Alterations of endophytic microbial community function in Spartina alterniflora as a result of crude oil exposure.

The 2010 Deepwater Horizon disaster remains one of the largest oil spills in history. This event caused significant damage to coastal ecosystems, the full extent of which has yet to be fully determined. Crude oil contains toxic heavy metals and substances such as polycyclic aromatic hydrocarbons that are detrimental to some microbial species and may be used as food and energy resources by others. As a result, oil spills have the potential to cause significant shifts in microbial communities. This study assessed the impact of oil contamination on the function of endophytic microbial communities associated with saltmarsh cordgrass (Spartina alterniflora). Soil samples were collected from two locations in coastal Louisiana, USA: one severely affected by the Deepwater Horizon oil spill and one relatively unaffected location. Spartina alterniflora seedlings were grown in both soil samples in greenhouses, and GeoChip 5.0 was used to evaluate the endophytic microbial metatranscriptome shifts in response to host plant oil exposure. Oil exposure was associated with significant shifts in microbial gene expression in functional categories related to carbon cycling, virulence, metal homeostasis, organic remediation, and phosphorus utilization. Notably, significant increases in expression were observed in genes related to metal detoxification with the exception of chromium, and both significant increases and decreases in expression were observed in functional gene subcategories related to hydrocarbon metabolism. These findings show that host oil exposure elicits multiple changes in gene expression from their endophytic microbial communities, producing effects that may potentially impact host plant fitness.

A novel PCR-clamping assay reducing plant host DNA amplification significantly improves prokaryotic endo-microbiome community characterization.

Due to the sequence homology between the bacterial 16S rRNA gene and plant chloroplast and mitochondrial DNA, the taxonomic characterization of plant microbiome using amplicon-based high throughput sequencing often results in the overwhelming presence of plant-affiliated reads, preventing the thorough description of plant-associated microbial communities. In this work we developed a PCR blocking primer assay targeting the taxonomically informative V5-V6 region of the 16S rRNA gene in order to reduce plant DNA co-amplification, and increase diversity coverage of associated prokaryotic communities. Evaluation of our assay on the characterization of the prokaryotic endophytic communities of Zea mays, Pinus taeda and Spartina alternifora leaves led to significantly reducing the proportion of plant reads, yielded 20 times more prokaryotic reads and tripled the number of detected OTUs compared to a commonly used V5-V6 PCR protocol. To expand the application of our PCR-clamping assay across a wider taxonomic spectrum of plant hosts, we additionally provide an alignment of chloroplast and mitochondrial DNA sequences encompassing more than 200 terrestrial plant families as a supporting tool for customizing our blocking primers.

Acetate promotes microbial reductive debromination of tetrabromobisphenol A during the startup phase of anaerobic wastewater sludge bioreactors.

The detection of increasing concentrations of tetrabromobisphenol A (TBBPA) in wastewater treatment plants is raising concerns as TBBPA has been identified as a potentially toxic flame retardant. The objectives of this study were to evaluate the effect of acetate biostimulation on TBBPA microbial reductive debromination, and the response of anaerobic sludge associated microbial communities repeatedly exposed to TBBPA. Results indicate that the bulk of the microbial community did not experience significant shifts as a result of TBBPA exposure, and that only a small fraction of the community responded to the presence of TBBPA. Taxa most likely responsible for TBBPA transformation affiliated to Clostridiales and the wastewater sludge group Blvii28. The biostimulating effect of acetate was only observed during reactor startup, when acetogenesis was likely not yet occurring. However, when acetate likely started to be microbially generated in the reactor, acetate addition resulted in a slight but significant inhibiting effect on TBBPA transformation. A significant increase of hydrogenotrophic methanogens in the TBBPA-spiked reactor overtime implies that TBBPA degraders were not in direct competition with methanogens for H2. These results strongly suggest that TBBPA degrading taxa might have been primarily using acetate as an electron donor for the reductive debromination of TBBPA.

Microbial community response to silver nanoparticles and Ag+ in nitrifying activated sludge revealed by ion semiconductor sequencing.

