Thermophilic Upflow Anaerobic Sludge Blanket Research Articles

Biomineralization of phosphorus during anaerobic treatment of distillery wastewaters

This study addresses the pressing environmental concerns associated with the rapidly growing distillery industry, which is a significant contributor to wastewater generation. By focusing on the treatment of distillery wastewater using anaerobic digestion, this research explores the potential to convert organic materials into biofuels (methane). Moreover, the study aims to recover both methane and phosphorus from distillery wastewater in a single anaerobic reactor, which represents a novel and unexplored approach. Laboratory-scale experiments were conducted using mesophilic and thermophilic upflow anaerobic sludge blanket reactors. A key aspect of the study involved the implementation of a unique strategy: the mixing of centrate and spent caustic wastewater streams. This approach was intended to enhance treatment performance, manipulate the microbial community structure, and thereby optimizing the overall treatment performance. The integration of the centrate and spent caustic streams yielded remarkable co-benefits, resulting in significant biomethane production and efficient phosphorus precipitation. The study demonstrated a phosphorus removal efficiency of ∼60 % throughout the 130–140 days operation period. The recovery of phosphorus via the reactor sludge offers exciting opportunities for its utilization as a fertilizer or as a raw material within the phosphorus refinery industry. The biomethane produced during the treatment exhibits significant energy potential, estimated at 0.5 GJ/(m3 distillery wastewater).

A new non-steady-state mass balance model for quantifying microbiome responses to disturbances in wastewater bioreactors

Herein we proposed an ecology model, based on a non-steady-state mass balance (16S rRNA MiSeq reads normalized by volatile suspended solids), to quantify microbiome responses to disturbances in wastewater bioreactors. Rather than focusing on the most abundant microbial groups commonly used in the literature, the goal of the model was to identify active species within the community. The model incorporated the temporal changes of operational taxonomic units following a disturbance, through considering the density and type of genotypes in the influent entering the bioreactor, in the effluent leaving the bioreactor, growing in the bioreactor, and in the waste sludge discharged from the bioreactor continuously or instantaneously, as well as the prior microbial community and the sludge characteristics. One application of this model demonstrated that significant differences existed between the key populations responding to an increasing organic loading rate and the dominant species in a high-rate thermophilic upflow anaerobic sludge blanket reactor.

Simultaneous Phosphorus Recovery in Energy Generation Reactor (SPRING): High Rate Thermophilic Blackwater Treatment

The present work studied the application of a thermophilic upflow anaerobic sludge blanket (UASB) reactor treating source-diverted blackwater for simultaneous energy (in the form of methane production) and nutrient (phosphorus) recovery. Our results showed that a methane production rate of 2.4 ± 0.1 NL CH4/(L d) can be achieved with an organic loading rate of 12.9 kg COD/(m3d) and a methane yield of 55.9 ± 6.2%; far exceeding previous yields reported for blackwater treatment. Most significant was the removal of phosphorus (77.7 ±8.5% PO4-P) in the form of Ca3(PO4)2 precipitates in the anaerobic granular sludge. Key factors thought to promote the observed performance included rapid protein and urea hydrolysis along with hydrogenotrophic methanogenesis (by Methanosarcina spp.), and localized pH increase enhancing granulation and phosphorous sequestration. The granule sludge rich-in phosphorus contained negligible heavy metal contamination, and could provide an alternative source for phosphorus refinery or direct agricultural use.

Correction to: Polyphenols from Sugarcane Vinasses, Quantification, and Removal Using Activated Carbon After Biochemical Treatment in Laboratory-Scale Thermophilic Upflow Anaerobic Sludge Blanket Reactors

Correction to: Polyphenols from Sugarcane Vinasses, Quantification, and Removal Using Activated Carbon After Biochemical Treatment in Laboratory-Scale Thermophilic Upflow Anaerobic Sludge Blanket Reactors

Blackwater biomethane recovery using a thermophilic upflow anaerobic sludge blanket reactor: Impacts of effluent recirculation on reactor performance

Thermophilic anaerobic digestion is a promising process for high-solid blackwater (BW) treatment due to improved hydrolysis rates, high methanogenesis efficiency, and pathogen removal, when compared with mesophilic treatment. In the present work, the effects of effluent recirculation (i.e., mixing) on thermophilic blackwater treatment were studied. A laboratory-scale thermophilic upflow anaerobic sludge blanket reactor was operated with and without effluent recirculation. The methanogenesis efficiency of the BW treatment increased from 45.0 ± 2.9% when effluent recirculation was applied to 56.7 ± 5.5% without effluent recirculation. Without effluent recirculation, the COD accumulation in the bioreactor was reduced from 17.2 to 3.8% and the effluent volatile fatty acids (VFA) concentration was reduced from 0.64 ± 0.18 to 0.15 ± 0.10 g/L. Further, both acetoclastic and hydrogenotrophic methanogenic activity increased from 101.3 ± 10.8 and 93.9 ± 6.1 to 120.4 ± 9.4 and 118.2 ± 13.2 mg CH4-COD/(gVSS⋅d), respectively, after effluent recirculation was discontinued. The predominant methanogens changed from Methanothermobacter (67%) with effluent recirculation to Methanosarcina (62%) without effluent recirculation. As compared to the effluent recirculation conditions, the enhanced biomethane recovery and treatment performance without effluent recirculation can be attributed to the close proximity of bacteria and archaea groups and the reduced VFA accumulation. Predicted functional gene comparison showed higher prevalence of function for intermediate metabolite transportation (transporters, ATP-binding cassette (ABC) transporters, and two-component system) after discontinuing effluent circulation, which contributed to improved syntrophic propionate oxidation and syntrophic acetate oxidization and enhanced hydrogenotrophic methanogenesis.

