Thermophilic treatment by anaerobic granular sludge as an effective approach to accelerate the electron transfer and improve the reductive decolorization of azo dyes in bioreactors.

The effects of temperature, hydraulic retention time (HRT), and the redox mediator, anthraquinone-2,6-disulfonate (AQDS), on electron transfer and subsequent reductive decolorization of dyes from textile wastewater was assessed in mesophilic and thermophilic anaerobic bioreactors. The results clearly show that compared to mesophilic anaerobic treatment, thermophilic treatment at 55 degrees C is an effective approach for increasing the electron transfer capacity in bioreactors, and thus improving the decolorization rates. At an HRT of 2.5 h and in the absence of AQDS, the color removal was 5.3-fold higher at 55 degrees C compared to 30 degrees C. Furthermore, similar decolorizations were found at 55 degrees C between the AQDS-free and AQDS-supplemented reactors, whereas a significant difference (up to 3.6-fold) on dye reduction occurred at 30 degrees C.     

Temperature effects on the trophic stages of perennial rye grass anaerobic digestion

Continuous Stirred Tank Reactors (CSTRs), operated in batch mode, were used to evaluate the feasibility of psychrophilic (low temperature) digestion of perennial rye grass in a long term experiment (150 days) for the first time. The reactors were operated in parallel at 3 different temperatures, 10, 15 and 37 degrees C. Hydrolysis, acidification and methanogenesis were assessed by VS degradation, by soluble chemical oxygen demand (SCOD) and volatile fatty acids (VFA) production, and by methane production, respectively. Hydrolysis was the rate-limiting step at all temperatures and the rates and extent of hydrolysis were considerably lower at 15 and 10 degrees C, than at 37 degrees C. The total VS degradation was 53%, 34% and 19% at 37, 15 and 10 degrees C, respectively. Acidification was not affected by temperature and VFA production and consumption was balanced in all cases, except at 10 degrees C. Methane yields were 0.215 m3 CH4 kg(-1) VS(-1) added, 0.160 m3 CH4 kg(-1) VS(-1) added and 0.125 m3 CH4 kg(-1) VS(-1) added at 37, 15 and 10 degrees C, respectively. Methanogenesis was not strongly affected at 15 C but it became rate-limiting at 10 degrees C. Overall, the solid degradation and methane production performance under psychrophilic conditions was encouraging and greater than previously reported. Considering the non-acclimated, mesophilic nature of the inoculum, there are grounds to believe that low-temperature anaerobic digestion of grass could be feasible if coupled to efficient hydrolysis of the biomass.     

Granular biofilm-based anaerobic digestion: molecular biomonitoring and high-rate psychrophilic treatment of phenolic wastewater.

Two pairs of expanded granular sludge bed (EGSB) bioreactors, R1/R2 and R3/R4, were designed. R1/R2 were used for mesophilic (37 degrees C) treatment of synthetic wastewater over a 100-day trial. A successful start-up was achieved by R1 and R2, with COD removal over 90%. Both reactors were operated under identical parameters; however, increased organic loading induced a reduction in COD removal by R1, while R2 maintained satisfactory performance throughout the experiment. R3/R4 were operated at 15 degrees C throughout a 422-day trial and were used for the stabilisation of volatile fatty acid-based wastewater. Phenol was introduced to R4 at an applied loading rate of 1 kg phenol m(-3)d(-1), which was increased to 2 kg phenol m(-3)d(-1). No phenol was supplied to R3. Efficient COD conversion was recorded in both R3 and R4, thus demonstrating the feasibility of high-rate phenol degradation under psychrophilic conditions. Terminal restriction fragment length polymorphism analysis was applied to the characterisation of microbial community dynamics within each of the reactors. The results indicated a microbiological basis for the deviation, in terms of operational performance, of R1 and R2. TRFLP analyses indicated stable microbial communities in R3 and R4, but detected changes in the abundance of specific ribotypes in response to phenol mineralisation.     

Thermal pre-treatment of primary and secondary sludge at 70 degrees C prior to anaerobic digestion.

