DNA Pyrosequencing Research Articles

Debromination of PBDEs in Arkansas Water Bodies Analyzed by Positive Matrix Factorization.

A previously generated data set for polybrominated diphenyl ethers (PBDEs) in dated sediment cores of West Lake of El Dorado (AED), Calion Lake (ACL), and the lagoon of Magnolia Wastewater Treatment Facility (AMW) from Southern Arkansas is examined by a weighted chemical mass balance (CMB) model and positive matrix factorization (PMF) in order to quantify PBDE sources and debromination. DNA extraction and pyrosequencing were done on several core sections in order to investigate microbial debromination. CMB and PMF analyses indicate that deca technical mixtures are the dominant PBDE input (>99% in mole fraction in AED and ACL, and 94.7% in AMW). Minor contributions of penta and octa technical mixtures were found in all three water bodies (<1% in AED and ACL; and 1.1% and 4.1% in AMW, respectively). Results suggest that debromination takes place in all three lakes, but is more intense in AMW. In-situ microbial debromination was supported by the microorganism analysis. The PMF results are validated by PBDE manufacturing records, and the operating history of AMW. Despite the high PBDE concentrations in these sediments near former manufacturing facilities, the extent of debromination is limited, possibly due to sorption to natural organic matter of the sediment.

Glutathione S-transferase pi polymorphism contributes to the treatment outcomes of advanced non-small cell lung cancer patients in a Chinese population.

We analyzed the association between polymorphisms in three glutathione S-transferase genes (GSTP1, GSTM1, and GSTT1) and the treatment outcome for advanced non-small cell lung cancer (NSCLC). We recruited 284 NSCLC patients at advanced stage from Department of Radiotherapy in Peace Hospital Attached to Changzhi Medical College between May 2009 and May 2011, who had received cisplatin-based chemotherapy. The GSTP1, GSTM1, and GSTT1 genotyping for was determined using DNA pyrosequencing on an ABI Prism 3100 DNA analyzer. In the Cox proportional hazards model, the IIe/Val and Val/Val genotypes of GSTP1 were associated with lower risk of disease progression compared with the IIe/IIe genotype, and the HRs (95%CIs) were 0.37 (0.18-0.74) and 0.15 (0.06-0.35), respectively. The IIe/Val and Val/Val genotypes significantly decreased risk of death from all causes in patients with NSCLC, and the HRs (95%CIs) were 0.52 (0.29-0.92) and 0.37 (0.17- 0.79), respectively No significant association was observed between GSTM1 and GSTT1 polymorphisms and progression-free survival and overall survival in the NSCLC patients. In summary, we suggest that GSTP1 polymorphisms might influence the treatment outcome of advanced NSCLC patients, and our results could help improve individualized therapy.

Pyrosequencing on a glass surface.

We demonstrate the use of open-surface microfluidics to sequence DNA by pyrosequencing at the plain hydrophobically coated surface of a microscope glass cover slip. This method offers significant advantages in terms of instrument size, simplicity, disposability, and functional integration, particularly when combined with the broad and flexible capabilities of open-surface microfluidics. The DNA was incubated on superparamagnetic particles and placed on a hydrophobically coated glass substrate. The particles with bound DNA were moved using magnetic force through microliter-sized droplets covered with mineral oil to prevent water evaporation from the droplets. These droplets served as reaction "stations" performing pyrosequencing as well as washing stations. The resequencing protocol with 34-mer single-stranded DNA (ssDNA) was used to determine the reaction performance. The de novo sequencing was performed with 51-mer and 81-mer ssDNA. The method can be integrated with previously shown sample preparation and PCR into a single sample-to-answer system on a plain glass surface.

