Impact of salinity origin on microbial communities in saline springs within the Illinois Basin, USA.

Saline springs within the Illinois Basin result from the discharge of deep-seated evaporated seawater (brine) and likely contain diverse and complex microbial communities that are poorly understood. In this study, seven saline/mineral springs with different geochemical characteristics and salinity origins were investigated using geochemical and molecular microbiological analyses to reveal the composition of microbial communities inhabiting springs and their key controlling factors. The 16S rRNA sequencing results demonstrated that each spring harbours a unique microbial community influenced by its geochemical properties and subsurface conditions. The microbial communities in springs that originated from Cambrian/Ordovician strata, which are deep confined units that have limited recharge from overlying formations, share a greater similarity in community composition and have a higher species richness and more overlapped taxa than those that originated from shallower Pennsylvanian strata, which are subject to extensive regional surface and groundwater recharge. The microbial distribution along the spring flow paths at the surface indicates that 59.8%-94.2% of total sequences in sedimentary samples originated from spring water, highlighting the role of springs in influencing microbiota in the immediate terrestrial environment. The results indicate that the springs introduce microbiota with a high biodiversity into surface terrestrial or aquatic ecosystems, potentially affecting microbial reservoirs in downstream ecosystems.

Meta-Omics-Supervised Characterization of Respiration Activities Associated with Microbial Immigrants in Anaerobic Sludge Digesters.

Immigration has been recently recognized as an important ecological process that affects the microbial community structure in diverse ecosystems. However, the fate of microbial immigrants in the new environment and their involvement in the local biochemical network remain unclear. In this study, we performed meta-omics-supervised characterization of immigrants' activities in anaerobic sludge digesters. Metagenomic analyses revealed that immigrants from the feed sludge accounted for the majority of populations capable of anaerobic respiration in a digester. Electron acceptors that were predicted to be respired, including nitrate, nitrite, sulfate, and elemental sulfur, were added to digester sludge in batch tests. Consumption of up to 91% of the added electron acceptors was observed within the experiment period. 16S rRNA sequencing detected populations that were stimulated by the electron acceptors, largely overlapping with respiration-capable immigrants identified by metagenomic analysis. Metatranscriptomic analysis of the batch tests provided additional evidence for upregulated expression of respiration genes and concomitant suppressed expression of methanogenesis. Anaerobic respiration activity was further evaluated in full-scale digesters in nine wastewater treatment plants. Although nitrate and sulfate respiration were ubiquitous, the expression level of respiration genes was generally 2-3 orders of magnitude lower than the expression of methanogenesis in most digesters, suggesting marginal ecological roles by immigrants in full-scale digester ecosystems.

360-Degree Distribution of Biofilm Quantity and Community in an Operational Unchlorinated Drinking Water Distribution Pipe.

In the present study, triplicate rings of 360° pipe surfaces of an operational drinking water distribution pipe were swabbed. Each ring was equally divided into 16 parts for swabbing. The collected swabs were grouped into 3 sections and compared with the biofilm samples sampled by sonication of specimens from the same pipe. The results showed that the biofilm is unevenly distributed over the 16 parts and the 3 sections of the pipe surface. Both the active biomass and the number of observed OTUs increased as the measurements proceeded from the top to the bottom of the pipe. The bacterial community was dominated in all sections by Proteobacteria. At the genus level, Nitrospira spp., Terrimonas spp., and Hyphomicrobium spp. were dominant in all sections. Gaiella spp. and Vicinamibacter spp. dominated in S-I, Blastopirellula spp. and Pirellula spp. dominated in S-II, while Holophaga spp. and Phaeodactylibacter spp. dominated in S-III. When swabbing and pipe specimen sonication were compared, the results showed that the sampling strategy significantly influences the obtained biofilm bacterial community. A consistent multisectional swabbing strategy is proposed for future biofilm sampling; it involves collecting swabs from all sections and comparing the swabs from the same position/section across locations.

Insights into the phylogeny and coding potential of microbial dark matter.