In general, mesophilic anaerobic digestion of sewage sludge is more widely used compared to thermophilic digestion, mainly because of the lower energy requirements and higher stability of the process. However, the thermophilic anaerobic digestion process is usually characterised by accelerated biochemical reactions and higher growth rate of microorganisms resulting in an increased methanogenic potential at lower hydraulic retention times. Furthermore, thermal pre-treatment is suitable for the improvement of stabilization and could be realized at relatively low cost especially at low temperatures. The present study investigates the effect of the pre-treatment at 70 degrees C on thermophilic (55 degrees C) anaerobic digestion of primary and secondary sludge in continuously operated digesters. Thermal pre-treatment of primary and secondary sludge at 70 degrees C enhanced the removal of organic matter and the methane production during the subsequent anaerobic digestion step at 55 degrees C. It also greatly contributed to the destruction of pathogens present in primary sludge. Finally it results in enhanced microbial activities of the subsequent anaerobic step suggesting that the same efficiencies in organic matter removal and methane recovery could be obtained at lower HRTs.     

Sequenced anaerobic-aerobic treatment of high strength, strong nitrogenous landfill leachates.

As a first step in treatment of high strength, strong nitrogenous landfill leachates (total COD--9.66-20.56 g/l, total nitrogen 780-1,080 mg/l), the performance of laboratory UASB reactors has been investigated under sub-mesophilic (19+/-3 degrees C) and psychrophilic (10+/-2 degrees C) conditions. Under hydraulic retention time (HRT) of around 1.2 days, when the average organic loading rate (OLR) was around 8.5 g COD/l/day, the total COD removal accounted for 71% (on average) for sub-mesophilic regime. The psychrophilic treatment conducted under the average HRT of 2.44 days and the average OLR of 4.2 g COD/l/day showed an average total COD removal of 58% giving effluents more suitable for subsequent biological nitrogen removal. Both anaerobic regimes were quite efficient for elimination of heavy metals by concomitant precipitation in the form of insoluble sulphides inside the sludge. The subsequent submesophilic aerobic-anoxic treatment of submesophilic anaerobic effluents led to only 75% of total inorganic N removal due to COD deficiency for denitrification created by too efficient anaerobic step. On the contrary, psychrophilic anaerobic effluents (richer in COD compared to the submesophilic ones) were more suitable for subsequent aerobic-anoxic treatment giving the total N removal of 95 and 92% at 19 and 10 degrees C, respectively.     

The effect of temperature on the performance and stability of thermophilic anaerobic digestion.

Sustainable operation of an anaerobic sewage sludge digester requires the effective shuttling of carbon from complex organic material to methane gas. The accumulation of intermediates and metabolic products such as volatile fatty acids and hydrogen gas not only reveal inefficiency within the digestion process, but can be detrimental to reactor operation at sufficiently high levels. Eight anaerobic digesters (1 mesophilic and 7 thermophilic) were operated in order to determine the effect of steady-state digestion temperature on the operational stability and performance of the digestion process. Replicate reactors operated at 57.5 degrees C, the highest temperature studied, were prone to accumulation of volatile fatty acids (4052 and 3411 mg/L as acetate) and gaseous hydrogen. Reactors operated at or below 55 degrees C showed no such accumulation of intermediate metabolites. Overall methanogenesis was also greatly reduced at 57.5 degrees C (0.09 L CH4/g VS fed) versus optimal methane formation at 53 degrees C (0.40 L CH4/g VS fed). Microbial community assessment and free energy calculations suggest that the accumulation of fatty acids and hydrogen, and relatively poor methanogenic performance at 57.5 degrees C are likely due to temperature limitations of thermophilic aceticlastic methanogens.     

Role of syntrophic microbial communities in high-rate methanogenic bioreactors

Anaerobic purification is a cost-effective way to treat high strength industrial wastewater. Through anaerobic treatment of wastewaters energy is conserved as methane, and less sludge is produced. For high-rate methanogenesis compact syntrophic communities of fatty acid-degrading bacteria and methanogenic archaea are essential. Here, we describe the microbiology of syntrophic communities in methanogenic reactor sludges and provide information on which microbiological factors are essential to obtain high volumetric methane production rates. Fatty-acid degrading bacteria have been isolated from bioreactor sludges, but also from other sources such as freshwater sediments. Despite the important role that fatty acid-degrading bacteria play in high-rate methanogenic bioreactors, their relative numbers are generally low. This finding indicates that the microbial community composition can be further optimized to achieve even higher rates.     

