Evaluation of EPS extraction protocols from anaerobic sludge for gel-based proteomic studies.

Despite the importance of anaerobic sludge extracellular polymeric substances (EPSs), their characterization is limited to information regarding their chemical classes and molecular size. This work explores the possibility of using proteomic techniques to study the proteins present in this matrix. Thus, this paper compares eight EPS extraction methods regarding extraction yield, protein/carbohydrate ratio, size distribution profile and suitability to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses. Despite the differences found in quantification and size exclusion chromatography assays, the band profile found for all methods was very similar. Considering the band pattern, extraction time and background level, heating method followed by ammonium sulfate precipitation proved to be the most appropriate method for gel-based analyses of anaerobic sludge EPS proteins.

Microbial dynamics during azo dye degradation in a UASB reactor supplied with yeast extract

The present work aimed to investigate the microbial dynamics during the anaerobic treatment of the azo dye blue HRFL in bench scale upflow anaerobic sludge bed (UASB) reactor operated at ambient temperature. Sludge samples were collected under distinct operational phases, when the reactor were stable (low variation of color removal), to assess the effect of glucose and yeast extract as source of carbon and redox mediators, respectively. Reactors performance was evaluated based on COD (chemical oxygen demand) and color removal. The microbial dynamics were investigated by PCR-DGGE (Polimerase Chain Reaction - Denaturing Gradient of Gel Electrophoresis) technique by comparing the 16S rDNA profiles among samples. The results suggest that the composition of microorganisms changed from the beginning to the end of the reactor operation, probably in response to the presence of azo dye and/or its degradation byproducts. Despite the highest efficiency of color removal was observed in the presence of 500 mg/L of yeast extract (up to 93%), there were no differences regarding the microbial profiles that could indicate a microbial selection by the yeast extract addition. On the other hand Methosarcina barkeri was detected only in the end of operation when the best efficiencies on color removal occurred. Nevertheless the biomass selection observed in the last stages of UASB operation is probably a result of the washout of the sludge in response of accumulation of aromatic amines which led to tolerant and very active biomass that contributed to high efficiencies on color removal.

Microbial dynamics during azo dye degradation in a UASB reactor supplied with yeast extract.

The present work aimed to investigate the microbial dynamics during the anaerobic treatment of the azo dye blue HRFL in bench scale upflow anaerobic sludge bed (UASB) reactor operated at ambient temperature. Sludge samples were collected under distinct operational phases, when the reactor were stable (low variation of color removal), to assess the effect of glucose and yeast extract as source of carbon and redox mediators, respectively. Reactors performance was evaluated based on COD (chemical oxygen demand) and color removal. The microbial dynamics were investigated by PCR-DGGE (Polimerase Chain Reaction - Denaturing Gradient of Gel Electrophoresis) technique by comparing the 16S rDNA profiles among samples. The results suggest that the composition of microorganisms changed from the beginning to the end of the reactor operation, probably in response to the presence of azo dye and/or its degradation byproducts. Despite the highest efficiency of color removal was observed in the presence of 500 mg/L of yeast extract (up to 93%), there were no differences regarding the microbial profiles that could indicate a microbial selection by the yeast extract addition. On the other hand Methosarcina barkeri was detected only in the end of operation when the best efficiencies on color removal occurred. Nevertheless the biomass selection observed in the last stages of UASB operation is probably a result of the washout of the sludge in response of accumulation of aromatic amines which led to tolerant and very active biomass that contributed to high efficiencies on color removal.

Degradation of a model azo dye in submerged anaerobic membrane bioreactor (SAMBR) operated with powdered activated carbon (PAC).

This work investigated the anaerobic degradation of the model azo dye Remazol Yellow Gold RNL in an upflow anaerobic sludge blanket reactor (UASB) and two submerged anaerobic membrane (SAMBR) bioreactors, one of which (SAMBR-1) was operated with powdered activated carbon (PAC) in its interior. The reactors were operated at 35 °C with a hydraulic retention time of 24 h in three operational phases, aimed to assess the effect of external sources of carbon (glucose) or redox mediator (yeast extract) on the removal or color and organic matter. The results showed that removal efficiencies of COD (73-94%) and color (90-94%) were higher for SAMBR-1 when compared to SAMBR-2 (operated without PAC) and UASB reactors. In addition, the presence of PAC in SAMBR-1 increased reactor stability, thereby leading to a lower accumulation of volatile fatty acids (VFA). The microfiltration membrane was responsible for an additional removal of ~50% of soluble residual COD in the form of VFA, thus improving permeate quality. On its turn, PAC exhibited the ability to adsorb byproducts (aromatic amines) of azo dye degradation as well as to act as source of immobilized redox mediator (quinone groups on its surface), thereby enhancing color removal.

Granular biomass selection in a double-stage biogas collection UASB reactor: effects on SMA, abundance and diversity of the methanogenic population.