Pyrosequencing reveals microbial community profile in anaerobic bio-entrapped membrane reactor for pharmaceutical wastewater treatment

In this study, pharmaceutical wastewater with high salinity and total chemical oxygen demand (TCOD) was treated by an anaerobic membrane bioreactor (AnMBR) and an anaerobic bio-entrapped membrane reactor (AnBEMR). The microbial populations and communities were analyzed using the 454 pyrosequencing method. The hydraulic retention time (HRT), membrane flux and mean cell residence time (MCRT) were controlled at 30.6h, 6L/m2h and 100d, respectively. The results showed that the AnBEMR achieved higher TCOD removal efficiency and greater biogas production compared to the AnMBR. Through DNA pyrosequencing analysis, both the anaerobic MBRs showed similar dominant groups of bacteria and archaea. However, phylum Elusimicrobia of bacteria was only detected in the AnBEMR; the higher abundance of dominant archaeal genus Methanimicrococcus found in the AnBEMR could play an important role in degradation of the major organic pollutant (i.e., trimethylamine) present in the pharmaceutical wastewater.

Odontogenic and rhinogenic chronic sinusitis: a modern microbiological comparison.

Odontogenic sinusitis and sinonasal complications of dental disease or treatment (SCDDT) play a relevant, often underappreciated role in paranasal sinus infections. Treating SCDDT patients requires tailored medical and surgical approaches in order to achieve acceptable success rates. These approaches differ from common rhinogenic sinusitis treatment protocols mostly because of the different etiopathogenesis. Our study comprehensively evaluated microbiology and antibiotic resistance in SCDDT patients and compared findings with a control group of patients affected by rhinogenic sinusitis. We performed microbiological sampling during surgery on 28 patients with SCDDT and 16 patients with chronic rhinosinusitis with nasal polyps (CRSwNP). Colonies were isolated, Gram-stained, and the species identified using classic biochemical methods. These results were confirmed by DNA pyrosequencing, and then the resistance profile of each SCDDT isolate to various antibiotics was tested. Microbial growth was observed in all SCDDT patients, whereas samples from 60% of patients in the control group failed to yield any bacterial growth (p < 0.001). Anaerobes grew in 14% of SCDDT patients as compared to 7% of CRSwNP patients (p = 0.42). Of the isolates from SCDDT patients, 70% were susceptible to amoxicillin/clavulanate, whereas all isolates were susceptible to levofloxacin, teicoplanin, and vancomycin. Of the staphylococci identified, 80% were capable of producing beta-lactamase. Given the extent of microbiological contamination within the maxillary sinus of SCDDT patients, these infections should be regarded as a different class of conditions from rhinogenic sinusitis. Our findings support the need for different approaches in the treatment of SCDDT patients.

Abstract 2959: Differential DNA methylation in peripheral blood DNA as a biomarker of hepatocellular carcinoma risk