Genome sequencing enhances our understanding of the biological world by providing blueprints for the evolutionary and functional diversity that shapes the biosphere. However, microbial genomes that are currently available are of limited phylogenetic breadth, owing to our historical inability to cultivate most microorganisms in the laboratory. We apply single-cell genomics to target and sequence 201 uncultivated archaeal and bacterial cells from nine diverse habitats belonging to 29 major mostly uncharted branches of the tree of life, so-called 'microbial dark matter'. With this additional genomic information, we are able to resolve many intra- and inter-phylum-level relationships and to propose two new superphyla. We uncover unexpected metabolic features that extend our understanding of biology and challenge established boundaries between the three domains of life. These include a novel amino acid use for the opal stop codon, an archaeal-type purine synthesis in Bacteria and complete sigma factors in Archaea similar to those in Bacteria. The single-cell genomes also served to phylogenetically anchor up to 20% of metagenomic reads in some habitats, facilitating organism-level interpretation of ecosystem function. This study greatly expands the genomic representation of the tree of life and provides a systematic step towards a better understanding of biological evolution on our planet.

IMG/VR: a database of cultured and uncultured DNA Viruses and retroviruses.

Viruses represent the most abundant life forms on the planet. Recent experimental and computational improvements have led to a dramatic increase in the number of viral genome sequences identified primarily from metagenomic samples. As a result of the expanding catalog of metagenomic viral sequences, there exists a need for a comprehensive computational platform integrating all these sequences with associated metadata and analytical tools. Here we present IMG/VR (https://img.jgi.doe.gov/vr/), the largest publicly available database of 3908 isolate reference DNA viruses with 264 413 computationally identified viral contigs from >6000 ecologically diverse metagenomic samples. Approximately half of the viral contigs are grouped into genetically distinct quasi-species clusters. Microbial hosts are predicted for 20 000 viral sequences, revealing nine microbial phyla previously unreported to be infected by viruses. Viral sequences can be queried using a variety of associated metadata, including habitat type and geographic location of the samples, or taxonomic classification according to hallmark viral genes. IMG/VR has a user-friendly interface that allows users to interrogate all integrated data and interact by comparing with external sequences, thus serving as an essential resource in the viral genomics community.

Novel energy conservation strategies and behaviour of Pelotomaculum schinkii driving syntrophic propionate catabolism.

Under methanogenic conditions, short-chain fatty acids are common byproducts from degradation of organic compounds and conversion of these acids is an important component of the global carbon cycle. Due to the thermodynamic difficulty of propionate degradation, this process requires syntrophic interaction between a bacterium and partner methanogen; however, the metabolic strategies and behaviour involved are not fully understood. In this study, the first genome analysis of obligately syntrophic propionate degraders (Pelotomaculum schinkii HH and P. propionicicum MGP) and comparison with other syntrophic propionate degrader genomes elucidated novel components of energy metabolism behind Pelotomaculum propionate oxidation. Combined with transcriptomic examination of P. schinkii behaviour in co-culture with Methanospirillum hungatei, we found that formate may be the preferred electron carrier for P. schinkii syntrophy. Propionate-derived menaquinol may be primarily re-oxidized to formate, and energy was conserved during formate generation through newly proposed proton-pumping formate extrusion. P. schinkii did not overexpress conventional energy metabolism associated with a model syntrophic propionate degrader Syntrophobacter fumaroxidans MPOB (i.e., CoA transferase, Fix and Rnf). We also found that P. schinkii and the partner methanogen may also interact through flagellar contact and amino acid and fructose exchange. These findings provide new understanding of syntrophic energy acquisition and interactions.

Impact of drinking water treatment and distribution on the microbiome continuum: an ecological disturbance's perspective.

While microbes are known to be present at different stages of a drinking water system, their potential functions and ability to grow in such systems are poorly understood. In this study, we demonstrated that treatment and distribution processes could be viewed as ecological disturbances exhibited over space on the microbiome continuum in a groundwater-derived system. Results from 16S rRNA gene amplicon analysis and metagenomics suggested that disturbances in the system were intense as the community diversity was substantially reduced during the treatment steps. Specifically, syntrophs and methanogens dominant in raw water (RW) disappeared after water abstraction, accompanied by a substantial decrease in both the abundance and number of functional genes related to methanogenesis. The softening effluent was dominated by an Exiguobacterium-related population, likely due to its ability to use the phosphotransferase system (PTS) as regulatory machinery to control the energy conditions of the cell. After disinfection and entering the distribution system, community-level functionality remained relatively stable, whereas the community structure differed from those taken in the treatment steps. The diversity and high abundance of some eukaryotic groups in the system suggested that predation could be a disturbance to the bacterial microbiome, which could further drive the diversification of the bacterial community.