Silver nanoparticles (AgNPs), which are known to act as biocides, are incorporated into medical and consumer products including athletic clothing, stuffed animals, liquid dietary supplements, and more. The increasing use of AgNPs in these products is likely to lead to their entry into both natural and engineered systems, which has the potential to disrupt bacterial processes including those involved in nutrient cycling in wastewater treatment. In the present study, sequencing batch reactors (SBR) mimicking secondary wastewater treatment were operated to determine the effects of AgNPs on the microbial communities contained within activated sludge of wastewater treatment plants (WWTP). SBRs were treated with 0.2 and 2ppm of either gum Arabic (GA)-coated AgNPs, citrate (Ca)-coated AgNPs, or Ag+ as AgNO3. Cell samples were collected and DNA isolated periodically throughout SBR operation. DNA was used for Ion Torrent Next Gen Sequencing of the V3 region of the 16S rDNA gene. Subsequent analyses revealed that the microbial community both shifted and recovered quickly in response to Ag+. Both AgNP treatments resulted in slower initial community shifts than that observed with the Ag+ treatment. GA-AgNPs elicited the longest lasting effect. Additional examination of nitrogen removal bacteria suggested the possibility of an increase in sludge bulking species with increased concentrations of AgNPs in WWTPs. This study supports the hypothesis that Ag+ release from AgNPs is largely coating-dependent and thus a key driver in dictating AgNP toxicity.

Biochar and activated carbon act as promising amendments for promoting the microbial debromination of tetrabromobisphenol A.

The increasing occurrence of tetrabromobisphenol A (TBBPA) in the environment is raising questions about its potential environmental health impacts as it has been shown to cause various deleterious effects in humans. The fact that the highest concentrations of TBBPA have been reported in wastewater sludge is concerning as effluent discharge and biosolids land application are likely a route by which TBBPA can be further disbursed to the environment. Our objectives in this study were to evaluate the effect of biochar (BC) and activated carbon (AC) in promoting the biodegradation of TBBPA, and characterize the response of anaerobic sludge microbial communities following amendments. Both carbonaceous amendments were found to promote the reductive debromination of TBBPA. Nearly complete transformation of TBBPA to BPA was observed in the amended reactors ∼20 days earlier than in the control reactors. In particular, the transformation of diBBPA to monoBBPA, which appears to be the rate-limiting step, was accelerated in the presence of either amendment. Overall, microbial taxa responding to the amendments, i.e., 'sensitive responders', represented a small proportion of the community (i.e., 7.2%), and responded positively. However, although both amendments had a similar effect on TBBPA degradation, the taxonomic profile of the sensitive responders differed greatly from one amendment to the other. BC had a taxonomically broader and slightly more pronounced effect than AC. This work suggests that BC and AC show great potential to promote the biodegradation of TBBPA in anaerobic sludge, and their integration into wastewater treatment processes may be helpful for removing TBBPA and possibly other emerging hydrophobic contaminants.

Distance decay relationships in foliar fungal endophytes are driven by rare taxa.

Foliar fungal endophytes represent a diverse and species-rich plant microbiome. Their biogeography provides essential clues to their cryptic relationship with hosts and the environment in which they disperse. We present species composition, diversity, and dispersal patterns of endophytic fungi associated with needles of Pinus taeda trees across regional scales in the absence of strong environmental gradients as well as within individual trees. An empirical designation of rare and abundant taxa enlightens us on the structure of endophyte communities. We report multiple distance-decay patterns consistent with effects of dispersal limitation, largely driven by community changes in rare taxa, those taxonomic units that made up less than 0.31% of reads per sample on average. Distance-decay rates and community structure also depended on specific classes of fungi and were predominantly influenced by rare members of Dothideomycetes. Communities separated by urban areas also revealed stronger effects of distance on community similarity, confirming that host density and diversity plays an important role in symbiont biogeography, which may ultimately lead to a mosaic of functional diversity as well as rare species diversity across landscapes.

Characterization and Adaptation of Anaerobic Sludge Microbial Communities Exposed to Tetrabromobisphenol A.

The increasing occurrence of tetrabromobisphenol A (TBBPA) in the environment is raising questions about its potential ecological and human health impacts. TBBPA is microbially transformed under anaerobic conditions to bisphenol A (BPA). However, little is known about which taxa degrade TBBPA and the adaptation of microbial communities exposed to TBBPA. The objectives of this study were to characterize the effect of TBBPA on microbial community structure during the start-up phase of a bench-scale anaerobic sludge reactor, and identify taxa that may be associated with TBBPA degradation. TBBPA degradation was monitored using LC/MS-MS, and the microbial community was characterized using Ion Torrent sequencing and qPCR. TBBPA was nearly completely transformed to BPA via reductive debromination in 55 days. Anaerobic reactor performance was not negatively affected by the presence of TBBPA and the bulk of the microbial community did not experience significant shifts. Several taxa showed a positive response to TBBPA, suggesting they may be associated with TBBPA degradation. Some of these taxa had been previously identified as dehalogenating bacteria including Dehalococcoides, Desulfovibrio, Propionibacterium, and Methylosinus species, but most had not previously been identified as having dehalogenating capacities. This study is the first to provide in-depth information on the microbial dynamics of anaerobic microbial communities exposed to TBBPA.