Abstract Hepatocellular carcinoma (HCC), one of the most prevalent types of primary liver cancer, is the sixth most common cancer worldwide and the third leading cause of cancer death with rising mortality and morbidity rates. As late onset of HCC accounts for late diagnosis and poor prognosis and early detection increases cure rate from 5% to 80%, identifying reliable and quantifiable biomarkers of risk prediction is of high interest. Aberrations in the DNA methylation patterns, an important early event in carcinogenesis, have been shown to differentiate HCC tumors from normal tissues. However, these changes as predictive markers would have a high application in clinics only if detectable by minimally invasive tests like blood test. In the present study, we performed a comprehensive evaluation of DNA methylation profiles in blood DNA collected before diagnosis with HCC. Aberrant methylation was investigated in DNA isolated from blood of 21 HCC patients (cases) who provided samples between 1-4 years prior to diagnosis and 21 controls enrolled by the Indiana Biobank of Indiana CTSI. Cases were matched with controls for gender, age, ethnicity, hepatitis C infection, and diabetes. We used Infinium Human Methylation 450K BeadChip array for genome-wide DNA methylation analysis and pyrosequencing for validation of DNA methylation differences. We found 966 probes differentially methylated between cases and controls with p&amp;lt;0.05 and intraclass correlation coefficient &amp;gt;0.5. Among these significant changes, 732 CpG sites are hypomethylated in HCC cases compared to matched controls and include 130 CpGs corresponding to 75 genes with delta beta value (differential methylation) ≥0.1. Forty six CpG sites out of 234 significantly hypermethylated probes show delta beta value ≥0.1 and correspond to 46 genes. Functional analyses using GO, KEGG and DAVID knowledgebase indicates that hypomethylated genes are associated with Wnt signaling, cell adhesion, blood coagulation, and regulation of transcription, whereas hypermethylated genes are enriched with cytoskeleton organization and small GTPase mediated signal transduction. One of the genes hypomethylated in blood DNA of HCC cases prior to diagnosis is TET1 that was found to be 8-fold overexpressed in HCC tumors in our previous studies and implicated in gene-specific hypomethylation. Interestingly, 75% of differentially methylated sites in blood DNA of HCC cases are hypomethylated prior to diagnosis. Validation by pyrosequencing of four hypomethylated probes including CpGs in the CpG island shore of BRUNOL5 that was linked to fatty liver disease in earlier reports and found demethylated and overexpressed in HCC tumors, suggests their predictive potential. Our results establish the possible predictive value of aberrant methylation, in particular DNA hypomethylation, in blood DNA for risk of HCC. This study was supported by Showalter Trust and Purdue Center for Cancer Research Awards granted to BS. Citation Format: Katarzyna Lubecka-Pietruszewska, Lucinda Kurzava, Hannah Buvala, Kirsty Flower, Samer Gawrieh, Jennifer Mansfield, Naga Chalasani, James M. Flanagan, Barbara Stefanska. Differential DNA methylation in peripheral blood DNA as a biomarker of hepatocellular carcinoma risk. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2959. doi:10.1158/1538-7445.AM2015-2959

A lineage-specific methylation pattern controls the transcription of the polycistronic mRNA coding MELOE melanoma antigens.

We recently characterized two melanoma antigens MELOE-1 and MELOE-2 derived from a polycistronic RNA overexpressed in the melanocytic lineage. This transcription profile was because of hypomethylation of the meloe proximal promoter in melanomas and melanocytes. Here, we investigate whether this demethylation was restricted to the meloe promoter or was linked to a general lack of methylation at the meloe locus in the melanocytic lineage. We establish the methylation pattern of the locus spanning more than 40 kbp, focusing on CpG islands, using DNA bisulfite conversion and pyrosequencing. The study was carried out on cultured cell lines (melanoma, melanocyte, colon cancer, and mesothelioma cell lines), healthy tissues (skin and colon), and melanoma tumors. Demethylation, specifically observed in the melanocytic lineage, involves a large promoter area and not the entire meloe locus. This enables updating a tight regulation of meloe transcription in this lineage, suggesting tissue-specific epigenetic mechanisms. Associated with the previously described translational mechanisms, leading to the specific expression of MELOE-1 and MELOE-2 in melanomas, this makes MELOE-derived antigens a relevant candidate for immunotherapy of melanoma.

Association between xanthine dehydrogenase tag single nucleotide polymorphisms and essential hypertension.