Thermodynamically diverse syntrophic aromatic compound catabolism.

Specialized organotrophic Bacteria 'syntrophs' and methanogenic Archaea 'methanogens' form a unique metabolic interaction to accomplish cooperative mineralization of organic compounds to CH4 and CO2 . Due to challenges in cultivation of syntrophs, mechanisms for how their organotrophic catabolism circumvents thermodynamic restrictions remain unclear. In this study, we investigate two communities hosting diverse syntrophic aromatic compound metabolizers (Syntrophus, Syntrophorhabdus, Pelotomaculum and an uncultivated Syntrophorhabdacaeae member) to uncover their catabolic diversity and flexibility. Although syntrophs have been generally presumed to metabolize aromatic compounds to acetate, CO2 , H2 and formate, combined metagenomics and metatranscriptomics show that uncultured syntrophs utilize unconventional alternative metabolic pathways in situ producing butyrate, cyclohexanecarboxylate and benzoate as catabolic byproducts. In addition, we also find parallel utilization of diverse H2 and formate generating pathways to facilitate interactions with partner methanogens. Based on thermodynamic calculations, these pathways may enable syntrophs to combat thermodynamic restrictions. In addition, when fed with specific substrates (i.e., benzoate, terephthalate or trimellitate), each syntroph population expresses different pathways, suggesting ecological diversification among syntrophs. These findings suggest we may be drastically underestimating the biochemical capabilities, strategies and diversity of syntrophic bacteria thriving at the thermodynamic limit.

Effect of Disinfectant Exposure on Legionella pneumophila Associated with Simulated Drinking Water Biofilms: Release, Inactivation, and Infectivity.

Legionella pneumophila, the most commonly identified causative agent in drinking water associated with disease outbreaks, can be harbored by and released from drinking water biofilms. In this study, the release of biofilm-associated L. pneumophila under simulated drinking water flow containing a disinfectant residual was examined. Meanwhile, the inactivation and infectivity (to amoebae) of the released L. pneumophila were studied. To simulate drinking water system conditions, biofilms were prepared under either disinfectant exposure (predisinfected biofilms) or disinfectant-free (untreated biofilms) conditions, respectively. For experiments with water flow containing a disinfectant to release the biofilm-associated L. pneumophila from these two types of biofilms, the L. pneumophila release kinetics values from predisinfected and untreated biofilms under flow condition were not statistically different (one-way ANOVA, p > 0.05). However, inactivation of the L. pneumophila released from predisinfected biofilms was 1-2 times higher and amoeba infectivity was 2-29 times lower than that from untreated biofilms. The higher disinfectant resistance of L. pneumophila released from untreated biofilms was presumably influenced by the detachment of a larger amount of biofilm material (determined by 16S rRNA qPCR) surrounding the released L. pneumophila. This study highlights the interaction among disinfectant residual, biofilms, and L. pneumophila, which provides guidelines to assess and control pathogen risk.

Complete Nutrient Removal Coupled to Nitrous Oxide Production as a Bioenergy Source by Denitrifying Polyphosphate-Accumulating Organisms.

Coupled aerobic-anoxic nitrous decomposition operation (CANDO) is a promising emerging bioprocess for wastewater treatment that enables direct energy recovery from nitrogen (N) in three steps: (1) ammonium oxidation to nitrite; (2) denitrification of nitrite to nitrous oxide (N2O); and (3) N2O conversion to N2 with energy generation. However, CANDO does not currently target phosphorus (P) removal. Here, we demonstrate that denitrifying polyphosphate-accumulating organism (PAO) enrichment cultures are capable of catalyzing simultaneous biological N and P removal coupled to N2O generation in a second generation CANDO process, CANDO+P. Over 7 months (>300 cycles) of operation of a prototype lab-scale CANDO+P sequencing batch reactor treating synthetic municipal wastewater, we observed stable and near-complete N removal accompanied by sustained high-rate, high-yield N2O production with partial P removal. A substantial increase in abundance of the PAO Candidatus Accumulibacter phosphatis was observed, increasing from 5% of the total bacterial community in the inoculum to over 50% after 4 months. PAO enrichment was accompanied by a strong shift in the dominant Accumulibacter population from clade IIC to clade IA, based on qPCR monitoring of polyphosphate kinase 1 (ppk1) gene variants. Our work demonstrates the feasibility of combining high-rate, high-yield N2O production for bioenergy production with combined N and P removal from wastewater, and it further suggests a putative denitrifying PAO niche for Accumulibacter clade IA.

