Microbial community functional structures in wastewater treatment plants as characterized by GeoChip
ABSTRACT: To understand microbial community functional structures of activated sludge in wastewater treatment plants (WWTPs) and the effects of environmental factors on their structure, 12 activated sludge samples were collected from four WWTPs in Beijing. GeoChip 4.2 was used to determine the microbial functional genes involved in a variety of biogeochemical processes. The results showed that, for each gene category, such as egl, amyA, nir, ppx, dsrA sox and benAB, there were a number of microorganisms shared by all 12 samples, suggestive of the presence of a core microbial community in the activated sludge of four WWTPs. Variance partitioning analyses (VPA) showed that a total of 53% of microbial community variation can be explained by wastewater characteristics (25%) and operational parameters (23%), respectively. This study provided an overall picture of microbial community functional structures of activated sludge in WWTPs and discerned the linkages between microbial communities and environmental variables in WWTPs. Four full-scale wastewater treatment systems located in Beijing were investigated. Triplicate samples were collected in each site.
Project description:Membrane bioreactor (MBR) systems are typically known different from conventional activated sludge (CAS) systems in operational parameters, while current knowledge of their microbial differentiations is barely sufficient. To this end, the current study was launched to address the differences of the overall functional genes of an oxidation ditch (OD) and an MBR running parallelly at full-scale using a functional gene array-GeoChip 4.2. Two full-scale wastewater treatment systems applying the processes of oxidation ditch (OD) and membrane bioreactor (MBR) were investigated. They treated identical wastewater at the same scale. 12 mixed-liquor suspended sludge (MLSS) samples collected daily on 12 consecutive days from each system were analyzed by GeoChip 4.2.
Project description:Wastewater treatment plants use a variety of bioreactor types and configurations to remove organic matter and nutrients. Little is known regarding the effects of different configurations and within-plant immigration on microbial community dynamics. Previously, we found that the structure of ammonia-oxidizing bacterial (AOB) communities in a full-scale dispersed growth activated sludge bioreactor correlated strongly with levels of NO2- entering the reactor from an upstream trickling filter (Wells et al 2009). Here, to further examine this puzzling association, we profile within-plant microbial biogeography (spatial variation) and test the hypothesis that substantial microbial immigration occurs along a transect (raw influent, trickling filter biofilm, trickling filter effluent, and activated sludge) at the same full-scale wastewater treatment plant. AOB amoA gene abundance increased >30-fold between influent and trickling filter effluent concomitant with NO2- production, indicating unexpected growth and activity of AOB within the trickling filter. Nitrosomonas europaea was the dominant AOB phylotype in trickling filter biofilm and effluent, while a distinct ‘Nitrosomonas-like’ lineage dominated in activated sludge. Prior time series indicated that this ‘Nitrosomonas-like’ lineage was dominant when NO2- levels in the trickling filter effluent (i.e., activated sludge influent) were low, while N. europaea became dominant in the activated sludge when NO2- levels were high. This is consistent with the hypothesis that NO2- production may co-occur with biofilm sloughing, releasing N. europaea from the trickling filter into the activated sludge bioreactor. Phylogenetic microarray (PhyloChip) analyses revealed significant spatial variation in taxonomic diversity, including a large excess of methanogens in the trickling filter relative to activated sludge and attenuation of Enterobacteriaceae across the transect, and demonstrated transport of a highly diverse microbial community via the trickling filter effluent to the activated sludge bioreactor. Our results provide compelling evidence that substantial immigration between coupled process units occurs and may exert significant influence over microbial community dynamics within staged bioreactors. Overall design: Samples described in this study were obtained at the Palo Alto Regional Water Quality Control Plant (PARWQCP) in Palo Alto, CA on 12 November 2008. Sampling along a transect within the PARWQCP occurred on 12 November 2008. Upon temporary shutdown of the rotating distributor arm in one of the trickling filters, a surface PVC media module (0.9 m deep) was removed from the reactor, and 1-3 g biofilm samples were obtained with a sterile spatula, placed in sterile 1.5 ml tubes, weighed, and stored at -20oC. Concurrently, samples of influent, trickling filter effluent, and activated sludge were obtained. Activated sludge samples represented a 24-h composite from the combined outlet of all four aeration basins using a Sigma 900 refrigerated automatic sampler (Hach, Loveland, CO). Eight 1.5 ml activated sludge samples were centrifuged onsite for 5 min at 5000 g, decanted, and stored at -20oC. Raw influent and trickling filter effluent samples were transported on ice to the laboratory, vacuum filtered in 10 ml aliquots onto 0.22 μm Hydrophilic Durapore PVDF filters (25 mm diameter, Millipore, Billerica, MA), and archived at -20oC. PhyloChip analyses were performed on biological triplicate samples from trickling filter biofilm, trickling filter effluent, and activated sludge, and biological duplicate samples (due to biomass limitations) from the plant influent.