The present study aimed to investigate the association between xanthine dehydrogenase (XDH) gene polymorphism and essential hypertension in the rural Han Chinese population of Fuxin, Liaoning. Han Chinese individuals, who had lived in rural areas of Fuxin, were selected as subjects for the present study. A total of 521 unrelated patients with hypertension were selected, along with a further 533 unrelated individuals with normal blood pressure, in order to serve as controls. Five tag single nucleotide polymorphisms (SNP) of the XDH gene were selected. An estimation of SNP allele frequency was determined using DNA pooling and pyrosequencing methods. Prior to Bonferroni correction, T allele frequency for rs206811 was significantly higher in patients with hypertension, as compared with the controls (64.1 vs. 59.4%; P=0.031); C allele frequency for rs1042039 was significantly higher in patients with hypertension, as compared with the controls (66.1 vs. 60.6%; P=0.011), C allele frequency for rs1054889 was significantly lower in patients with hypertension, as compared with the controls (38.8 vs. 44.8%; P=0.007); and A allele frequency for rs2073316 was significantly lower in patients with hypertension, as compared with the controls (29.2 vs. 34.4%; P=0.013). However, once a Bonferroni correction for multiple testing was applied, the XDH gene polymorphisms rs1042039, rs1054889, and rs2073316 were shown to be associated with hypertension (P=0.044, 0.035, and 0.039, respectively). These results suggest that the XDH gene polymorphisms rs1042039, rs1054889, and rs2073316 may be associated with hypertension in the rural Han Chinese population.

Gut dysbiosis in acute-on-chronic liver failure and its predictive value for mortality.

Bacterial translocation from the gut plays an important role in the pathophysiology of acute-on-chronic liver failure (ACLF). However, gut dysbiosis in ACLF was not widely documented in previous studies. This research characterized the fecal microbiota in patients with ACLF and analyzed the temporal stability of gut microbiota during illness. Fecal microbiota of 79 ACLF patients (42 patients were followed in the next 4 weeks after the first visit for longitudinal study) and 50 healthy controls was analyzed by 16S ribosomal DNA pyrosequencing. There was a marked difference between the ACLF group and the control group. The overall microbial diversity and richness were significantly lower in ACLF than in controls. ACLF patients had lower abundance of Bacteroidaceae, Ruminococcaceae, and Lanchnospiraceae, but higher abundance of Pasteurellaceae, Streptococcaceae, and Enterecoccaceae. The relative abundance of Lachnospiraceae was obviously decreased in ACLF patients with hepatic encephalopathy. The gut microbiota kept relatively stable in a short term after the onset of ACLF. The use of antibiotics only showed moderate impacts on the gut microbiota. The relative abundance of Pasteurellaceae and Model of End Stage Liver Disease score were independent factors predicting mortality rate. Network analysis comparison showed robust correlations between specific bacterial families (Ruminococcaceae and Lachnospiraceae) and inflammatory cytokines (interleukin [IL]-6, tumor necrosis factor alpha, IL-2) in ACLF patients. These data suggest gut dysbiosis in ACLF and its predictive value for mortality. The results thus open up the possibility of designing diagnostic biomarkers and targeted probiotics aimed at decreasing mortality in ACLF.

An Integrated Prognostic Classifier for Stage I Lung Adenocarcinoma Based on mRNA, microRNA, and DNA Methylation Biomarkers

Up to 30% stage I lung cancer patients suffer recurrence within 5 years of curative surgery. We sought to improve existing protein-coding gene and microRNA expression prognostic classifiers by incorporating epigenetic biomarkers. Genome-wide screening of DNA methylation and pyrosequencing analysis of HOXA9 promoter methylation were performed in two independently collected cohorts of stage I lung adenocarcinoma. The prognostic value of HOXA9 promoter methylation alone and in combination with mRNA and miRNA biomarkers was assessed by Cox regression and Kaplan-Meier survival analysis in both cohorts. Promoters of genes marked by polycomb in embryonic stem cells were methylated de novo in tumors and identified patients with poor prognosis. The HOXA9 locus was methylated de novo in stage I tumors (p < 0.0005). High HOXA9 promoter methylation was associated with worse cancer-specific survival (hazard ratio [HR], 2.6; p = 0.02) and recurrence-free survival (HR, 3.0; p = 0.01), and identified high-risk patients in stratified analysis of stages IA and IB. Four protein-coding gene (XPO1, BRCA1, HIF1α, and DLC1), miR-21 expression, and HOXA9 promoter methylation were each independently associated with outcome (HR, 2.8; p = 0.002; HR, 2.3; p = 0.01; and HR, 2.4; p = 0.005, respectively), and when combined, identified high-risk, therapy naive, stage I patients (HR, 10.2; p = 3 × 10). All associations were confirmed in two independently collected cohorts. A prognostic classifier comprising three types of genomic and epigenomic data may help guide the postoperative management of stage I lung cancer patients at high risk of recurrence.