Automated equipment for anaerobic sludge parameters determination.

Methanogenic activity, anaerobic biodegradability and toxicity are key parameters in the design and operation of anaerobic bioreactors. A large variety of methods exist for the determination of these parameters but a normalized method has not been established so far. This paper presents the development of an automated manometric system for the determination of these anaerobic sludge parameters. The system is based on monitoring the production of methane by using a pressure transducer that measures the pressure in a gas-collecting chamber of known adjustable volume, which is independent of the space where biogas production takes place. The evolution of pressure generated by the accumulation of methane relates to the conversion of COD. In this way, the methanogenic activity of the sludge can be determined, as well as the biodegradability of solids and liquid, as well as the methanogenic toxicity of compounds. The equipment permits gas sampling, as well as extraction and introduction of liquid, without losing the anaerobic conditions. Various assays have been conducted to test the reliability and reproducibility of the obtained results, showing a high level of both. The methanogenic activities obtained in the assays ranged between 0.1 and 1.8 g COD g(-1) VSS d(-1), and the biodegradability of the organic compounds tested ranged between 20 and 90%.     

Effect of nitrate on the reduction of Reactive Red 2 by mesophilic anaerobic sludge.

This research aimed to evaluate the effect of nitrate on anaerobic azo dye reduction by using mesophilic bioreactors, in the absence (reactor R2) and in the presence (reactor R1) of redox mediators. The azo dye Reactive Red 2 (RR2) and the redox mediator anthraquinone-2,6-disulphonate (AQDS) were selected as model compounds. The results showed that the bioreactors were efficient on RR2 reduction, in which ethanol showed to be a good electron donor to sustain dye reduction under anaerobic conditions. The redox mediator AQDS increased the rates of reductive decolourisation, but its effect was not so remarkable compared to the previous experiments conducted. Contrary to the raised hypothesis that the nitrate addition could decrease decolourisation rates and catalytic properties of the redox mediators, no effect of nitrate was observed in the bioreactors, suggesting that the presence of nitrate in textile wastewaters will not decrease the capacity of anaerobic reactors supplemented or not with redox mediators to decolourize azo dyes.     

Relative kinetics of anaerobic digestion under thermophilic and mesophilic conditions

With several advantages over the conventional mesophilic anaerobic digestion, such as better sludge quality and higher biogas production, thermophilic anaerobic digestion is regarded as a promising alternative for sludge digestion. Primary and activated sludges are complex materials, and historically, analysis of kinetics has been largely on whole sludge, without analysis of individual components. This paper analyses relative digestion kinetics of pure substrates designed to target main stages of sludge digestion under thermophilic and mesophilic conditions. Hydrolysis rate of cellulose was significantly influenced by temperature with hydrolysis coefficients of--at 55 degrees C (0.7 +/- 0.1 day(-1)), 60 degrees C (0.8 +/- 0.2 day(-1)), 65 degrees C (1.1 +/- 0.2 day(-1)) and 70 degrees C (1.2 +/- 0.2 day(-1)) over 38 degrees C (0.4 +/- 0.1 day(-1)). This strongly follows the Arrhenius relationship, with an activation energy (E(A)) of 31 +/- 4 kJ mol(-1), corresponding to an increase of 1.5x for each 10 degrees C of temperature increase. Glucose uptake was rapid with a wide variety of fermentation products detected under mesophilic conditions, while uptake was slower under thermophilic conditions with acetate and propionate being dominant products. Propionate acetogenesis and acetate-utilizing methanogenesis kinetics were not influenced by temperatures. Hydrolysis is widely regarded as a rate-limiting step in sludge digestion, thus improvements in hydrolysis rates as measured during this study have the potential for significant improvements in overall apparent sludge digestion rates.     