A review of the environmental implications of in situ remediation by nanoscale zero valent iron (nZVI): Behavior, transport and impacts on microbial communities.

The increasing use of strategies incorporating nanoscale zero valent iron (nZVI) for soil and groundwater in situ remediation is raising some concerns regarding the potential adverse effects nZVI could have on indigenous microbial communities and ecosystem functioning. This review provides an overview of the current literature pertaining to the impacts of nZVI applications on microbial communities. Toxicity studies suggest that cell membrane disruption and oxidative stress through the generation of Fe(2+) and reactive oxygen species by nZVI are the main mechanisms contributing to nZVI cytotoxicity. In addition, nZVI has been shown to substantially alter the taxonomic and functional composition of indigenous microbial communities. However, because the physico-chemical conditions encountered in situ highly modulate nZVI toxicity, a better understanding of the environmental factors affecting nZVI toxicity and transport in the environment is of primary importance in evaluating the ecological consequences that could result from a more extensive use of nZVI.

Comparison of bacterial and archaeal communities in depth-resolved zones in an LNAPL body.

Advances in our understanding of the microbial ecology at sites impacted by light non-aqueous phase liquids (LNAPLs) are needed to drive development of optimized bioremediation technologies, support longevity models, and develop culture-independent molecular tools. In this study, depth-resolved characterization of geochemical parameters and microbial communities was conducted for a shallow hydrocarbon-impacted aquifer. Four distinct zones were identified based on microbial community structure and geochemical data: (i) an aerobic, low-contaminant mass zone at the top of the vadose zone; (ii) a moderate to high-contaminant mass, low-oxygen to anaerobic transition zone in the middle of the vadose zone; (iii) an anaerobic, high-contaminant mass zone spanning the bottom of the vadose zone and saturated zone; and (iv) an anaerobic, low-contaminant mass zone below the LNAPL body. Evidence suggested that hydrocarbon degradation is mediated by syntrophic fermenters and methanogens in zone III. Upward flux of methane likely contributes to promoting anaerobic conditions in zone II by limiting downward flux of oxygen as methane and oxygen fronts converge at the top of this zone. Observed sulfate gradients and microbial communities suggested that sulfate reduction and methanogenesis both contribute to hydrocarbon degradation in zone IV. Pyrosequencing revealed that Syntrophus- and Methanosaeta-related species dominate bacterial and archaeal communities, respectively, in the LNAPL body below the water table. Observed phylotypes were linked with in situ anaerobic hydrocarbon degradation in LNAPL-impacted soils.

A comparison of the community diversity of foliar fungal endophytes between seedling and adult loblolly pines (Pinus taeda).

Fungal endophytes represent one of the most ubiquitous plant symbionts on Earth and are phylogenetically diverse. The structure and diversity of endophyte communities have been shown to depend on host taxa and climate, but there have been relatively few studies exploring endophyte communities throughout host maturity. We compared foliar fungal endophyte communities between seedlings and adult trees of loblolly pines (Pinus taeda) at the same seasons and locations by culturing and culture-independent methods. We sequenced the internal transcribed spacer region and adjacent partial large subunit nuclear ribosomal RNA gene (ITS-LSU amplicon) to delimit operational taxonomic units and phylogenetically characterize the communities. Despite the lower infection frequency in seedlings compared to adult trees, seedling needles were receptive to a more diverse community of fungal endophytes. Culture-free method confirmed the presence of commonly cultured OTUs from adult needles but revealed several new OTUs from seedling needles that were not found with culturing methods. The two most commonly cultured OTUs in adults were rarely cultured from seedlings, suggesting that host age is correlated with a selective enrichment for specific endophytes. This shift in endophyte species dominance may be indicative of a functional change between these fungi and their loblolly pine hosts.

Unique odd-chain polyenoic phospholipid fatty acids present in chytrid fungi.