Project description:Manufactured nanomaterials (MNMs) are increasingly incorporated into consumer products that are disposed into sewage. In wastewater treatment, MNMs adsorb to activated sludge biomass where they may impact biological wastewater treatment performance, including nutrient removal. Here, we studied MNM effects on bacterial polyhydroxyalkanoate (PHA), specifically polyhydroxybutyrate (PHB), biosynthesis because of its importance to enhanced biological phosphorus (P) removal (EBPR). Activated sludge was sampled from an anoxic selector of a municipal wastewater treatment plant (WWTP), and PHB-containing bacteria were concentrated by density gradient centrifugation. After starvation to decrease intracellular PHB stores, bacteria were nutritionally augmented to promote PHB biosynthesis while being exposed to either MNMs (TiO2 or Ag) or to Ag salts (each at a concentration of 5 mg L-1). Cellular PHB concentration and PhyloChip community composition were analyzed. The final bacterial community composition differed from activated sludge, demonstrating that laboratory enrichment was selective. Still, PHB was synthesized to near-activated sludge levels. Ag salts altered final bacterial communities, although MNMs did not. PHB biosynthesis was diminished with Ag (salt or MNMs), indicating the potential for Ag-MNMs to physiologically impact EBPR through the effects of dissolved Ag ions on PHB producers. 18 samples; Triplicate PHB-enriched bacterial communities recovered from activated sludge were exposed to nanoparticle (TiO2 or Ag) or AgNO3 (as a silver control) or were not exposed to an nanoparticles (control) to determine if the naoparticles affected PHB production.
Project description:Understanding microbial community diversity is thought to be crucial for improving process functioning and stabilities of wastewater treatment systems. However, current studies largely focus on taxonomic groups based on 16S rRNA, which are not necessarily linked to functioning, or a few selected functional genes. Here we launched a study to profile the overall functional genes of microbial communities in three full-scale wastewater treatment systems. Triplicate activated sludge samples from each system were analyzed using a high-throughput metagenomics tool named GeoChip 4.2, resulting in the detection of 38,507 to 40,647 functional genes. A high similarity of 75.5% to 79.7% shared genes was noted among the nine samples. Moreover, correlation analyses showed that the abundances of a wide array of functional genes were associated with system performances. For example, the abundances of overall nitrogen cycling genes had a strong correlation to total nitrogen (TN) removal rates (r = 0.7647, P < 0.01). The abundances of overall carbon cycling genes were moderately correlated with COD removal rates (r = 0.6515, P < 0.01). Lastly, we found that influent chemical oxygen demand (COD inf) and total phosphorus concentrations (TP inf), and dissolved oxygen (DO) concentrations were key environmental factors shaping the overall functional genes. Together, the results revealed vast functional gene diversity and some links between the functional gene compositions and microbe-mediated processes. Overall design: Three full-scale wastewater treatment systems located in the same city were investigated. Triplicate samples were collected in each site.