Heavy metal pollution from Russian landfill leachates and its elimination together with other contaminants.

Systematic monitoring of raw leachates (RL) from the operating landfill "Khmet'yevo" during December, 2001--June, 2002 with regard to heavy metals (HM) revealed that these RL were moderately contaminated with Fe, Zn, Pb and Cd (Cu is present in non-dangerous concentrations). This contamination depends on season--the winter leachates are less polluted compared to the summer ones. For removal of HM together with removal of bulk COD, the UASB reactors were applied where, besides elimination of the major part of organic matter, concomitant precipitation of HM in the form of insoluble sulphides inside the sludge bed occurred due to development of the process of biological sulphate reduction. Both removal processes were quite efficient even during operation under submesophilic and psychrophilic conditions (20-10 degrees C). The subsequent submesophilic aerobic-anoxic treatment of submesophilic anaerobic effluents led to only 75% of total inorganic N removal due to COD deficiency for denitrification created by a too efficient anaerobic step. On the contrary, psychrophilic anaerobic effluents (richer in COD compared to the submesophilic ones) were more suitable for subsequent aerobic-anoxic treatment giving the total N removal of 95 and 92% at 20 and 10 degrees C, respectively. The final effluent is approaching the current national standards for direct discharge of treated wastewater.     

Thermal pretreatment of the solid fraction of manure: impact on the biogas reactor performance and microbial community.

Application of thermal treatment at 100-140 degrees C as a pretreatment method prior to anaerobic digestion of a mixture of cattle and swine manure was investigated. In a batch test, biogasification of manure with thermally pretreated solid fraction proceeded faster and resulted in the increase of methane yield. The performances of two thermophilic continuously stirred tank reactors (CSTR) treating manure with solid fraction pretreated for 40 minutes at 140 degrees C and non-treated manure were compared. The digester fed with the thermally pretreated manure had a higher methane productivity and an improved removal of the volatile solids (VS). The properties of microbial communities of both reactors were analysed. The specific methanogenic activity (SMA) test showed that both biomasses had significant activity towards hydrogen and formate, while the activity with the VFA - acetate, propionate and butyrate - was low. The kinetic parameters of the VFA conversion revealed a reduced affinity of the microbial community from the CSTR fed with thermally pre-treated manure for acetate, propionate and butyrate. The bacterial and archaeal populations identified by t-RLFP analysis of 16S rRNA genes were found to be identical in both systems. However, a change in the abundance of the species present was detected.     

Use of microwave pretreatment for enhanced anaerobiosis of secondary sludge.

This work elucidates the effects of pretreatment of secondary sludge by microwave irradiation on anaerobic digestion. The soluble chemical oxygen demand (COD) concentration increased up to 22% as microwave irradiation time increased, which indicated the sludge particles disintegrated. Three identical automated bioreactors with working volume of 5 l were used as anaerobic digesters at mesophilic temperature (35 degrees C). The reactors were separately fed with sludge with microwave pretreated- and control- sludge at different hydraulic retention times (HRT). The volatile solid (VS) reduction in the control operation was approximately 23.2 +/- 1.3%, while it was 25.7 +/- 0.8% for the reactors with the pretreated sludge. The average biogas production rate with the pretreated sludge at 8, 10, 12, and 15 days HRTs was 240 +/- 11, 183 +/- 9, 147 +/- 8, and 117 +/- 7 ml/l/d respectively, while those with the control sludge were 134 +/- 12 and 94 +/- 7 ml/l/d at 10 and 15 days HRTs. Maximum rates of COD removal and methane production with the pretreated sludge were 64% and 79% higher than those of the control system, respectively.     

