Molecular Diversity of Bacteroidales in Fecal and Environmental Samples and Swine-Associated Subpopulations

Several swine-specific microbial source tracking methods are based on PCR assays targeting Bacteroidales 16S rRNA gene sequences. The limited application of these assays can be explained by the poor understanding of their molecular diversity in fecal sources and environmental waters. In order to address this, we studied the diversity of 9,340 partial (\u3e600bp in length) Bacteroidales 16S rRNA gene sequences from 13 fecal sources and nine feces-contaminated watersheds. The compositions of major Bacteroidales populations were analyzed to determine which host and environmental sequences were contributing to each group. This information allowed us to identify populations which were both exclusive to swine fecal sources and detected in swine-contaminated waters. Phylogenetic and diversity analyses revealed that some markers previously believed to be highly specific to swine populations are shared by multiple hosts, potentially explaining the cross-amplification signals obtained with nontargeted hosts. These data suggest that while many Bacteroidales populations are cosmopolitan, others exhibit a preferential host distribution and may be able to survive different environmental conditions. This study further demonstrates the importance of elucidating the diversity patterns of targeted bacterial groups to develop more inclusive fecal source tracking applications

Protease and hemicellulase assisted extraction of dietary fiber from wastes of Cynara cardunculus

6057-6075The action of protease and hemicellulase for the extraction of fractions enriched in soluble fiber from bracts and stems of Cynara cardunculus was evaluated. Using a two-factor simplex design comprising protease amounts of 0-200 ?L and hemicellulase amounts of 0-200 mg for 5 g of material, we explored the effect of a 5 h enzymatic treatment at 40 °C on the chemical composition and yield of the fractions isolated. The fractions contained inulin and pectin. In general, the protein, inulin, and polyphenol contents and also the yields were higher for fractions obtained from stems. The most marked effects were observed when enzymes were used at higher concentrations, especially for hemicellulase. The inclusion of a pre-heating step increased the yield and the inulin content for fractions isolated from bracts and stems and decreased the protein and polyphenol contents, and the galacturonic acid for bracts. These fractions, in general, contained the polyphenolic compounds monocaffeoylquinic acid, apigenin, and pinoresinol

Evaluation of Swine-Specific PCR Assays Used for Fecal Source Tracking and Analysis of Molecular Diversity of Swine-Specific bacteroidales Populations

In this study, we evaluated the specificity, distribution, and sensitivity of Prevotella strain-based (PF163 and PigBac1) and methanogen-based (P23-2) PCR assays proposed to detect swine fecal pollution in environmental waters. The assays were tested against 222 fecal DNA extracts derived from target and nontarget animal hosts and against 34 groundwater and 15 surface water samples from five different sites. We also investigated the phylogenetic diversity of 1,340 Bacteroidales 16S rRNA gene sequences derived from swine feces, swine waste lagoons, swine manure pits, and waters adjacent to swine operations. Most swine fecal samples were positive for the host-specific Prevotella-based PCR assays (80 to 87%), while fewer were positive with the methanogen-targeted PCR assay (53%). Similarly, the Prevotella markers were detected more frequently than the methanogen-targeted assay markers in waters historically impacted with swine fecal contamination. However, the PF163 PCR assay cross-reacted with 23% of nontarget fecal DNA extracts, although Bayesian statistics suggested that it yielded the highest probability of detecting pig fecal contamination in a given water sample. Phylogenetic analyses revealed previously unknown swine-associated clades comprised of clones from geographically diverse swine sources and from water samples adjacent to swine operations that are not targeted by the Prevotella assays. While deeper sequencing coverage might be necessary to better understand the molecular diversity of fecal Bacteroidales species, results of sequence analyses supported the presence of swine fecal pollution in the studied watersheds. Overall, due to nontarget cross amplification and poor geographic stability of currently available host-specific PCR assays, development of additional assays is necessary to accurately detect sources of swine fecal pollution

COREXIT 9500 enhances oil biodegradation and changes microbial community structure of oil-enriched microcosms

To better understand the impacts of Corexit 9500 on the structure and activity levels of hydrocarbon-degrading microbial communities, we analyzed next-generation 16S rRNA gene sequencing libraries of hydrocarbon enrichments grown at 5 and 25°C using both DNA and RNA extracts as the sequencing templates. Oil biodegradation patterns in both 5 and 25°C enrichments were consistent with those reported in the literature (i.e., aliphatics were degraded faster than aromatics). Slight increases in biodegradation were observed in the presence of Corexit at both temperatures. Differences in community structure were observed between treatment conditions in the DNA-based libraries. The 25°C consortia were dominated by Vibrio, Idiomarina, Marinobacter, Alcanivorax, and Thalassospira species, while the 5°C consortia were dominated by several species of the genera Flavobacterium, Alcanivorax, and Oleispira. Most of these genera have been linked to hydrocarbon degradation and have been observed after oil spills. Colwellia and Cycloclasticus, known aromatic degraders, were also found in these enrichments. The addition of Corexit did not have an effect on the active bacterial community structure of the 5°C consortia, while at 25°C, a decrease in the relative abundance of Marinobacter was observed. At 25°C, Thalassospira, Marinobacter, and Idiomarina were present at higher relative abundances in the RNA than DNA libraries, suggesting that they were active in degradation. Similarly, Oleispira was greatly stimulated by the addition of oil at 5°C

