the metagenomic data set, 14 near-complete genomes were reconstructed and functionally characterized. On the basis of their genetic content, a metabolic and geochemical model was proposed. The organisms represented by draft genomes had the capability to oxidize ammonium, nitrite, hydrogen sulfide, methane, potentially iron and manganese as well as to assimilate organic compounds. A composite Nitrospira genome was recovered, and amo-containing Nitrospira genome contigs were identified. This finding, together with the high Nitrospira abundance, and the abundance of atypical amo and hao genes, suggests the potential for complete ammonium oxidation by Nitrospira, and a major role of Nitrospira in the investigated RSFs and potentially other nitrifying environments.

Ecological patterns, diversity and core taxa of microbial communities in groundwater-fed rapid gravity filters.

Here, we document microbial communities in rapid gravity filtration units, specifically serial rapid sand filters (RSFs), termed prefilters (PFs) and after- filters (AFs), fed with anoxic groundwaters low in organic carbon to prepare potable waters. A comprehensive 16S rRNA-based amplicon sequencing survey revealed a core RSF microbiome comprising few bacterial taxa (29-30 genera) dominated by Nitrospirae, Proteobacteria and Acidobacteria, with a strikingly high abundance (75-87±18%) across five examined waterworks in Denmark. Lineages within the Nitrospira genus consistently comprised the second most and most abundant fraction in PFs (27±23%) and AFs (45.2±23%), respectively, and were far more abundant than typical proteobacterial ammonium-oxidizing bacteria, suggesting a physiology beyond nitrite oxidation for Nitrospira. Within the core taxa, sequences closely related to types with ability to oxidize ammonium, nitrite, iron, manganese and methane as primary growth substrate were identified and dominated in both PFs (73.6±6%) and AFs (61.4±21%), suggesting their functional importance. Surprisingly, operational taxonomic unit richness correlated strongly and positively with sampling location in the drinking water treatment plant (from PFs to AFs), and a weaker negative correlation held for evenness. Significant spatial heterogeneity in microbial community composition was detected in both PFs and AFs, and was higher in the AFs. This is the first comprehensive documentation of microbial community diversity in RSFs treating oligotrophic groundwaters. We have identified patterns of local spatial heterogeneity and dispersal, documented surprising energy-diversity relationships, observed a large and diverse Nitrospira fraction and established a core RSF microbiome.

An improved method to set significance thresholds for ß diversity testing in microbial community comparisons.

Exploring the variation in microbial community diversity between locations (ß diversity) is a central topic in microbial ecology. Currently, there is no consensus on how to set the significance threshold for ß diversity. Here, we describe and quantify the technical components of ß diversity, including those associated with the process of subsampling. These components exist for any proposed ß diversity measurement procedure. Further, we introduce a strategy to set significance thresholds for ß diversity of any group of microbial samples using rarefaction, invoking the notion of a meta-community. The proposed technique was applied to several in silico generated operational taxonomic unit (OTU) libraries and experimental 16S rRNA pyrosequencing libraries. The latter represented microbial communities from different biological rapid sand filters at a full-scale waterworks. We observe that ß diversity, after subsampling, is inflated by intra-sample differences; this inflation is avoided in the proposed method. In addition, microbial community evenness (Gini > 0.08) strongly affects all ß diversity estimations due to bias associated with rarefaction. Where published methods to test ß significance often fail, the proposed meta-community-based estimator is more successful at rejecting insignificant ß diversity values. Applying our approach, we reveal the heterogeneous microbial structure of biological rapid sand filters both within and across filters.

Internal porosity of mineral coating supports microbial activity in rapid sand filters for groundwater treatment.

A mineral coating develops on the filter grain surface when groundwater is treated via rapid sand filtration in drinking water production. The coating changes the physical and chemical properties of the filter material, but little is known about its effect on the activity, colonization, diversity, and abundance of microbiota. This study reveals that a mineral coating can positively affect the colonization and activity of microbial communities in rapid sand filters. To understand this effect, we investigated the abundance, spatial distribution, colonization, and diversity of all and of nitrifying prokaryotes in filter material with various degrees of mineral coating. We also examined the physical and chemical characteristics of the mineral coating. The amount of mineral coating correlated positively with the internal porosity, the packed bulk density, and the biologically available surface area of the filter material. The volumetric NH4 (+) removal rate also increased with the degree of mineral coating. Consistently, bacterial 16S rRNA and amoA abundances positively correlated with increased mineral coating levels. Microbial colonization could be visualized mainly within the outer periphery (60.6 ± 35.6 µm) of the mineral coating, which had a thickness of up to 600 ± 51 µm. Environmental scanning electron microscopic (E-SEM) observations suggested an extracellular polymeric substance-rich matrix and submicron-sized bacterial cells. Nitrifier diversity profiles were similar irrespective of the degree of mineral coating, as indicated by pyrosequencing analysis. Overall, our results demonstrate that mineral coating positively affects microbial colonization and activity in rapid sand filters, most likely due to increased volumetric cell abundances facilitated by the large surface area of internal mineral porosity accessible for microbial colonization.

Sequentially aerated membrane biofilm reactors for autotrophic nitrogen removal: microbial community composition and dynamics.

Membrane-aerated biofilm reactors performing autotrophic nitrogen removal can be successfully applied to treat concentrated nitrogen streams. However, their process performance is seriously hampered by the growth of nitrite oxidizing bacteria (NOB). In this work we document how sequential aeration can bring the rapid and long-term suppression of NOB and the onset of the activity of anaerobic ammonium oxidizing bacteria (AnAOB). Real-time quantitative polymerase chain reaction analyses confirmed that such shift in performance was mirrored by a change in population densities, with a very drastic reduction of the NOB Nitrospira and Nitrobacter and a 10-fold increase in AnAOB numbers. The study of biofilm sections with relevant 16S rRNA fluorescent probes revealed strongly stratified biofilm structures fostering aerobic ammonium oxidizing bacteria (AOB) in biofilm areas close to the membrane surface (rich in oxygen) and AnAOB in regions neighbouring the liquid phase. Both communities were separated by a transition region potentially populated by denitrifying heterotrophic bacteria. AOB and AnAOB bacterial groups were more abundant and diverse than NOB, and dominated by the r-strategists Nitrosomonas europaea and Ca. Brocadia anammoxidans, respectively. Taken together, the present work presents tools to better engineer, monitor and control the microbial communities that support robust, sustainable and efficient nitrogen removal.