Project description:Waste decomposition in landfills is a complex and microbe-mediated process. Understanding the microbial community composition and structure is critical for accelerating decomposition and reducing adverse impact on the environment. Here, we examined the microbial communities along with landfill depth and age (LDA) in a sanitary landfill in Beijing, China using 16s rRNA Illumina sequencing and GeoChip 4.6. We found that Clostridiales and Methanofollis were the predominant bacteria and archaea in the present landfill, respectively. Interestingly, in contrast with the decreasing trend of microbial diversity in soil, both phylogenetic and functional diversities were higher in deeper and older refuse in the landfill. Phylogenetic compositions were obviously different in the refuse with the same LDA and such difference is mainly attributed to the heterogeneity of refuse instead of random process. Nevertheless, functional structures were similar within the same LDA, indicating that microbial community assembly in the landfill may be better reflected by functional genes rather than phylogenetic identity. Mantel test and canonical correspondence analysis suggested that environmental variables had significant impacts on both phylogenetic composition and functional structure. Higher stress genes, genes for degrading toxic substances and endemic genes in deeper and older refuse indicated that they were needed for the microorganisms to survive in the more severe environments. This study suggests that landfills are a repository of stress-resistant and contaminant-degrading microorganisms, which can be used for accelerating landfill stabilization and enhancing in situ degradation. Fifteen refuse samples with five landfill depths and ages (6m/2a, 12m/4a, 18m/6a, 24m/8a and 30m/10a) were collected from a sanitary landfill in Beijing, China. Three replicates in every landfill depth and age
Project description:Understanding the bacterial community structure, and their functional analysis for active bioremediation process is essential to design better and cost effective strategies. Microarray analysis enables us to simultaneously study the functional and phylogenetic markers of hundreds of microorganisms which are involved in active bioremediation process in an environment. We have previously described development of a hybrid 60-mer multibacterial microarray platform (BiodegPhyloChip) for profiling the bacterial communities and functional genes simultaneously in environments undergoing active bioremediation process (Pathak et al; Appl Microbiol Biotechnol,Vol. 90, 1739-1754). The present study involved profiling the status of bacterial communities and functional (biodegradation) genes using the developed 60-mer oligonucleotide microarray BiodegPhyloChip at five contaminated hotspots in the state of Gujarat, in western India. The expression pattern of functional genes (coding for key enzymes in active bioremediation process) at these sites was studied to understand the dynamics of biodegradation in the presence of diverse group of chemicals. The results indicated that the nature of pollutants and their abundance greatly influence the structure of bacterial communities and the extent of expression of genes involved in various biodegradation pathways. In addition, site specific factors also play a pivotal role to affect the microbial community structure as was evident from results of 16S rRNA gene profiling of the five contaminated sites, where the community structure varied from one site to another drastically. Agilent one-color CGH experiment and one-color Gene Expresssion expereiment,Organism: Genotypic designed Agilent-17159 Genotypic designed Agilent Multibacterial 8x15k Array , Labeling kits: Agilent Genomic DNA labeling Kit (Part Number: 5190-0453) and Agilent Quick Amp Kit PLUS (Part number: 5190-0442).
Project description:The transcriptome analysis by the human DNA microarray was applied to evaluate the impacts of whole wastewater effluents from the membrane bioreactors (MBRs) and the activated sludge process (AS), on the biological processes of human hepatoma HepG2 cells. The three conventional bioassays (i.e., cytotoxicity tests and bioluminescence inhibition test) and chemical analysis of the domestic effluent standards were conducted in parallel since they are well-established methods with previous applications to wastewater. A significant variation of effluent quality was sdemonstrated among the tested effluents despite that all effluents met the 40 national effluent standards. The three conventional bioassays supported the result of the transcriptome analysis, indicating the comparable or even higher sensitivity of the new assay. The most superior effluent quality was found in the MBR operated at a relatively long sludge retention time (i.e., 40 days) and small membrane pore size (i.e., 0.03 μm). In addition, functional analysis of the differentially expressed genes revealed that the effluents made various impacts on the cellular functions, suggesting the transcriptome analysis by DNA microarray as more comprehensive, rapid and sensitive tool to detect multiple impacts of the whole effluents. Moreover, the potential genetic markers were proposed to quantitatively evaluate the treatability of the wastewater effluents. In this study, we examined the gene expression alteration in human hepatoma cell line, HepG2 exposed to the raw wastewater, effluents from three types of membrane bioreactors (MBRs), and the activated sludge process. Wastewater DNA microarray with 8795 human genes. MQ water was used as control. For duplicate, two dishes were prepared for each sample and individually treated in parallel.