Direct treatment of high-strength soft drink wastewater using a down-flow hanging sponge reactor: performance and microbial community dynamics.

A stand-alone down-flow hanging sponge (DHS) system with a two-stage configuration was operated for 700 days to treat synthetic soft drink wastewater at 3000 mg/L chemical oxygen demand (COD). Throughout the operation, >90% COD and total organic carbon (TOC) removal efficiency was obtained by the first stage, and a final effluent of COD <60 mg/L (TOC <20 mg/L) was consistently maintained with the second stage. Lower organic removal efficiency was observed to closely correlate with lower pH, higher volatile fatty acid (VFA) concentration, and higher suspended solid (SS) in the effluent. Occasionally, biomass sloughing was observed as a cause to unstable reactor performance in the first stage. The microbial community of the retained biomass on the sponges differed significantly based on spatial locations of sponges, sampling time points, and loading shocks. In general, Proteobacteria were found to be more abundant in the reactor at an organic removal efficiency >80% than that at <50%. Specifically, operational taxonomic units closely related to Tolumonas auensis and Rivicola pingtungensis were identified as important populations that were responsible for degrading the major substrate in the soft drink wastewater toward to the end of the reactor operation. In addition, high abundance of Bacteroidetes in the reactor was speculated to be responsible for the VFA accumulation in the effluent. This study demonstrated that stand-alone DHS reactor could be used in treating high-strength wastewater efficiently.

Characterization of bacterial community dynamics in a full-scale drinking water treatment plant.

Understanding the spatial and temporal dynamics of microbial communities in drinking water systems is vital to securing the microbial safety of drinking water. The objective of this study was to comprehensively characterize the dynamics of microbial biomass and bacterial communities at each step of a full-scale drinking water treatment plant in Beijing, China. Both bulk water and biofilm samples on granular activated carbon (GAC) were collected over 9months. The proportion of cultivable cells decreased during the treatment processes, and this proportion was higher in warm season than cool season, suggesting that treatment processes and water temperature probably had considerable impact on the R2A cultivability of total bacteria. 16s rRNA gene based 454 pyrosequencing analysis of the bacterial community revealed that Proteobacteria predominated in all samples. The GAC biofilm harbored a distinct population with a much higher relative abundance of Acidobacteria than water samples. Principle coordinate analysis and one-way analysis of similarity indicated that the dynamics of the microbial communities in bulk water and biofilm samples were better explained by the treatment processes rather than by sampling time, and distinctive changes of the microbial communities in water occurred after GAC filtration. Furthermore, 20 distinct OTUs contributing most to the dissimilarity among samples of different sampling locations and 6 persistent OTUs present in the entire treatment process flow were identified. Overall, our findings demonstrate the significant effects that treatment processes have on the microbial biomass and community fluctuation and provide implications for further targeted investigation on particular bacteria populations.

Response of Simulated Drinking Water Biofilm Mechanical and Structural Properties to Long-Term Disinfectant Exposure.