Chytrid fungi are ubiquitous components of aquatic and terrestrial ecosystems yet they remain understudied. To investigate the use of phospholipid fatty acids as phenotypic characteristics in taxonomic studies and biomarkers for ecological studies, 18 chytrid fungi isolated from soil to freshwater samples were grown in defined media and their phospholipid fatty acid profile determined. Gas chromatographic/mass spectral analysis indicated the presence of fatty acids typically associated with fungi, such as 16:1(n-7), 16:0, 18:2(n-6), 18:3(n-3) 18:1(n-9), and 18:0, as well as, a number of odd-chain length fatty acids, including two polyunsaturated C-17 fatty acids. Conversion to their 3-pyridylcarbinol ester facilitated GC-MS determination of double-bond positions and these fatty acid were identified as 6,9-17:2 [17:2(n-8)] and 6,9,12-17:3 [17:3(n-5)]. To the best of our knowledge, this is the first report of polyunsaturated C-17 fatty acids isolated from the phospholipids of chytrid fungi. Cluster analysis of PLFA profiles showed sufficient correlation with chytrid phylogeny to warrant inclusion of lipid analysis in species descriptions and the presence of several phospholipid fatty acids of restricted phylogenetic distributions suggests their usefulness as biomarkers for ecological studies.

Molecular assessment of the sensitivity of sulfate-reducing microbial communities remediating mine drainage to aerobic stress.

Sulfate-reducing permeable reactive zones (SR-PRZs) are microbially-driven anaerobic systems designed for the removal of heavy metals and sulfate in mine drainage. Environmental perturbations, such as oxygen exposure, may adversely affect system stability and long-term performance. The objective of this study was to examine the effect of two successive aerobic stress events on the performance and microbial community composition of duplicate laboratory-scale lignocellulosic SR-PRZs operated using the following microbial community management strategies: biostimulation with ethanol or carboxymethylcellulose; bioaugmentation with sulfate-reducing or cellulose-degrading enrichments; inoculation with dairy manure only; and no inoculation. A functional gene-based approach employing terminal restriction fragment length polymorphism and quantitative polymerase chain reaction targeting genes of sulfate-reducing (dsrA), cellulose-degrading (cel5, cel48), fermentative (hydA), and methanogenic (mcrA) microbes was applied. In terms of performance (i.e., sulfate removal), biostimulation with ethanol was the only strategy that clearly had an effect (positive) following exposure to oxygen. In terms of microbial community composition, significant shifts were observed over the course of the experiment. Results suggest that exposure to oxygen more strongly influenced microbial community shifts than the different microbial community management strategies. Sensitivity to oxygen exposure varied among different populations and was particularly pronounced for fermentative bacteria. Although the community structure remained altered after exposure, system performance recovered, indicating that SR-PRZ microbial communities were functionally redundant. Results suggest that pre-exposure to oxygen might be a more effective strategy to improve the resilience of SR-PRZ microbial communities relative to bioaugmentation or biostimulation.

Three new genera in Chytridiales from aquatic habitats in Argentina.

Sampling for chytrids in a variety of habitats has resulted in pure cultures that when analyzed have yielded hypotheses of relationships based on molecular and zoospore ultrastructural markers. To extend our understanding of diversity of Chytridiales in eastern Argentina and USA, we isolated and examined the morphology, ultrastructure and 28S and ITS1-5.8S-ITS2 rDNA sequences of numerous chytrids from aquatic habitats from these two regions. Three family-level lineages (Chytridiaceae, Chytriomycetaceae, family incertae sedis) are represented in our molecular phylogeny, and three new genera (Avachytrium, Odontochytrium in Chytriomycetaceae, Delfinachytrium in family incertae sedis) are described. These findings of new genera and species emphasize the potential for discovery of additional diversity.

Strategy to find molecular signatures in a small series of rare cancers: validation for radiation-induced breast and thyroid tumors.

Methods of classification using transcriptome analysis for case-by-case tumor diagnosis could be limited by tumor heterogeneity and masked information in the gene expression profiles, especially as the number of tumors is small. We propose a new strategy, EMts_2PCA, based on: 1) The identification of a gene expression signature with a great potential for discriminating subgroups of tumors (EMts stage), which includes: a) a learning step, based on an expectation-maximization (EM) algorithm, to select sets of candidate genes whose expressions discriminate two subgroups, b) a training step to select from the sets of candidate genes those with the highest potential to classify training tumors, c) the compilation of genes selected during the training step, and standardization of their levels of expression to finalize the signature. 2) The predictive classification of independent prospective tumors, according to the two subgroups of interest, by the definition of a validation space based on a two-step principal component analysis (2PCA). The present method was evaluated by classifying three series of tumors and its robustness, in terms of tumor clustering and prediction, was further compared with that of three classification methods (Gene expression bar code, Top-scoring pair(s) and a PCA-based method). Results showed that EMts_2PCA was very efficient in tumor classification and prediction, with scores always better that those obtained by the most common methods of tumor clustering. Specifically, EMts_2PCA permitted identification of highly discriminating molecular signatures to differentiate post-Chernobyl thyroid or post-radiotherapy breast tumors from their sporadic counterparts that were previously unsuccessfully classified or classified with errors.