Ribosomal Tag Pyrosequencing of DNA and RNA Reveals “Rare” Taxa with High Protein Synthesis Potential in the Sediment of a Hypersaline Lake in Western Australia

ABSTRACTLittle is known about the potential activity of microbial communities in hypersaline sediment ecosystems. Ribosomal tag libraries of DNA and RNA extracted from the sediment of Lake Strawbridge (Western Australia) revealed bacterial and archaeal operational taxonomic units (OTUs) with high RNA/DNA ratios providing evidence for the presence of ‘rare’ but potentially “active” taxa. Among the ‘rare’ bacterial taxa Halomonas, Salinivibrio and Idiomarina showed the highest protein synthesis potential. Rare but ‘active’ archaeal OTUs were related to the KTK 4A cluster and the Marine-Benthic-Groups B and D. We present the first molecular analysis of the microbial diversity and protein synthesis potential of rare microbial taxa in a hypersaline sediment ecosystem.

Benthic microbial communities of coastal terrestrial and ice shelf Antarctic meltwater ponds.

The numerous perennial meltwater ponds distributed throughout Antarctica represent diverse and productive ecosystems central to the ecological functioning of the surrounding ultra oligotrophic environment. The dominant taxa in the pond benthic communities have been well described however, little is known regarding their regional dispersal and local drivers to community structure. The benthic microbial communities of 12 meltwater ponds in the McMurdo Sound of Antarctica were investigated to examine variation between pond microbial communities and their biogeography. Geochemically comparable but geomorphologically distinct ponds were selected from Bratina Island (ice shelf) and Miers Valley (terrestrial) (<40 km between study sites), and community structure within ponds was compared using DNA fingerprinting and pyrosequencing of 16S rRNA gene amplicons. More than 85% of total sequence reads were shared between pooled benthic communities at different locations (OTU0.05), which in combination with favorable prevailing winds suggests aeolian regional distribution. Consistent with previous findings Proteobacteria and Bacteroidetes were the dominant phyla representing over 50% of total sequences; however, a large number of other phyla (21) were also detected in this ecosystem. Although dominant Bacteria were ubiquitous between ponds, site and local selection resulted in heterogeneous community structures and with more than 45% of diversity being pond specific. Potassium was identified as the most significant contributing factor to the cosmopolitan community structure and aluminum to the location unique community based on a BEST analysis (Spearman's correlation coefficient of 0.632 and 0.806, respectively). These results indicate that the microbial communities in meltwater ponds are easily dispersed regionally and that the local geochemical environment drives the ponds community structure.

Noteworthy Facts about a Methane-Producing Microbial Community Processing Acidic Effluent from Sugar Beet Molasses Fermentation

