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.

Occurrence of pharmaceuticals and endocrine disruptors in raw sewage and their behavior in UASB reactors operated at different hydraulic retention times.

This work investigated the occurrence of pharmaceuticals and endocrine disrupting compounds (EDCs) in raw sewage (from Belo Horizonte city, Minas Gerais state, Brazil) and assessed their behavior in demo-scale upflow anaerobic sludge blanket reactors (UASB reactors) operated at different hydraulic retention times (HRT). The dissolved concentration of the studied micropollutants in the raw and treated sewage was obtained using solid phase extraction (SPE) followed by analysis in a liquid chromatography system coupled to a hybrid high resolution mass spectrometer consisting of an ion-trap and time of flight (LC-MS-IT-TOF). The natural (estradiol) and synthetic (ethinylestradiol) estrogens were hardly detected; when present, however, their concentrations were lower than the method quantification limits. The concentrations of bisphenol A and miconazole in raw sewage were similar to that reported in the literature (around 200 ng L⁻¹ and hardly detected, respectively). The antibiotics sulfamethoxazole (median 13.0 ng L⁻¹) and trimethoprim (median 61.5 ng L⁻¹), and the other pharmaceutical compounds (diclofenac and bezafibrate, with median 99.9 and 94.4 ng L⁻¹, respectively) were found in lower concentrations when compared with reports in the literature, which might indicate a lower consumption of such drugs in Brazil. The UASB reactors were inefficient in the removal of bisphenol A, and led to an increased concentration of nonylphenol in the effluent. The anaerobic reactors were also inefficient in the removal of diclofenac, and led to a partial removal of bezafibrate; whereas, for sulfamethoxazole there seemed to be a direct relationship between the HRT and removal efficiencies. For trimethoprim the sludge retention time (SRT) seemed to play an important role, although it was only partially removed in the UASB reactors.

Use of submerged anaerobic membrane bioreactor (SAMBR) containing powdered activated carbon (PAC) for the treatment of textile effluents.

This work investigated the use of submerged anaerobic membrane bioreactors (SAMBRs) in the presence and absence of powdered activated carbon (PAC) for the treatment of genuine textile wastewater. The reactors were operated at 35 °C with an HRT of 24 h and the textile effluent was diluted (1:10) with nutrient solution containing yeast extract as the source of the redox mediation riboflavin. The results showed that although both SAMBRs exhibited an excellent performance, the presence of PAC inside SAMBR-1 enhanced reactor stability and removal efficiency of chemical oxygen demand (COD), volatile fatty acids (VFA), turbidity and color. The median removal efficiencies of COD and color in SAMBR-1 were, 90 and 94% respectively; whereas for SAMBR-2 (without PAC) these values were 79 and 86%, In addition, the median values of turbidity and VFA were 8 NTU and 8 mg/L for SAMBR-1 and 14 NTU and 26 mg/L for SAMBR-2, indicating that the presence of PAC inside SAMBR-1 led to the production of an anaerobic effluent of high quality regarding such parameters.

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.

Quantification of dissolved methane in UASB reactors treating domestic wastewater under different operating conditions.

This paper aimed at measuring the concentration of methane dissolved in effluents from different UASB reactors (pilot-, demo- and full-scale) treating domestic wastewater, in order to calculate the degree of saturation of such greenhouse gas and evaluate the losses of energetic potential in such systems. The results showed that methane saturation degrees, calculated according to Henry's law, varied from ∼1.4 to 1.7 in the different reactors, indicating that methane was oversaturated in the liquid phase. The overall results indicated that the losses of dissolved methane in the anaerobic effluents were considerably high, varying from 36 to 41% of total methane generated in the reactor. These results show that there is considerable uncontrolled loss of methane in anaerobic wastewater treatment plants, implying the need of research on technologies aimed at recovering such energetic greenhouse gas.

Conceptual analysis of the UASB/polishing pond system regarding the removal of surfactants, micropollutants and control of gaseous emissions.

A conceptual and integrated analysis of the UASB/Polishing Pond system is presented in relation to the removal of specific constituents not normally covered in the pond's literature, namely surfactants (linear alkylbenzene sulphonate, LAS), micropollutants (phthalates and bisphenol A) and sulfide. Experimental studies carried out on a small full-scale UASB/PP system have shown limited overall removal efficiencies for some constituents (53% to 70% for phthalates and 50% for LAS), but excellent removal levels for bisphenol A (around 99%) and sulfide (no trace concentrations were detected in the second and third ponds of the series). Further research is needed to investigate possible limitations and to identify means to improve the performance of the UASB/PP system.

Methodologies for determining the bioavailability and biodegradability of sludges.