Activity from SAX J1747.0-2853 and KS 1741-293 detected by INTEGRAL Galactic Bulge Monitoring

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Bioremediation of aqueous pollutants using biomass embedded in hydrophilic foam. Final report

The major objective of this project was to examine the potential of a novel hydrophilic polyurethane foam as an immobilization medium for algal, bacteria, and other types of biomass, and to test the resulting foam/biomass aggregates for their use in cleaning up waters contaminated with heavy metals, radionuclides and toxic organic compounds. Initial investigations focused on the bioremoval of heavy metals from wastewaters at SRS using immobilized algal biomass. This effort met with limited success for reasons which included interference in the binding of biomass and target metals by various non-target constituents in the wastewater, lack of an appropriate wastewater at SRS for testing, and the unavailability of bioreactor systems capable of optimizing contact of target pollutants with sufficient biomass binding sites. Subsequent studies comparing algal, bacterial, fungal, and higher plant biomass demonstrated that other biomass sources were also ineffective for metal bioremoval under the test conditions. Radionuclide bioremoval using a Tc-99 source provided more promising results than the metal removal studies with the various types of biomass, and indicated that the alga Cyanidium was the best of the tested sources of biomass for this application. However, all of the biomass/foam aggregates tested were substantially inferior to a TEVA resin for removing Tc-99 in comparative testing. The authors also explored the use of hydrophilic polyurethane foam to embed Burkholderia cepacia, which is an efficient degrader of trichloroethylene (TCE), a contaminant of considerable concern at SRS and elsewhere. The embedded population proved to be incapable of growth on nutrient media, but retained respiratory activity. Lastly, the degradative capabilities of embedded G4 were examined. Phenol- or benzene-induced bacteria retained the ability to degrade TCE and benzene. The authors were successful in inducing enzyme activity after the organisms had already been embedded

Further INTEGRAL observations of the transient X-ray burster EXO 1745-248

INTEGRAL performed further observations as part of the Galactic Bulge monitoring program (see ATel #438) on 2010 October 11 between (UTC) 02:25 and 06:07. The X-ray source in outburst in the direction of the globular cluster Terzan 5, EXO 1745-248 (ATels #2919, #2920 and #2922), is detected with both JEM-X 1&2, and with IBIS/ISGRI. A re-analysis of the observations performed on 2010 October 10 from (UTC) 08:45 to 12:26 (ATel #2919), shows that the source was detected in the JEM-X monitor at a position (RA=267.023, DEC=-24.781, with a 90% confidence error of 1 arcmin) consistent with the known X-ray burster EXO 1745-248 (Heinke et al. 2003, ApJ, 590, 809). At that time the flux was 9 +/- 5 mCrab (3-10 keV) and 33 +/-9 mCrab (10-25 keV). The observations on October 11 shows that within a day the flux has increased to 34 +/- 7 mCrab (3-10 keV), 49 +/- 4 mCrab (10-25 keV), 52 +/- 2 mCrab (18-40 keV), and 49 +/- 5 mCrab (40-100 keV). We detected one type I X-ray burst, on 2010 October 11 06:27, which lasted for about 20 sec. This strengthens EXO 1745-248 to be in outburst, as it is a known (transient) X-ray burster

Evaluation of Swine-Specific PCR Assays Used for Fecal Source Tracking and Analysis of Molecular Diversity of Swine-Specific “Bacteroidales” Populations

In this study, we evaluated the specificity, distribution, and sensitivity of Prevotella strain-based (PF163 and PigBac1) and methanogen-based (P23-2) PCR assays proposed to detect swine fecal pollution in environmental waters. The assays were tested against 222 fecal DNA extracts derived from target and nontarget animal hosts and against 34 groundwater and 15 surface water samples from five different sites. We also investigated the phylogenetic diversity of 1,340 “Bacteroidales” 16S rRNA gene sequences derived from swine feces, swine waste lagoons, swine manure pits, and waters adjacent to swine operations. Most swine fecal samples were positive for the host-specific Prevotella-based PCR assays (80 to 87%), while fewer were positive with the methanogen-targeted PCR assay (53%). Similarly, the Prevotella markers were detected more frequently than the methanogen-targeted assay markers in waters historically impacted with swine fecal contamination. However, the PF163 PCR assay cross-reacted with 23% of nontarget fecal DNA extracts, although Bayesian statistics suggested that it yielded the highest probability of detecting pig fecal contamination in a given water sample. Phylogenetic analyses revealed previously unknown swine-associated clades comprised of clones from geographically diverse swine sources and from water samples adjacent to swine operations that are not targeted by the Prevotella assays. While deeper sequencing coverage might be necessary to better understand the molecular diversity of fecal Bacteroidales species, results of sequence analyses supported the presence of swine fecal pollution in the studied watersheds. Overall, due to nontarget cross amplification and poor geographic stability of currently available host-specific PCR assays, development of additional assays is necessary to accurately detect sources of swine fecal pollution