Project description:Studies investigating the feasibility of new, or improved, biotechnologies, such as wastewater treatment digesters, inevitably start with laboratory-scale trials. However, it is rarely determined whether laboratory-scale results reflect full-scale performance or microbial ecology. The Expanded Granular Sludge Bed (EGSB) bioreactor, which is a high-rate anaerobic digester configuration, was used as a model to address that knowledge gap in this study. Two laboratory-scale idealizations of the EGSB-a one-dimensional and a three- dimensional scale-down of a full-scale design-were built and operated in triplicate under near-identical conditions to a full-scale EGSB. The laboratory-scale bioreactors were seeded using biomass obtained from the full-scale bioreactor, and, spent water from the distillation of whisky from maize was applied as substrate at both scales. Over 70 days, bioreactor performance, microbial ecology, and microbial community physiology were monitored at various depths in the sludge-beds using 16S rRNA gene sequencing (V4 region), specific methanogenic activity (SMA) assays, and a range of physical and chemical monitoring methods. SMA assays indicated dominance of the hydrogenotrophic pathway at full-scale whilst a more balanced activity profile developed during the laboratory-scale trials. At each scale, Methanobacterium was the dominant methanogenic genus present. Bioreactor performance overall was better at laboratory-scale than full-scale. We observed that bioreactor design at laboratory-scale significantly influenced spatial distribution of microbial community physiology and taxonomy in the bioreactor sludge-bed, with 1-D bioreactor types promoting stratification of each. In the 1-D laboratory bioreactors, increased abundance of Firmicutes was associated with both granule position in the sludge bed and increased activity against acetate and ethanol as substrates. We further observed that stratification in the sludge-bed in 1-D laboratory-scale bioreactors was associated with increased richness in the underlying microbial community at species (OTU) level and improved overall performance.
Project description:The microbial community of a laboratory-scale bioreactor based on the anammox process was investigated by using metagenomic approaches and fluorescent in situ hybridization (FISH). The bioreactor was initially inoculated with activated sludge from the denitrifying bioreactor of a municipal wastewater treatment station. By constantly increasing the ammonium and nitrite load, a microbial community containing the novel species of anammox bacteria "Candidatus Jettenia ecosi" developed in the bioreactor after 5 years when the maximal daily nitrogen removal rate reached 8.5 g/L. Sequencing of the metagenome of anammox granules and the binning of the contigs obtained, allowed a high quality draft genome of the dominant anammox bacterium, "Candidatus Jettenia ecosi" to be assembled. Annotation of the 3.9 Mbp long genome revealed 3970 putative protein-coding genes, 45 tRNA genes, and genes for 16S/23S rRNAs. Analysis of the genome of "Candidatus Jettenia ecosi" revealed genes involved in anammox metabolism, including nitrite and ammonium transporters, copper-containing nitrite reductase, a nitrate reductase complex, hydrazine synthase, and hydrazine dehydrogenase. Autotrophic carbon fixation could be accomplished through the Wood Ljungdahl pathway. The composition of the community was investigated through a search of 16S rRNA sequences in the metagenome and FISH analysis of the anammox granules. The presence of the members of Ignavibacteriae, Betaproteobacteria, Chloroflexi and other microbial lineages reflected the complexity of the microbial processes in the studied bioreactor performed by anammox Planctomycetes, fermentative bacteria, and denitrifiers.
Project description:The effects of a precipitous decrease in the inlet organic loading rate on sludge reductions and the microbial community in a membrane bioreactor were investigated. The sludge biomass was markedly reduced to 47.4% of the initial concentration (approximately 15,000 mg L(-1)) within 7 d after the organic loading rate was decreased by half (450 to 225 mg chemical oxygen demand L(-1) d(-1)). An analysis of the microbial community structure using high-throughput sequencing revealed an increase in the abundance of facultative predatory bacteria-related operational taxonomic units as well as microorganisms tolerant to environmental stress belonging to the classes Deinococci and Betaproteobacteria.
Project description:The formation of biofilm in a membrane bioreactor depends on the production of various signaling molecules like N-acyl homoserine lactones (AHLs). In the present study, a total of 200 bacterial strains were isolated from membrane bioreactor activated sludge and screened for AHLs production using two biosensor systems, Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136. A correlation between AHLs production and biofilm formation has been made among screened AHLs producing strains. The 16S rRNA gene sequence analysis revealed the dominance of Aeromonas and Enterobacter sp. in AHLs production; however few a species of Serratia, Leclercia, Pseudomonas, Klebsiella, Raoultella and Citrobacter were also identified. The chromatographic characterization of sludge extract showed the presence of a broad range of quorum sensing signal molecules. Further identification of sludge AHLs by thin layer chromatography bioassay and high performance liquid chromatography confirms the presence of C4-HSL, C6-HSL, C8-HSL, 3-oxo-C8-HSL, C10-HSL, C12-HSL, 3-oxo-C12-HSL and C14-HSL. The occurrence of AHLs in sludge extract and dominance of Aeromonas and Enterobacter sp. in activated sludge suggests the key role of these bacterial strains in AHLs production and thereby membrane fouling.