Project description:Anaerobic digestion is a popular and effective microbial process for waste treatment. The performance of anaerobic digestion processes is contingent on the balance of the microbial food web in utilizing various substrates. Recently, co-digestion, i.e., supplementing the primary substrate with an organic-rich co-substrate has been exploited to improve waste treatment efficiency. Yet the potential effects of elevated organic loading on microbial functional gene community remains elusive. In this study, functional gene array (GeoChip 5.0) was used to assess the response of microbial community to the addition of poultry waste in anaerobic digesters treating dairy manure. Consistent with 16S rRNA gene sequences data, GeoChip data showed that microbial community compositions were significantly shifted in favor of copiotrophic populations by co-digestion, as taxa with higher rRNA gene copy number such as Bacilli were enriched. The acetoclastic methanogen Methanosarcina was also enriched, while Methanosaeta was unaltered but more abundant than Methanosarcina throughout the study period. The microbial functional diversity involved in anaerobic digestion were also increased under co-digestion. Overall design: There were two sets of anaerobic digesters. Three control digesters were fed with dairy manure and the organic loading rate kept constant. The three treatment digesters were fed with poultry waste in addition to dairy manure, resulting in step-wise increase in the organic loading rate. Sludge samples were taken at different time points from the six digesters.
Project description:In this study the impact of protein fractionation techniques prior to LC/MS analysis was investigated on activated sludge samples derived at winter and summer condition from a full-scale wastewater treatment plant (WWTP). For reduction of the sample complexity, different fractionation techniques including RP-LC (1D-approach), SDS-PAGE and RP-LC (2D-approach) as well as RP-LC, SDS-PAGE and liquid IEF (3D-approach) were carried out before subsequent ITMS analysis. The derived spectra were identified by MASCOT search using a combination of the public UniProtKB/Swiss-Prot protein database and metagenome data from a WWTP (data are available via ProteomeXchange with identifier PXD001547). The results showed a significant increase of identified spectra, enabled by applying IEF and SDS-PAGE to the proteomic workflow. Based on meta-proteins, a core metaproteome and the corresponding taxonomic profile of the wastewater activated sludge was revealed and functional aspects using the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway library were analyzed. Hereby, the KEGG Orthology identifiers (KO numbers) of protein hits were plotted into pathway maps of the central carbon (map01200) and nitrogen metabolism (map00910). Using the 3D-approach, most proteins involved in glycolysis and citrate cycle and nearly all proteins of the nitrogen removal were identified, qualifying this approach as the most promising for future studies.
Project description:Samples of oil and production water were collected from five wells of the Qinghai Oilfield, China, and subjected to GeoChip hybridization experiments for microbial functional diversity profiling. Unexpectedly, a remarkable microbial diversity in oil samples, which was higher than that in the corresponding water samples, was observed, thus challenging previously believed assumptions about the microbial diversity in this ecosystem. Hierarchical clustering separated oil and water samples, thereby indicating distinct functional structures in the samples. Genes involved in the degradation of hydrocarbons, organic remediation, stress response, and carbon cycling were significantly abundant in crude oil, which is consistent with their important roles in residing in oil. Association analysis with environmental variables suggested that oil components comprising aromatic hydrocarbons, aliphatic hydrocarbons, and a polar fraction with nitrogen-, sulfur-, and oxygen-containing compounds were mainly influential on the structure of the microbial community. Furthermore, a comparison of microbial communities in oil samples indicated that the structures were depth/temperature-dependent. To our knowledge, this is the first thorough study to profile microbial functional diversity in crude oil samples. From the Qinghai Oilfield located in the Tibetan Plateau, northwest China, oil production mixtures were taken from four oil production wells (No. 813, 516, 48 and 27) and one injection well (No. 517) in the Yue-II block. The floating oil and water phases of the production mixtures were separated overnight by gravitational separation. Subsequently, the microbial community and the characteristics of the water solution (W813, W516, W48, and W27) and floating crude oil (O813, O516, O48, and O27) samples were analyzed. A similar analysis was performed with the injection water solution (W517).