Anaerobic digestion is a complex process involving hydrolysis, acidogenesis, acetogenesis and methanogenesis. The separation of the hydrogen-yielding (dark fermentation) and methane-yielding steps under controlled conditions permits the production of hydrogen and methane from biomass. The characterization of microbial communities developed in bioreactors is crucial for the understanding and optimization of fermentation processes. Previously we developed an effective system for hydrogen production based on long-term continuous microbial cultures grown on sugar beet molasses. Here, the acidic effluent from molasses fermentation was used as the substrate for methanogenesis in an upflow anaerobic sludge blanket bioreactor. This study focused on the molecular analysis of the methane-yielding community processing the non-gaseous products of molasses fermentation. The substrate for methanogenesis produces conditions that favor the hydrogenotrophic pathway of methane synthesis. Methane production results from syntrophic metabolism whose key process is hydrogen transfer between bacteria and methanogenic Archaea. High-throughput 454 pyrosequencing of total DNA isolated from the methanogenic microbial community and bioinformatic sequence analysis revealed that the domain Bacteria was dominated by Firmicutes (mainly Clostridia), Bacteroidetes, δ- and γ-Proteobacteria, Cloacimonetes and Spirochaetes. In the domain Archaea, the order Methanomicrobiales was predominant, with Methanoculleus as the most abundant genus. The second and third most abundant members of the Archaeal community were representatives of the Methanomassiliicoccales and the Methanosarcinales. Analysis of the methanogenic sludge by scanning electron microscopy with Energy Dispersive X-ray Spectroscopy and X-ray diffraction showed that it was composed of small highly heterogeneous mineral-rich granules. Mineral components of methanogenic granules probably modulate syntrophic metabolism and methanogenic pathways. A rough functional analysis from shotgun data of the metagenome demonstrated that our knowledge of methanogenesis is poor and/or the enzymes responsible for methane production are highly effective, since despite reasonably good sequencing coverage, the details of the functional potential of the microbial community appeared to be incomplete.

Detecting predictive androgen receptor modifications in circulating prostate cancer cells.

Molecular modifications of the androgen receptor (AR) can cause resistance to androgen deprivation therapy (ADT) in prostate cancer patients. Since lack of representative tumor samples hinders therapy adjustments according to emerging AR-modifications, we evaluated simultaneous detection of the two most common AR modifications (AR-V7 splice variant and AR point mutations) in circulating tumor cells (CTCs). We devised a single-tube assay to detect AR-V7 splice variants and AR point mutations in CTCs using immunomagnetic cell isolation, followed by quantitative real-time PCR and DNA pyrosequencing. We prospectively investigated 47 patients with PSA progression awaiting therapy switch. Comparison of response to newly administered therapy and CTC-AR-status allowed effect size estimation. Nineteen (51%) of 37 patients with detectable CTCs carried AR-modifications. Seventeen patients carried the AR-V7 splice variant, one harbored a p.T878A point mutation and one harbored both AR-V7 and a p.H875Y mutation. We estimated a positive predictive value for response and non-response to therapy by AR status in CTCs of ~94%. Based on a conservative calculation, we estimated the effect size for molecularly-informed therapy switches for prospective clinical trial planning to ~27%. In summary, the ability to determine key resistance-mediating AR modifications in CTCs has the potential to considerably improve prostate cancer treatment.

Dynamics and distribution of bacterial and archaeal communities in oil-contaminated temperate coastal mudflat mesocosms.

Mudflats are ecologically important habitats that are susceptible to oil pollution, but intervention is difficult in these fine-grained sediments, and so clean-up usually relies on natural attenuation. Therefore, we investigated the impact of crude oil on the bacterial, diatom and archaeal communities within the upper parts of the diatom-dominated sediment and the biofilm that detached from the surface at high tide. Biodegradation of petroleum hydrocarbons was rapid, with a 50 % decrease in concentration in the 0-2-mm section of sediment by 3 days, indicating the presence of a primed hydrocarbon-degrading community. The biggest oil-induced change was in the biofilm that detached from the sediment, with increased relative abundance of several types of diatom and of the obligately hydrocarbonoclastic Oleibacter sp., which constituted 5 % of the pyrosequences in the oiled floating biofilm on day 3 compared to 0.6 % in the non-oiled biofilm. Differences in bacterial community composition between oiled and non-oiled samples from the 0-2-mm section of sediment were only significant at days 12 to 28, and the 2-4-mm-sediment bacterial communities were not significantly affected by oil. However, specific members of the Chromatiales were detected (1 % of sequences in the 2-4-mm section) only in the oiled sediment, supporting other work that implicates them in anaerobic hydrocarbon degradation. Unlike the Bacteria, the archaeal communities were not significantly affected by oil. In fact, changes in community composition over time, perhaps caused by decreased nutrient concentration and changes in grazing pressure, overshadowed the effect of oil for both Bacteria and Archaea. Many obligate hydrocarbonoclastic and generalist oil-degrading bacteria were isolated, and there was little correspondence between the isolates and the main taxa detected by pyrosequencing of sediment-extracted DNA, except for Alcanivorax, Thalassolituus, Cycloclasticus and Roseobacter spp., which were detected by both methods.