Mechanical and structural properties of biofilms influence the accumulation and release of pathogens in drinking water distribution systems (DWDS). Thus, understanding how long-term residual disinfectants exposure affects biofilm mechanical and structural properties is a necessary aspect for pathogen risk assessment and control. In this study, elastic modulus and structure of groundwater biofilms was monitored by atomic force microscopy (AFM) and optical coherence tomography (OCT) during three months of exposure to monochloramine or free chlorine. After the first month of disinfectant exposure, the mean stiffness of monochloramine- or free-chlorine-treated biofilms was 4 to 9 times higher than those before treatment. Meanwhile, the biofilm thickness decreased from 120 ± 8 µm to 93 ± 6-107 ± 11 µm. The increased surface stiffness and decreased biofilm thickness within the first month of disinfectant exposure was presumably due to the consumption of biomass. However, by the second to third month during disinfectant exposure, the biofilm mean stiffness showed a 2- to 4-fold decrease, and the biofilm thickness increased to 110 ± 7-129 ± 8 µm, suggesting that the biofilms adapted to disinfectant exposure. After three months of the disinfectant exposure process, the disinfected biofilms showed 2-5 times higher mean stiffness (as determined by AFM) and 6-13-fold higher ratios of protein over polysaccharide, as determined by differential staining and confocal laser scanning microscopy (CLSM), than the nondisinfected groundwater biofilms. However, the disinfected biofilms and nondisinfected biofilms showed statistically similar thicknesses (t test, p > 0.05), suggesting that long-term disinfection may not significantly remove net biomass. This study showed how biofilm mechanical and structural properties vary in response to a complex DWDS environment, which will contribute to further research on the risk assessment and control of biofilm-associated-pathogens in DWDS.

Global metabolomic responses of Nitrosomonas europaea 19718 to cold stress and altered ammonia feeding patterns.

The model ammonia-oxidizing bacterium Nitrosomonas europaea represents one of the environmentally and biotechnologically significant microorganisms. Genome-based studies over the last decade have led to many intriguing discoveries about its cellular biochemistry and physiology. However, knowledge regarding the regulation of overall metabolic routes in response to various environmental stresses is limited due to a lack of comprehensive, time-resolved metabolomic analyses. In this study, gas chromatography-mass spectrometry (GC-MS)-based metabolic profiling was performed to characterize the temporal variations of N. europaea 19718 intercellular metabolites in response to varied temperature (23 and 10 °C) and ammonia feeding patterns (shock loading and continuous feeding of 20 mg N/L). Approximately 87 metabolites were successfully identified and mapped to the existing pathways of N. europaea 19718, allowing interpretation of the influence of temperature and feeding pattern on metabolite levels. In general, varied temperature had a more profound influence on the overall metabolism than varied feeding patterns. Total extracellular metabolite concentrations (relative to internal standards and normalized to biomass weight) were lower under cold stress and shock loading conditions compared with the control (continuous feeding at 23 °C). Cold stress caused the widespread downregulation of metabolites involved in central carbon metabolism, amino acid, and lipid synthesis (e.g., malonic acid, succinic acid, putrescine, and phosphonolpyruvate). Metabolites that showed differences under varied feeding patterns were mainly involved in nucleotide acid, amino acid, and lipid metabolism (e.g., adenine, uracil, and spermidine). This study highlighted the roles of central carbon and nitrogen metabolism in countering cold stress and altered ammonia availability. In addition, transcriptomic, proteomic, and metabolomic data from three studies on N. europaea were compared to achieve a holistic view of some important synergy and interconnectivity among different cellular components (RNA, protein, and metabolites) during ammonia starvation.

Effects of hydraulic retention time on aerobic granulation and granule growth kinetics at steady state with a fast start-up strategy.

A hydraulic retention time (HRT) of 4, 6, and 8 h was employed, respectively, in three reactors to study the effects of HRT on granulation with a newly developed fast granulation strategy, i.e., a strategy by combining strong hydraulic selection pressure with high organic loading rate (OLR). Granules with clear boundary appeared within 24 h after reactor start-up and all reactors reached a pseudo steady state after 6-day operation. A 4-h HRT resulted in a relatively higher increasing rate in terms of granule size during granule development period, i.e., 208 µm day(-1), and the bigger granule size and the higher sludge volume index at the pseudo steady state. For HRT of 6 or 8 h, no obvious difference was observed. However, it was found that HRT influenced sludge retention time (SRT) and kinetics significantly. A HRT changing from 4 to 8 h led to an increased SRT from 3 to 21 days, a decreased observed specific biomass growth rate (µ obs) and an decreased observed biomass yield (Y obs) of stable granules from 0.37 to 0.062 days(-1), and 0.177 to 0.055 g MLVSS g(-1) COD, respectively. Both µ obs and Y obs had a linear relationship with the reciprocal of HRT. In addition, the great difference of microbial community between seed sludge, sludge retained in the reactors, and sludge washed out indicated a strong microbial selection for fast granulation within 24 h. However, during the granule development period from day 1 to 6, no more microbial selection was observed except an adjustment of microbial community. Little influence of HRT on microbial population in granular sludge indicated a minor role of HRT played for granulation with the fast start-up strategy adopted in this study. The results demonstrated that hydraulic selection pressure for granulation was mainly from short settling time, which led to strong microbial selection during the granulation period. Meanwhile, although HRT did not affect granulation with the fast start-up strategy, it played an important role on sludge retention time and excess sludge production. Therefore, HRT should be carefully optimized to balance benefits and shortfalls it brings to aerobic granular sludge system.