The present work aimed at investigating biomass selection in a pilot-scale double-stage biogas collection (DSBC) upflow anaerobic sludge bed (USAB) reactor treating domestic wastewater. Specific methanogenic activity (SMA) measurements and FISH countings were applied to sludge samples collected during 102 days of operation of the DSBC-UASB and of a control reactor. Results showed that both reactors presented similar SMA values in early stages of operation however the UASB-DSBC reactor showed much higher SMA after day 45, when the biomass was in granular stage. In terms of archaeal abundance, no statistical difference was observed between the reactors. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) revealed a similar composition of the archaeal communities in the two reactors and during the operational period, mainly constituted by Methanosaeta concilii. The results suggest that cell activity rather than archaeal abundance or diversity drive the methane production in the UASB reactors.

Biological oxidation of sulphides by microorganisms present in the scum layer of UASB reactors treating domestic wastewater.

This study aimed at the identification of microorganisms present in the scum layer of the settler compartment of upflow anaerobic sludge blanket (UASB) reactors, and to evaluate their role in the biological oxidation of sulphides. The experiments were conducted using scum samples taken from two pilot-scale UASB reactors, both treating domestic wastewater. Microorganisms similar to Beggiatoa sp., Thiotrix sp. and species of cyanobacteria were identified based on their morphology, and most of them have been shown to be capable of carrying out sulphur oxidation. Tests of biological oxidation of sulphides using scum and cultures of the cyanobacteria Phormidium and Pseudoanabaena showed a significant decrease in the concentrations of the sulphides, suggesting that the microorganisms present in the scum layer can play a role in the minimization of odour emissions.

Anaerobic degradation of azo dye Drimaren blue HFRL in UASB reactor in the presence of yeast extract a source of carbon and redox mediator.

This paper presents results on anaerobic degradation of the azo dye blue HFRL in a bench scale Upflow anaerobic sludge blanket (UASB) reactor operated at ambient temperature. The results show that the addition of yeast extract (500 mg/L) increased color removal (P < 0.05) from 62 to 93% despite the low chemical oxygen demand (COD) removal (~35%) which happened due to volatile fatty acids (VFA) accumulation. There were no differences in color removal (~91%) when yeast extract (500 mg/L) was used in the presence or absence of glucose, suggesting that yeast extract acted as source of redox mediator (riboflavin) and carbon. The specific rate of dye removal increased along the operational phases and depended on the presence of yeast extract, suggesting progressive biomass acclimatization. Analysis of bacterial diversity by Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) method showed there was biomass selection along the bioreactor operation and no evidence of azo dye degrading bacteria predominance. This strengthens the hypothesis that color removal happens extracellularly by the reduction of azo bond by reduced redox mediators, such as riboflavin, which is present in high amount in the yeast extract.

Characterisation of pathogenic bacteria in a UASB-polishing pond system using molecular techniques.

Molecular techniques have been commonly used to detect and quantify pathogenic bacteria in food, clinical and environmental samples, but in wastewater treatment plants few studies have been carried out. This work applied PCR with a specific set of primers to investigate pathogenic bacteria in a wastewater plant comprised of a UASB reactor followed by polishing ponds. In addition, in-situ hybridisation technique (FISH) was used to estimate the abundance of Escherichia coli in the system. According to the PCR results it was observed that Escherichia coli and Salmonella enterica subsp. enterica were not completely removed in the system, since they were detected either in the raw sewage or UASB and pond effluents. Shigella dysenteriae and Enterococcus spp. were detected in raw sewage and UASB, but not in the pond effluent. In contrast Staphylococcus aureus and Helicobacter pylori were not detected in any samples. The quantification of E. coli using FISH revealed values in the range of 10(7) cells/100 mL for raw sewage and 10(6) cells/100 mL for pond effluent, slightly higher than values obtained by traditional techniques. Finally the results show the applicability of PCR method for monitoring pathogenic bacteria in wastewater systems; however, more samples need to be analysed in order to certify the applicability of FISH to estimate pathogenic bacteria in WWT effluents.

Production and characterization of scum and its role in odour control in UASB reactors treating domestic wastewater.

There are few studies in the literature that have aimed at characterizing the physical, chemical, and microbial aspects of scum produced in UASB reactors. In addition, there is little information on the influence of operational conditions of UASB reactors on scum formation, and the present work addresses these issues. Three demo-scale UASB reactors, fed on domestic wastewater, were employed to monitor the formation and its characteristics. Scum production was periodically assessed during different operational phases, and its characterization involved analyses of BOD, COD, solids, sulfide, sulfate, microscopic observations, as well as biodegradability tests. The results show that the scum formed was physically, chemically, and microscopically similar in both geminated reactors, being comprised mainly of organic material of low biodegradability. Several bacterial morphotypes, mainly filaments and rods, with internal sulfur granules, were observed, and the aerobic microorganisms that developed at the scum layer as a result of photosynthetic activity of cyanobacteria, seemed to play an important role in sulfide removal and odour control. Scum production rates were similar in both reactors, but the imposed higher upflow velocities resulted in a higher production rate and in a reduced biodegradability of the scum.