How host gender affects the bacterial community in pig feces and its correlation to skatole production

To clarify the role of intestinal microbiota on skatole production and the influences of host gender, individual fecal samples were collected from female, castrated and entire male pigs at the ages of 20, 85, and 185 days. Concentrations of skatole in feces were determined by high performance liquid chromatography (HPLC), and the bacterial community in the pig feces was analyzed using 454-pyrosequencing based on 16S rRNA genes. Results showed that, compared to the gilts, the phyla Firmicutes was lower in boars and barrows, and the abundance of Proteobacteria was higher in barrows. The host gender significantly affected the proportion of ten and 11 genera in total community at the ages of 85 and 185 days, respectively. Skatole concentrations in entire male pigs were higher than in gilts at 20 days. No significant differences in the feces of different-gendered pigs remained at 85 and 185 days. The fecal skatole concentrations were significantly related to the presence of some genera of Bacteroidetes and Firmicutes in the feces, including Clostridium and Oscillibacter. This is the first study using a barcoded DNA pyrosequencing method to survey pigs’ fecal microbiota composition by gender. This gender-related microbiota correlated with the fecal concentration of indolic compounds. These results provide a new vision for pig welfare, as well as the possibility of controlling boar taints by modulating the gut microbiota instead of castration.

Evaluation of simultaneous nutrient and COD removal with polyhydroxybutyrate (PHB) accumulation using mixed microbial consortia under anoxic condition and their bioinformatics analysis.

Simultaneous nitrate-N, phosphate and COD removal was evaluated from synthetic waste water using mixed microbial consortia in an anoxic environment under various initial carbon load (ICL) in a batch scale reactor system. Within 6 hours of incubation, enriched DNPAOs (Denitrifying Polyphosphate Accumulating Microorganisms) were able to remove maximum COD (87%) at 2g/L of ICL whereas maximum nitrate-N (97%) and phosphate (87%) removal along with PHB accumulation (49 mg/L) was achieved at 8 g/L of ICL. Exhaustion of nitrate-N, beyond 6 hours of incubation, had a detrimental effect on COD and phosphate removal rate. Fresh supply of nitrate-N to the reaction medium, beyond 6 hours, helped revive the removal rates of both COD and phosphate. Therefore, it was apparent that in spite of a high carbon load, maximum COD and nutrient removal can be maintained, with adequate nitrate-N availability. Denitrifying condition in the medium was evident from an increasing pH trend. PHB accumulation by the mixed culture was directly proportional to ICL; however the time taken for accumulation at higher ICL was more. Unlike conventional EBPR, PHB depletion did not support phosphate accumulation in this case. The unique aspect of all the batch studies were PHB accumulation was observed along with phosphate uptake and nitrate reduction under anoxic conditions. Bioinformatics analysis followed by pyrosequencing of the mixed culture DNA from the seed sludge revealed the dominance of denitrifying population, such as Corynebacterium, Rhodocyclus and Paraccocus (Alphaproteobacteria and Betaproteobacteria). Rarefaction curve indicated complete bacterial population and corresponding number of OTUs through sequence analysis. Chao1 and Shannon index (H’) was used to study the diversity of sampling. “UCI95” and “LCI95” indicated 95% confidence level of upper and lower values of Chao1 for each distance. Values of Chao1 index supported the results of rarefaction curve.

Analysis of microbial community composition in a lab-scale membrane distillation bioreactor.