Syntrophic acetate oxidation in two-phase (acid-methane) anaerobic digesters

The microbial processes involved in two-phase anaerobic digestion were investigated by operating a laboratory-scale acid-phase (AP) reactor and analyzing two full-scale, two-phase anaerobic digesters operated under mesophilic (35 °C) conditions. The digesters received a blend of primary sludge and waste activated sludge (WAS). Methane levels of 20% in the laboratory-scale reactor indicated the presence of methanogenic activity in the AP. A phylogenetic analysis of an archaeal 16S rRNA gene clone library of one of the full-scale AP digesters showed that 82% and 5% of the clones were affiliated with the orders Methanobacteriales and Methanosarcinales, respectively. These results indicate that substantial levels of aceticlastic methanogens (order Methanosarcinales) were not maintained at the low solids retention times and acidic conditions (pH 5.2-5.5) of the AP, and that methanogenesis was carried out by hydrogen-utilizing methanogens of the order Methanobacteriales. Approximately 43, 31, and 9% of the archaeal clones from the methanogenic phase (MP) digester were affiliated with the orders Methanosarcinales, Methanomicrobiales, and Methanobacteriales, respectively. A phylogenetic analysis of a bacterial 16S rRNA gene clone library suggested the presence of acetate-oxidizing bacteria (close relatives of Thermacetogenium phaeum, 'Syntrophaceticus schinkii,' and Clostridium ultunense). The high abundance of hydrogen consuming methanogens and the presence of known acetate-oxidizing bacteria suggest that acetate utilization by acetate oxidizing bacteria in syntrophic interaction with hydrogen-utilizing methanogens was an important pathway in the second-stage of the two-phase digestion, which was operated at high ammonium-N concentrations (1.0 and 1.4 g/L). A modified version of the IWA Anaerobic Digestion Model No. 1 (ADM1) with extensions for syntrophic acetate oxidation and weak-acid inhibition adequately described the dynamic profiles of volatile acid production/degradation and methane generation observed in the laboratory-scale AP reactor. The model was validated with historical data from the full-scale digesters.     

Phosphate inhibition on thermophilic acetoclastic methanogens: a warning.

The inhibitory effect of phosphate on acetoclastic-methanogens was investigated for three different thermophilic (55 degrees C) anaerobic consortia. When 70 mM of phosphate was tested, acetoclastic methanogens was completely inhibited in "Eerbeek" sludge which is dominated by Methanosaeta-like methanogens. For the "Hoogezand" sludge the specific methanogenic activity dropped by 79%, indicating that any of the acetate-consuming methanogens present in the sludge was more resistant to the phosphate applied. The results demonstrated that the inhibitory effect of phosphate may affect both methane production rate and final methane concentration and might also be time dependent. This study indicates that the degree of inhibition is species-dependent, and even more resistant species may be affected during long-term experiments. Such inhibition is a matter of concern for researchers since misleading conclusions might be taken from growth and specific methanogenic activity tests when considerable concentrations of phosphate buffer are used and no interference is expected.     

Two-stage thermophilic-mesophilic anaerobic digestion of waste activated sludge from a biological nutrient removal plant.

A two-stage thermophilic-mesophilic anaerobic digestion pilot-plant was operated solely on waste activated sludge (WAS) from a biological nutrient removal (BNR) plant. The first-stage thermophilic reactor (HRT 2 days) was operated at 47, 54 and 60 degrees C. The second-stage mesophilic digester (HRT 15 days) was held at a constant temperature of 36-37 degrees C. For comparison with a single-stage mesophilic process, the mesophilic digester was also operated separately with an HRT of 17 days and temperature of 36-37 degrees C. The results showed a truly thermophilic stage (60 degrees C) was essential to achieve good WAS degradation. The lower thermophilic temperatures examined did not offer advantages over single-stage mesophilic treatment in terms of COD and VS removal. At a thermophilic temperature of 60 degrees C, the plant achieved 35% VS reduction, representing a 46% increase compared to the single-stage mesophilic digester. This is a significant level of degradation which could make such a process viable in situations where there is no primary sludge generated. The fate of the biologically stored phosphorus in this BNR sludge was also investigated. Over 80% of the incoming phosphorus remained bound up with the solids and was not released into solution during the WAS digestion. Therefore only a small fraction of phosphorus would be recycled to the main treatment plant with the dewatering stream.     

Membrane-coupled anaerobic digestion of municipal sewage sludge.

