16S rRNA Gene Amplicon Profiling of Anaerobic Bulking-Associated Prokaryotic Microbiota in a Mesophilic Expanded Granular Sludge Bed Reactor for Beverage Wastewater Treatment.
ABSTRACT: Information regarding prokaryotic microbiota associated with anaerobic bulking is limited. Here, we provide 16S rRNA gene-based prokaryotic diversity profiles for anaerobic bulking and healthy granular sludge in a mesophilic expanded granular sludge bed (EGSB) reactor. These data were tabulated at the phylum level based on high-quality reads.
Project description:We analyzed the prokaryotes in bulking and healthy sludge from a mesophilic expanded granular sludge bed reactor treating wastewater with high organic content by 16S rRNA gene amplicon sequencing. We tabulated the microbiota at the phylum level, providing a framework for avoiding sludge bulking.
Project description:A filamentous bulking of a methanogenic granular sludge caused by uncultured filamentous bacteria of the candidate phylum KSB3 in an upflow anaerobic sludge blanket (UASB) system has been reported. To characterize the physiological traits of the filaments, a polyphasic approach consisting of rRNA-based activity monitoring of the KSB3 filaments using the RNase H method and substrate uptake profiling using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) was conducted. On the basis of rRNA-based activity, the monitoring of a full-scale UASB reactor operated continuously revealed that KSB3 cells became active and predominant (up to 54% of the total 16S rRNA) in the sludge when the carbohydrate loading to the system increased. Batch experiments with a short incubation of the sludge with maltose, glucose, fructose, and maltotriose at relatively low concentrations (approximately 0.1 mM) in the presence of yeast extract also showed an increase in KSB3 rRNA levels under anaerobic conditions. MAR-FISH confirmed that the KSB3 cells took up radioisotopic carbons from [(14)C]maltose and [(14)C]glucose under the same incubation conditions in the batch experiments. These results suggest that one of the important ecophysiological characteristics of KSB3 cells in the sludge is carbohydrate degradation in wastewater and that high carbohydrate loadings may trigger an outbreak of KSB3 bacteria, causing sludge bulking in UASB systems.
Project description:Activated sludge from wastewater treatment plants was seeded into a sequencing batch reactor (SBR) in which synthetic wastewater was used as the influent. The sludge was bulked by decreasing the concentration of dissolved oxygen (DO). By adding a 30?min step of anaerobic stirring after the water inflow, the sludge bulking was rapidly inhibited after 10 running cycles, and the sludge volume index (SVI) decreased from 222 to 74?mL·g-1. The results of high-throughput sequencing showed that the relative abundance of bacteria Thiothrix, bacteria norank_o_Sphingobacteriales and fungi Trichosporon was increased by 6.3, 4.3 and 81.2%, after initial SBR stages, but these bacteria were inhibited by the addition of an anaerobic step, as their relative abundances decreased by 0.7, 0.8 and 14.7%, respectively. The proliferation of Thiothrix, norank_o_Sphingobacteriales and Trichosporon was the primary reason for the observed sludge bulking in the reactor. After the anaerobic step was added, the sludge extracellular polymeric substances (EPS) concentration was increased from 84.4 to 104.0?mg·(gMLSS)-1 (grams of mixed liquor suspended solids). Thus, the addition of an anaerobic step can inhibit the growth of filamentous bacteria, increasing the sludge EPS concentration and promoting the precipitation of activated sludge.
Project description:Methanogenic community structure and dynamics were investigated in two different, replicated anaerobic wastewater treatment reactor configurations [inverted fluidized bed (IFB) and expanded granular sludge bed (EGSB)] treating synthetic dairy wastewater, during operating temperature transitions from 37°C to 25°C, and from 25°C to 15°C, over a 430-day trial. Non-metric multidimensional scaling (NMS) and moving-window analyses, based on quantitative real-time PCR data, along with denaturing gradient gel electrophoresis (DGGE) profiling, demonstrated that the methanogenic communities developed in a different manner in these reactor configurations. A comparable level of performance was recorded for both systems at 37°C and 25°C, but a more dynamic and diverse microbial community in the IFB reactors supported better stability and adaptative capacity towards low temperature operation. The emergence and maintenance of particular bacterial genotypes (phylum Firmicutes and Bacteroidetes) was associated with efficient protein hydrolysis in the IFB, while protein hydrolysis was inefficient in the EGSB. A significant community shift from a Methanobacteriales and Methanosaetaceae towards a Methanomicrobiales-predominated community was demonstrated during operation at 15°C in both reactor configurations.
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:Predation by protists is top-down pressure that regulates prokaryotic abundance, community function, structure, and diversity in natural and artificial ecosystems. Although the effects of predation by protists have been studied in aerobic ecosystems, they are poorly understood in anoxic environments. We herein studied the influence of predation by Metopus and Caenomorpha ciliates-ciliates frequently found in anoxic ecosystems-on prokaryotic community function, structure, and diversity. Metopus and Caenomorpha ciliates were cocultivated with prokaryotic assemblages (i.e., anaerobic granular sludge) in an up-flow anaerobic sludge blanket (UASB) reactor for 171 d. Predation by these ciliates increased the methanogenic activities of granular sludge, which constituted 155% of those found in a UASB reactor without the ciliates (i.e., control reactor). Sequencing of 16S rRNA gene amplicons using Illumina MiSeq revealed that the prokaryotic community in the UASB reactor with the ciliates was more diverse than that in the control reactor; 2,885-3,190 and 2,387-2,426 operational taxonomic units (>97% sequence similarities), respectively. The effects of predation by protists in anaerobic engineered systems have mostly been overlooked, and our results show that the influence of predation by protists needs to be examined and considered in the future for a better understanding of prokaryotic community structure and function.