AimsMembrane distillation bioreactors (MDBR) have potential for industrial applications where wastewater is hot or waste heat is available, but the role of micro-organisms in MDBRs has never been determined, and thus was the purpose of this study.Methods and ResultsMicrobial communities were characterized by bacterial and archaeal 16S and eukaryotic 18S rRNA gene tag-encoded pyrosequencing of DNA obtained from sludge. Taxonomy-independent analysis revealed that bacterial communities had a relatively low richness and diversity, and community composition strongly correlated with conductivity, total nitrogen and bound extracellular polymeric substances (EPS). Taxonomy-dependent analysis revealed that Rubrobacter and Caldalkalibacillus were abundant members of the bacterial community, but no archaea were detected. Eukaryotic communities had a relatively high richness and diversity, and both changes in community composition and abundance of the dominant genus, Candida, correlated with bound EPS.ConclusionsThermophilic MDBR communities were comprised of a low diversity bacterial community and a highly diverse eukaryotic community with no archea detected. Communities exhibited low resilience to changes in operational parameters. Specifically, retenatate nutrient composition and concentration was strongly correlated with the dominant species.Significance and Impact of the StudyThis study provides an understanding of microbial community diversity in an MDBR, which is fundamental to the optimization of reactor performance.

Intragenomic distribution of RTE retroelements suggests intrachromosomal movement.

Much is known about the abundance of transposable elements (TEs) in eukaryotic genomes, but much is still unknown on their behaviour within cells. We employ here a combination of cytological, molecular and genomic approaches providing information on the intragenomic distribution and behaviour of non-long terminal repeat (LTR) retrotransposon-like elements (RTE). We microdissected every chromosome in a single first meiotic metaphase cell of the grasshopper Eyprepocnemis plorans and polymerase chain reaction (PCR) amplified a fragment of the RTE reverse transcriptase gene with specific primers. PCR products were cloned and 139 clones were sequenced. Analysis of molecular variance (AMOVA) showed significant intragenomic structure for these elements, with 4.6% of molecular variance being found between chromosomes. A maximum likelihood tree built with the RTE sequences revealed the frequent presence of two or more elements showing very high similarity and being located on the same chromosome, thus suggesting intrachromosome movement. The 454 pyrosequencing of genomic DNA gave strong support to the microdissection results and provided evidence for the existence of 5' truncated elements. Our results thus indicate a tendency of RTE elements to reinsert into the same chromosome from where they were transcribed, which could be achieved if retrotranscription and insertion takes place immediately after transcription.

Prokaryotic and diazotrophic population dynamics within a large oligotrophic inverse estuary

The ecology of microbial assemblages inhabiting classical (positive) estuaries has been well documented. However, we know relatively little about the microbial ecology of inverse (negative) estuaries, which exhibit different physical and hydrodynamic properties, including oligotrophy and hypersalinity. We investigated the dynamics of bacterioplankton communities in Spencer Gulf, an inverse estuary in temperate South Australia. We characterised patterns in the overall diversity and composition of the resident microbial assemblage, and tested the hypothesis that pelagic nitrogen-fixing bacteria (diazotrophs) could be an important functional group in the nutrient limited waters of the region. Prokaryotic and diazotrophic communities were evaluated using 16S ribosomal DNA and nifH amplicon tag pyrosequencing, respectively. Significant heterogeneity in microbial community composition and diazotrophic population structure was observed, which was driven by shifts in the relative importance of temperate vs. subtropical and oceanic vs. coastal ecotypes of Cyanobacteria throughout the inverse estuary. The globally significant unicellular cyanobacterium, UCYN-A 'Candidatus Atelocyano-bacterium thalassa', was the dominant diazotrophic phylotype. Temperature, chlorophyll a and nitrogen availability were all significant drivers of bacterioplankton dynamics within the gulf. These results demonstrate the heterogeneous microbiology of inverse estuaries, indicating that specific abiotic and biotic characteristics select for discrete microbial communities, and that pelagic nitrogen fixation may be important in this temperate oligotrophic system.