Membrane-coupled anaerobic digestion utilizes a concept of simultaneous sludge digestion and thickening. Membranes may successfully be applied to eliminate the need for thickening polymers and avoid their likely inhibitory effect on anaerobic biomass. A 550 L completely mixed anaerobic digester was operated under mesophilic conditions (35 degrees C). Two ultrafiltration membrane systems were evaluated for their potential in membrane-coupled anaerobic digestion: vibrating and cross flow. A volatile solids reduction of 590% was achieved at an average mixed liquor suspended solids concentration of 1.8%. The substrate utilization rate was 1.3 d(-1). The vibrating membrane operated at a flux of 1.6-2.0 m3/m2-d and the tubular membrane fluxes in the range 3.4-3.6 m3/m2-d.     

Integrated biological treatment of recalcitrant effluents from pulp mills.

This work aimed at determining the degree of depuration of a recalcitrant effluent (weak black liquor, WBL) achieved in a series treatment consisting of a first stage methanogenic fluidised bed reactor followed by a second stage aerobic, upflow reactor packed with "biocubes" of Trametes versicolor immobilised onto small cubes of holm oak wood. The mesophilic, lab scale methanogenic fluidised bed reactor contained a microbial consortium immobilised onto granular activated carbon 500 microm average size. The process removed decreasing amounts of organic matter at decreasing hydraulic retention times (HRT), eventually reaching an average of 50% at 0.5 day HRT. Colour and ligninoid removals also decreased with decreasing HRT. Although the methanogenic fluidised bed reactor provided an effective treatment for the degradable organic matter, important concentrations of recalcitrant organic matter and colour still remained in the anaerobic effluent. This anaerobic effluent was fed to the aerobic packed bed reactor. Two HRT were tested in this unit, namely 5 and 2.5 days. The reactor averaged an organic matter removal in the range of 32% COD basis, during an experimental run of 95 days. Colour and ligninoid contents were removed in high percentages (69% and 54%, respectively). There was no significant difference in reactor performance at 5- and 2.5-day HRT. There was a positive correlation between pollutant removal efficiencies and Laccase activity in crude extracts of the reactor liquor. No supplemental soluble carbohydrate was required to sustain the fungus activity and the consistent reactor performance. Overall, the two-stage treatment achieved approximately a 78% removal of the original organic matter of the WBL (COD basis) and ca. 75% of colour and ligninoid contents.     

Factors affecting measurement of specific methanogenic activity.

Specific methane production (SMP) tests have been used to determine the potential loading rate capacity of anaerobic reactors, to characterize biomass prior to its use as an inoculum for new anaerobic reactors, to detect changes in biomass activity during operation, or to assess the occurrence of toxic conditions. SMP tests also provide a basis for estimating specific methanogenic activity in mixed anaerobic cultures. SMP protocols used to date have varied widely in both procedure and objective. Tests conducted by the present authors indicated that biomass concentration, substrate type and concentration, and mixing intensity are factors that can affect the results of SMP tests.     

Effect of didecyl dimethyl ammonium chloride on nitrate reduction in a mixed methanogenic culture.

The effect of the quaternary ammonium compound, didecyl dimethyl ammonium chloride (DDAC), on nitrate reduction was investigated at concentrations up to 100 mg/L in a batch assay using a mixed, mesophilic (35 degrees C) methanogenic culture. Glucose was used as the carbon and energy source and the initial nitrate concentration was 70 mg N/L. Dissimilatory nitrate reduction to ammonia (DNRA) and to dinitrogen (denitrification) were observed at DDAC concentrations up to 25 mg/L. At and above 50 mg DDAC/L, DNRA was inhibited and denitrification was incomplete resulting in accumulation of nitrous oxide. At DDAC concentrations above 10 mg/L, production of nitrous oxide, even transiently, resulted in complete, long-term inhibition of methanogenesis and accumulation of volatile fatty acids. Fermentation was inhibited at and above 75 mg DDAC/L. DDAC suppressed microbial growth and caused cell lysis at a concentration 50 mg/L or higher. Most of the added DDAC was adsorbed on the biomass. Over 96% of the added DDAC was recovered from all cultures at the end of the 100-days incubation period, indicating that DDAC did not degrade in the mixed methanogenic culture under the conditions of this study.