Project description:Filamentous bulking caused by Thiothrix species is responsible for sludge washout and loss of performance in dairy wastewater treatment plants. A long-term study was conducted over 1.5 years to test three different mitigation strategies in a full-scale plant composed of two parallel sequential batch reactors (SBR1 and 2). Strategies based on polyaluminium chloride addition and volatile fatty acids reduction were ineffective to permanently solve the problem. On the contrary, modification of the reactor cycle based on the implementation of a periodic starvation proved efficient to solve the biomass wash-out and drastically reduce the sludge volume index in both reactors. Bacterial diversity analysis using 16S amplicon sequencing and quantitative PCR indicated a reduction of Thiothrix abundance from 51.9 to 1.0% in SBR1 and from 71.8 to 0.6% in SBR2. Simultaneously, the abundance of the glycogen-accumulating bacterium Candidatus Competibacter increased in both reactors. Microscopy analysis confirmed the transition between a bulking sludge towards a granular-like sludge. This study confirms the applicability of a periodic starvation to (1) solve recurring Thiothrix bulking, (2) convert loose aggregates into dense and compact granular-like structures and (3) considerably reduce energy demand for aeration.
Project description:The microbial characteristics of granular sludge during the rapid start of an enhanced external circulating anaerobic reactor were studied to improve algae-laden water treatment efficiency. Results showed that algae laden water was effectively removed after about 35 d, and the removal rates of chemical oxygen demand (COD) and algal toxin were around 85% and 92%, respectively. Simultaneously, the gas generation rate was around 380 mL/gCOD. The microbial community structure in the granular sludge of the reactor was complicated, and dominated by coccus and filamentous bacteria. Methanosphaera, Methanolinea, Thermogymnomonas, Methanoregula, Methanomethylovorans, and Methanosaeta were the major microorganisms in the granular sludge. The activities of protease and coenzyme F420 were high in the granular sludge. The intermittent stirring device and the reverse-flow system were further found to overcome the disadvantage of the floating and crusting of cyanobacteria inside the reactor. Meanwhile, the effect of mass transfer inside the reactor can be accelerated to help give the reactor a rapid start.
Project description:Activated sludge process has been widely adopted to remove pollutants in wastewater treatment plants (WWTPs). However, stable operation of activated sludge process is often compromised by the occurrence of filamentous bulking. The aim of this study is to build a proper model for timely diagnosis and prediction of filamentous sludge bulking in an activated sludge process. This study developed a state-based Gaussian Process Regression (GPR) model to monitor the filamentous sludge bulking related parameter, sludge volume index (SVI), in such a way that the evolution of SVI can be predicted over multi-step ahead. This methodology was validated with SVI data collected from one full-scale WWTP. Online diagnosis and prediction of filamentous bulking sludge with real-time SVI prediction was tested through a simulation study. The results showed that the proposed methodology was capable of predicting future SVIs with good accuracy, thus providing sufficient time for predicting and controlling filamentous sludge bulking.
Project description:A sensor-mediated strategy was applied to a laboratory-scale granular sludge reactor (GSR) to demonstrate that energy-efficient inorganic nitrogen removal is possible with a dilute mainstream wastewater. The GSR was fed a dilute wastewater designed to simulate an A-stage mainstream anaerobic treatment process. DO, pH, and ammonia/nitrate sensors measured water quality as part of a real-time control strategy that resulted in low-energy nitrogen removal. At a low COD (0.2 kg m-3 day-1 ) and ammonia (0.1 kg-N m-3 day-1 ) load, the average degree of ammonia oxidation was 86.2 ± 3.2% and total inorganic nitrogen removal was 56.7 ± 2.9% over the entire reactor operation. Aeration was controlled using a DO setpoint, with and without residual ammonia control. Under both strategies, maintaining a low bulk oxygen level (0.5 mg/L) and alternating aerobic/anoxic cycles resulted in a higher level of nitrite accumulation and supported shortcut inorganic nitrogen removal by suppressing nitrite oxidizing bacteria. Furthermore, coupling a DO setpoint aeration strategy with residual ammonia control resulted in more stable nitritation and improved aeration efficiency. The results show that sensor-mediated controls, especially coupled with a DO setpoint and residual ammonia controls, are beneficial for maintaining stable aerobic granular sludge. PRACTITIONER POINTS: Tight sensor-mediated aeration control is need for better PN/A. Low DO intermittent aeration with minimum ammonium residual results in a stable N removal. Low DO aeration results in a stable NOB suppression. Using sensor-mediated aeration control in a granular sludge reactor reduces aeration cost.