The nexus of syntrophy-associated microbiota in anaerobic digestion revealed by long-term enrichment and community survey.

Anaerobic digestion (AD) processes are known to effectively convert organic waste to CO2 and CH4 , but much of the microbial ecology remains unclear. Specifically, we have limited insights into symbiotic syntroph and methanogen ('syntrophy') acid degradation, although they are essential for preventing process deterioration. Also, we often observed many uncharacterized or uncultivated organisms, but poorly understood their role(s) in relation to syntrophy. To define syntrophy-associated populations, this study enriched methanogenic communities with propionate, butyrate, benzoate, acetate, formate and H2 from two different inocula over 3 years. 16S pyrotag analysis revealed core populations of known syntrophs (six clades) and methanogens (nine clades) associated with acid degradation, and evidence for substrate- and/or inoculum-dependent specificity in syntrophic partnerships. Based on comprehensive re-evaluation of publically available microbial community data for AD, the known syntrophs and methanogens identified were clearly representatives of the AD-associated syntrophs and methanogens. In addition, uncultivated clades related to Bacteroidetes, Firmicutes, Actinobacteria and Chloroflexi were ubiquitously found in AD and enrichments. These organisms may be universally involved in AD syntrophic degradation, but only represented <23% of the yet-to-be-cultivated organisms (89 of 390 clades). Thus, the contribution of these uncultured organisms in AD remains unclear and warrants further investigation.

The genome of Syntrophorhabdus aromaticivorans strain UI provides new insights for syntrophic aromatic compound metabolism and electron flow.

How aromatic compounds are degraded in various anaerobic ecosystems (e.g. groundwater, sediments, soils and wastewater) is currently poorly understood. Under methanogenic conditions (i.e. groundwater and wastewater treatment), syntrophic metabolizers are known to play an important role. This study explored the draft genome of Syntrophorhabdus aromaticivorans strain UI and identified the first syntrophic phenol-degrading phenylphosphate synthase (PpsAB) and phenylphosphate carboxylase (PpcABCD) and syntrophic terephthalate-degrading decarboxylase complexes. The strain UI genome also encodes benzoate degradation through hydration of the dienoyl-coenzyme A intermediate as observed in Geobacter metallireducens and Syntrophus aciditrophicus. Strain UI possesses electron transfer flavoproteins, hydrogenases and formate dehydrogenases essential for syntrophic metabolism. However, the biochemical mechanisms for electron transport between these H2 /formate-generating proteins and syntrophic substrate degradation remain unknown for many syntrophic metabolizers, including strain UI. Analysis of the strain UI genome revealed that heterodisulfide reductases (HdrABC), which are poorly understood electron transfer genes, may contribute to syntrophic H2 and formate generation. The genome analysis further identified a putative ion-translocating ferredoxin : NADH oxidoreductase (IfoAB) that may interact with HdrABC and dissimilatory sulfite reductase gamma subunit (DsrC) to perform novel electron transfer mechanisms associated with syntrophic metabolism.

Phenotypic and Phylogenetic Identification of Coliform Bacteria Obtained Using 12 Coliform Methods Approved by the U.S. Environmental Protection Agency.