Project description:The large diversity of viruses that exist in human populations are potentially excreted into sewage collection systems and concentrated in sewage sludge. In the U.S., the primary fate of processed sewage sludge (class B biosolids) is application to agricultural land as a soil amendment. To characterize and understand infectious risks associated with land application, and to describe the diversity of viruses in human populations, shotgun viral metagenomics was applied to 10 sewage sludge samples from 5 wastewater treatment plants throughout the continental U.S, each serving between 100,000 and 1,000,000 people. Nearly 330 million DNA sequences were produced and assembled, and annotation resulted in identifying 43 (26 DNA, 17 RNA) different types of human viruses in sewage sludge. Novel insights include the high abundance of newly emerging viruses (e.g., Coronavirus HKU1, Klassevirus, and Cosavirus) the strong representation of respiratory viruses, and the relatively minor abundance and occurrence of Enteroviruses. Viral metagenome sequence annotations were reproducible and independent PCR-based identification of selected viruses suggests that viral metagenomes were a conservative estimate of the true viral occurrence and diversity. These results represent the most complete description of human virus diversity in any wastewater sample to date, provide engineers and environmental scientists with critical information on important viral agents and routes of infection from exposure to wastewater and sewage sludge, and represent a significant leap forward in understanding the pathogen content of class B biosolids.
Project description:Anaerobic digestion (AD) plays an important role in waste activated sludge (WAS) treatment; however, conventional AD (CAD) process needs substantial improvements, especially for the treatment of WAS with low solids content and poor anaerobic biodegradability. Herein, we propose a submerged anaerobic dynamic membrane bioreactor (AnDMBR) for simultaneous WAS thickening and digestion without any pretreatment. During the long-term operation, the AnDMBR exhibited an enhanced sludge reduction and improved methane production over CAD process. Moreover, the biogas generated in the AnDMBR contained higher methane content than CAD process. Stable carbon isotopic signatures elucidated the occurrence of combined methanogenic pathways in the AnDMBR process, in which hydrogenotrophic methanogenic pathway made a larger contribution to the total methane production. It was also found that organic matter degradation was enhanced in the AnDMBR, thus providing more favorable substrates for microorganisms. Pyrosequencing revealed that Proteobacteria and Bacteroidetes were abundant in bacterial communities and Methanosarcina and Methanosaeta in archaeal communities, which played an important role in the AnDMBR system. This study shed light on the enhanced digestion of WAS using AnDMBR technology.
Project description:With the emergence of Next Generation Sequencing, major advances were made with regard to identifying viruses in natural environments. However, bioinformatical research on viruses is still limited because of the low amounts of viral DNA that can be obtained for analysis. To overcome this limitation, DNA is often amplified with multiple displacement amplification (MDA), which may cause an unavoidable bias. Here, we describe a case study in which the virome of a bioreactor is sequenced using Ion Torrent technology. DNA-spiking of samples is compared with MDA-amplified samples. DNA for spiking was obtained by amplifying a bacterial 16S rRNA gene. After sequencing, the 16S rRNA gene reads were removed by mapping to the Silva database. Three samples were tested, a whole genome from Enterobacteria P1 Phage and two viral metagenomes from an infected bioreactor. For one sample, the new DNA-spiking protocol was compared with the MDA technique. When MDA was applied, the overall GC content of the reads showed a bias towards lower GC%, indicating a change in composition of the DNA sample. Assemblies using all available reads from both MDA and the DNA-spiked samples resulted in six viral genomes. All six genomes could be almost completely retrieved (97.9%-100%) when mapping the reads from the DNA-spiked sample to those six genomes. In contrast, 6.3%-77.7% of three viral genomes was covered by reads obtained using the MDA amplification method and only three were nearly fully covered (97.4%-100%). This case study shows that DNA-spiking could be a simple and inexpensive alternative with very low bias for sequencing of metagenomes for which low amounts of DNA are available.
Project description:Micropowder (20-250 µm) made from ground dry waste sludge from a municipal sewage treatment plant was added in a sequencing batch reactor (R2), which was fed by synthetic wastewater with acetate as carbon source. Compared with the traditional SBR (R1), aerobic sludge granulation time was shortened 15 days in R2. Furthermore, filamentous bacteria in bulking sludge were controlled to accelerate aerobic granulation and form large granules. Correspondingly, the SVI decreased from 225 mL/g to 37 mL/g. X-ray Fluorescence (XRF) analysis demonstrated that Al and Si from the micropowder were accumulated in granules. A mechanism hypotheses for the acceleration of aerobic granulation by adding dry sludge micropowder is proposed: added micropowder acts as nuclei to induce bacterial attachment; dissolved matters from the micropowder increase abruptly the organic load for starved sludge to control overgrown filamentous bacteria as a framework for aggregation; increased friction from the movement of micropowder forces the filaments which extend outwards to shrink for shaping granules.
Project description:We sequenced the metagenome of a granular sludge in a nitritation/anammox bioreactor used for the treatment of ammonium-rich wastewater. Proteobacteria, Planctomycetes, Bacteroidetes, Chloroflexi, Ignavibacteriae, and Acidobacteria were the predominant phyla in the studied bioreactor. Binning of contigs yielded a near-complete genome of the dominant anammox bacterium assigned to the candidate genus Brocadia.
Project description:A chlorate (ClO(3)(-)) reducing microbial consortium oxidized arsenite (As(III)) to arsenate (As(V)) in an upflow anaerobic sludge-bed bioreactor over 550 days operation. As(III) was converted with high conversion efficiencies (>98%) at volumetric loadings ranging from 0.45 to 1.92 mmol As/(L(reactor)d). The oxidation of As(III) was linked to the complete reduction of ClO(3)(-) to Cl(-) and H(2)O, as demonstrated by a molar ratio of approximately 3.0 mol As(III) oxidized per mole of Cl(-) formed and by the greatly lowered ClO(3)(-)-reducing capacity without As(III) feeding. An autotrophic enrichment culture was established from the bioreactor biofilm. A 16S rRNA gene clone library indicated that the culture was dominated by Dechloromonas, and Stenotrophomonas as well as genera within the family Comamonadaceae. The results indicate that the oxidation of As(III) to less mobile As(V) utilizing ClO(3)(-) as a terminal electron acceptor provides a sustainable bioremediation strategy for arsenic contamination in anaerobic environments.