The measurement of sludge bioavailability and biodegradability is important to assess the sludge potential as a source of carbon and energy during its biological stabilization. As the majority of biological sludge (secondary sludge) consists of biomass, an increase in sludge bioavailability can be achieved by using different techniques for cell lysis, which is aimed at releasing intracellular organics into the bulk solution and enhancing hydrolysis of particulate organic matter. This paper reviews the main methodologies currently used for cell lysis, which include thermal, ultrasonic and chemical techniques and a combination of these, as well as the analytical procedures that can be employed to assess the degree of lysis and sludge solubilization. In addition, this paper also discusses common techniques that can be used for measuring the biodegradability of sludge under aerobic and anaerobic conditions.

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.

Chromatographic characterization of dissolved organics in effluents from two anaerobic reactors treating synthetic wastewater.

This paper presents results on the quantification and chromatographic characterization of soluble microbial products (SMP) accumulated in two laboratory-scale reactors: a submerged anaerobic membrane reactor (SAMBR or MBR), and an anaerobic CSTR. The results obtained under steady-state conditions show that 2.1% of the substrate was channelled into the production of SMP in the CSTR, whilst in the SAMBR this was estimated to be 25%. Chromatographic characterization showed that more hydrophobic and high MW organics that absorb at 254 nm were detected in the SAMBR supernatant than in the CSTR. A comparison of chromatograms suggest that the release of extracellular polymers (ECP) and cell lysis may be important sources of SMP in the SAMBR. Electrophoresis results confirmed that there was more soluble protein inside the SAMBR, and showed that the release of ECP by shear or hydrolysis seemed to have contributed to the production of protein-like SMP in both systems.

Soluble microbial products formation in anaerobic chemostats in the presence of toxic compounds.

Anaerobic chemostats fed on glucose (approximately 10 g chemical oxygen demand (COD)/L) were used to investigate the effects of toxicity on soluble microbial product (SMP) formation. Addition of the toxic compounds chloroform and chromium increased the net accumulation of SMP, despite reducing the percentage of SMP in the effluent due to the overwhelming production of volatile fatty acids (VFAs). In the reactor spiked with chloroform the normalized accumulation of SMP (SMP/So) increased from 2% to 8%, whereas in the reactor spiked with Cr (VI) the SMP/So ratio reached as high as 20% after the spike, and in both cases SMP net accumulation was proportional to the concentration of toxicant. After the chloroform and chromium spikes biomass seemed to produce more extra cellular polymeric substances (EPS) suggesting that this might have helped them to cope with the stress. Chromatography results indicate that some of the high MW compounds present in the SMP might have been due to EPS release into the bulk solution, and that other compounds, probably released as a result of cell lysis, were also present. Hydrolysis of EPS did not seem to contribute to SMP accumulation in the presence of toxic compounds, and DNA analysis suggested that cell lysis products was an important contribution to SMP accumulation, in the presence of chromium.

The effect of organic and hydraulic shock loads on the production of soluble microbial products in anaerobic digesters.

Anaerobic chemostats were used to investigate the effects of organic and hydraulic shock loads on the production of soluble microbial products (SMP). Production of SMP was found to increase during glucose spikes, reaching up 15% of the influent chemical oxygen demand. These SMP appear to be utilization-associated products produced as a result of the temporarily high organic load, and chemical analysis and ultrafiltration experiments revealed that most of these compounds are difficult to identify and that the majority of them are present in the low molecular weight (MW) range. Production of SMP also increased when the hydraulic retention time was reduced from 15 to 3 days, and an increase in DNA concentration in the bulk solution suggested enhanced cell lysis. Although the cause of lysis was not clear, it is believed that most of the SMP produced under such conditions were biomass-associated products following cell death. While the majority of these compounds lay in the low MW range, as much as 35% were found to have MWs greater than 1 kDa. During the period when the anaerobic chemostat was fed no alkalinity and the pH remained lower than 6.5 for more than a week, a slightly higher production of SMP and a shift in the MW distribution towards the production of higher MW SMP was observed.

Characterization of soluble microbial products (SMP) in effluents from anaerobic reactors.

The residual COD from anaerobic treatment processes is usually too high to comply with legislative discharge levels. It has been shown that in well operated systems the majority of the effluent COD originates from soluble microbial products (SMP) produced by the system itself, hence the characteristics of these compounds become important when assessing post-treatment systems to remove the residual COD. The molecular weight (MW) distribution and the identification of SMP in the effluents from three different anaerobic reactors will be presented. It has been found that the bulk of SMP lies in the low MW range, though compounds with MW as high as 300 kDa were also present in all anaerobic effluents. Preliminary results on the identification of such compounds using GC/MS surprisingly revealed the presence of long chain alkenes (C12-C24) and alkanes (C12-C16), as well as some aromatic compounds. These compounds that likely come from cell lysis and endogenous decay may not be easily biodegradable, hence their presence in the effluent is likely to cause the residual COD.