The current definition of coliform bacteria is method dependent, and when different culture-based methods are used, discrepancies in results can occur and affect the accuracy of identification of true coliforms. This study used an alternative approach to the identification of true coliforms by combining the phenotypic traits of the coliform isolates and the phylogenetic affiliation of 16S rRNA gene sequences with the use of lacZ and uidA genes. A collection of 1,404 isolates detected by 12 U.S. Environmental Protection Agency-approved coliform-testing methods were characterized based on their phylogenetic affiliations and responses to their original isolation media and lauryl tryptose broth, m-Endo, and MI agar media. Isolates were phylogenetically classified into 32 true-coliform, or targeted Enterobacteriaceae (TE), groups and 14 noncoliform, or nontargeted Enterobacteriaceae (NTE), groups. It was shown statistically that detecting true-positive (TP) events is more challenging than detecting true-negative (TN) events. Furthermore, most false-negative (FN) events were associated with four TE groups (i.e., Serratia group I and the Providencia, Proteus, and Morganella groups) and most false-positive (FP) events with two NTE groups, the Aeromonas and Plesiomonas groups. In Escherichia coli testing, 18 out of 145 E. coli isolates identified by enzymatic methods were validated as FN. The reasons behind the FP and FN reactions could be explained through analysis of the lacZ and uidA genes. Overall, combining the analyses of the 16S rRNA, lacZ, and uidA genes with the growth responses of TE and NTE on culture-based media is an effective way to evaluate the performance of coliform detection methods.

Responses of Bacterial Communities to Simulated Climate Changes in Alpine Meadow Soil of the Qinghai-Tibet Plateau.

The soil microbial community plays an important role in terrestrial carbon and nitrogen cycling. However, microbial responses to climate warming or cooling remain poorly understood, limiting our ability to predict the consequences of future climate changes. To address this issue, it is critical to identify microbes sensitive to climate change and key driving factors shifting microbial communities. In this study, alpine soil transplant experiments were conducted downward or upward along an elevation gradient between 3,200 and 3,800 m in the Qinghai-Tibet plateau to simulate climate warming or cooling. After a 2-year soil transplant experiment, soil bacterial communities were analyzed by pyrosequencing of 16S rRNA gene amplicons. The results showed that the transplanted soil bacterial communities became more similar to those in their destination sites and more different from those in their "home" sites. Warming led to increases in the relative abundances in Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria and decreases in Acidobacteria, Betaproteobacteria, and Deltaproteobacteria, while cooling had opposite effects on bacterial communities (symmetric response). Soil temperature and plant biomass contributed significantly to shaping the bacterial community structure. Overall, climate warming or cooling shifted the soil bacterial community structure mainly through species sorting, and such a shift might correlate to important biogeochemical processes such as greenhouse gas emissions. This study provides new insights into our understanding of soil bacterial community responses to climate warming and cooling.

Role of biofilm roughness and hydrodynamic conditions in Legionella pneumophila adhesion to and detachment from simulated drinking water biofilms.

Biofilms in drinking water distribution systems (DWDS) could exacerbate the persistence and associated risks of pathogenic Legionella pneumophila (L. pneumophila), thus raising human health concerns. However, mechanisms controlling adhesion and subsequent detachment of L. pneumophila associated with biofilms remain unclear. We determined the connection between L. pneumophila adhesion and subsequent detachment with biofilm physical structure characterization using optical coherence tomography (OCT) imaging technique. Analysis of the OCT images of multispecies biofilms grown under low nutrient condition up to 34 weeks revealed the lack of biofilm deformation even when these biofilms were exposed to flow velocity of 0.7 m/s, typical flow for DWDS. L. pneumophila adhesion on these biofilm under low flow velocity (0.007 m/s) positively correlated with biofilm roughness due to enlarged biofilm surface area and local flow conditions created by roughness asperities. The preadhered L. pneumophila on selected rough and smooth biofilms were found to detach when these biofilms were subjected to higher flow velocity. At the flow velocity of 0.1 and 0.3 m/s, the ratio of detached cell from the smooth biofilm surface was from 1.3 to 1.4 times higher than that from the rough biofilm surface, presumably because of the low shear stress zones near roughness asperities. This study determined that physical structure and local hydrodynamics control L. pneumophila adhesion to and detachment from simulated drinking water biofilm, thus it is the first step toward reducing the risk of L. pneumophila exposure and subsequent infections.