Development of a Real-Time PCR assay for quantitative assessment of uncultured freshwater zoosporic fungi.

Recently, molecular environmental surveys of the eukaryotic microbial community in lakes have revealed a high diversity of sequences belonging to uncultured zoosporic fungi. Although they are known as saprobes and algal parasites in freshwater systems, zoosporic fungi have been neglected in microbial food web studies. Recently, it has been suggested that zoosporic fungi, via the consumption of their zoospores by zooplankters, could transfer energy from large inedible algae and particulate organic material to higher trophic levels. However, because of their small size and their lack of distinctive morphological features, traditional microscopy does not allow the detection of fungal zoospores in the field. Hence, quantitative data on fungal zoospores in natural environments is missing. We have developed a quantitative PCR (qPCR) assay for the quantification of fungal zoospores in lakes. Specific primers were designed and qPCR conditions were optimized using a range of target and non-target plasmids obtained from previous freshwater environmental 18S rDNA surveys. When optimal DNA extraction protocol and qPCR conditions were applied, the qPCR assay developed in this study demonstrated high specificity and sensitivity, with as low as 100 18S rDNA copies per reaction detected. Although the present work focuses on the design and optimization of a new qPCR assay, its application to natural samples indicated that qPCR offers a promising tool for quantitative assessment of fungal zoospores in natural environments. We conclude that this will contribute to a better understanding of the ecological significance of zoosporic fungi in microbial food webs of pelagic ecosystems.

The molecular diversity of freshwater picoeukaryotes reveals high occurrence of putative parasitoids in the plankton.

Eukaryotic microorganisms have been undersampled in biodiversity studies in freshwater environments. We present an original 18S rDNA survey of freshwater picoeukaryotes sampled during spring/summer 2005, complementing an earlier study conducted in autumn 2004 in Lake Pavin (France). These studies were designed to detect the small unidentified heterotrophic flagellates (HF, 0.6-5 microm) which are considered the main bacterivores in aquatic systems. Alveolates, Fungi and Stramenopiles represented 65% of the total diversity and differed from the dominant groups known from microscopic studies. Fungi and Telonemia taxa were restricted to the oxic zone which displayed two fold more operational taxonomic units (OTUs) than the oxycline. Temporal forcing also appeared as a driving force in the diversification within targeted organisms. Several sequences were not similar to those in databases and were considered as new or unsampled taxa, some of which may be typical of freshwater environments. Two taxa known from marine systems, the genera Telonema and Amoebophrya, were retrieved for the first time in our freshwater study. The analysis of potential trophic strategies displayed among the targeted HF highlighted the dominance of parasites and saprotrophs, and provided indications that these organisms have probably been wrongfully regarded as bacterivores in previous studies. A theoretical exercise based on a new 'parasite/saprotroph-dominated HF hypothesis' demonstrates that the inclusion of parasites and saprotrophs may increase the functional role of the microbial loop as a link for carbon flows in pelagic ecosystems. New interesting perspectives in aquatic microbial ecology are thus opened.

Unveiling fungal zooflagellates as members of freshwater picoeukaryotes: evidence from a molecular diversity study in a deep meromictic lake.

This study presents an original 18S rRNA PCR survey of the freshwater picoeukaryote community, and was designed to detect unidentified heterotrophic picoflagellates (size range 0.6-5 microm) which are prevalent throughout the year within the heterotrophic flagellate assemblage in Lake Pavin. Four clone libraries were constructed from samples collected in two contrasting zones in the lake. Computerized statistic tools have suggested that sequence retrieval was representative of the in situ picoplankton diversity. The two sampling zones exhibited similar diversity patterns but shared only about 5% of the operational taxonomic units (OTUs). Phylogenetic analysis clustered our sequences into three taxonomic groups: Alveolates (30% of OTUs), Fungi (23%) and Cercozoa (19%). Fungi thus substantially contributed to the detected diversity, as was additionally supported by direct microscopic observations of fungal zoospores and sporangia. A large fraction of the sequences belonged to parasites, including Alveolate sequences affiliated to the genus Perkinsus known as zooparasites, and chytrids that include host-specific parasitic fungi of various freshwater phytoplankton species, primarily diatoms. Phylogenetic analysis revealed five novel clades that probably include typical freshwater environmental sequences. Overall, from the unsuspected fungal diversity unveiled, we think that fungal zooflagellates have been misidentified as phagotrophic nanoflagellates in previous studies. This is in agreement with a recent experimental demonstration that zoospore-producing fungi and parasitic activity may play an important role in aquatic food webs.