Polyphenols from Sugarcane Vinasses, Quantification, and Removal Using Activated Carbon After Biochemical Treatment in Laboratory-Scale Thermophilic Upflow Anaerobic Sludge Blanket Reactors

Studies on the removal of polyphenols from sugarcane vinasses using three upflow anaerobic sludge blanket (UASB) reactors operating isothermally at temperatures of 45, 55, and 65 °C are presented. The procedure started evaluating the stability of the reactors through the quantification of four important parameters of the process: temperature, pH, alkalinity factor, and acetic acid production. The physical and chemical characterizations of the vinasses were made to evaluate the applied treatment. Total polyphenols were quantified before and after the biochemical treatment using the Folin-Ciocalteu’s method. The obtained results showed that this biochemical treatment was not enough by itself to remove the phenolic compounds present in the vinasses feed. The use of complementary treatment for the removal of polyphenols with activated carbon was proposed and evaluated. The effectiveness of the adsorption process was analyzed and compared through the models of Langmuir, Freundlich, and Dubinin-Radushkevich, using the obtained experimental data. The model that provided the best fit of the experimental data was the Langmuir model with a correlation coefficient of 0.991. The adsorption process achieved to reduce the concentration of polyphenols in the vinasses from 3600 mg of gallic acid equivalent (GAE)/L to 1072 mg GAE/L by single-step adsorption. That is equivalent to an overall reduction of 70.2%.

Anaerobic Treatment of Diluted Decanted Palm Oil Mill Effluent in a High-Rate Thermophilic Upflow Anaerobic Sludge Blanket Reactor: Microbial Selection and Enhanced Population of Hydrogenotrophic Methanogen

This paper presents a study of the startup and operation of a high-rate upflow anaerobic sludge blanket (UASB) treating diluted decanted palm oil mill effluent (POME) under thermophilic conditions. The operating temperature was increased in one-step from 25 to 55 °C. The organic loading rate (OLR) of POME was increased stepwise from 2 g/L day of COD to 23.8 g/L day. High-rate anaerobic digestion with methane (CH4) productivity of 8 L/L day was achieved. The development of microbial populations in the sludge involved an increase in both H2 producers, specifically the genus Clostridium, and H2 consumers, specifically the order Methanobacteriales. A gene quantification based on real-time polymerase chain reaction (PCR) showed that the archaeal rRNA gene concentration increased from 3.48 × 107 copies/mL in the original digested sludge to 1.89 × 109 copies/mL in the acclimated sludge. The increase in archaeal rRNA gene concentration was accompanied by a 3700 times increase in Methanobacteriales gene concentration. However, the Methanobacteriales was found to be vulnerable to substrate over-loading with its gene concentration decreasing 35 fold from that in the original digested sludge at an OLR of 23.8 g/L day.

Improving the energy balance of ethanol industry with methane production from vinasse and molasses in two-stage anaerobic reactors

Abstract Thermophilic anaerobic digestion is a viable option for processing sugarcane vinasse that recovers energy in the form of methane. However, restarting real-scale vinasse-treating reactors, at the resumption of the sugarcane harvest season, can lead to difficulties, making it desirable to keep them in continuous operation in the off-season. Thus, was evaluated the thermophilic anaerobic conversion of sugarcane vinasse in a two-stage upflow anaerobic sludge blanket (UASB) reactor (reactors R1 and R2), employing sugarcane molasses in the off-season for methane production, followed again by vinasse in the following harvest season. UASB reactor R2 yielded a higher methane percentage in biogas and higher volumetric methane production rates and methane yield than R1, accounting for a 58% increase in energy production in the two-stage system compared to the single-stage system using vinasse. It is observed that the molasses is an important source of carbon for the energy production using the thermophilic UASB reactors (18.01 MJ L−1 molasses), compared to alcoholic fermentation (8.30 MJ L−1 molasses). The methane produced in the reactors system (R1 + R2), after the off-season, can generate 5.1 kWh d−1 per m3 of vinasse, or 17% of the electricity needed to process a ton of sugarcane.

Reduction of alkalinity supplementation for acid-based wastewater treatment using a thermophilic multi-feed upflow anaerobic sludge blanket reactor

ABSTRACT Generally, Shochu distillery wastewater treatment is required the addition of alkalinity agents for an increase of pH in the UASB reactor. However, to reduce the cost of alkalinity supplementation, cost-effective reactor operation method has been desired. This study aimed to reduce the alkalinity supplementation for a thermophilic (55°C) multi-feed up-flow anaerobic sludge blanket (MF UASB) reactor for the low-cost treatment of the wastewater from the production of the Japanese distilled alcohol called shochu. Shochu distillery wastewater contains high concentrations of organics (46,500–57,600 mgCOD L−1; COD: chemical oxygen demand) and volatile fatty acids (16,200–25,000 mgCOD L−1), and low pH (4.1–4.5). With alkalinity supplementation of 0.045 mgCaCO3 mgCOD−1 using 24% NaOH, the MF UASB reactor achieved an 87 ± 2% COD removal rate with an organic loading rate of 24 kgCOD m−3 day−1 for 554 days reactor operation (hydraulic retention time of 10 h and influent concentration of 10,000 mgCOD L−1). The organic removal rate decreased to 19 ± 3% in the MF UASB reactor when alkalinity supplementation was reduced to 0.015 mgCaCO3·mgCOD−1. In this study , the minimum alkalinity supplementation was 0.045 mgCaCO3 mgCOD−1 at an organic loading rate of 24 kgCOD m−3 day−1.

Effect of temperature on selenium removal from wastewater by UASB reactors

The effect of temperature on selenium (Se) removal by upflow anaerobic sludge blanket (UASB) reactors treating selenate and nitrate containing wastewater was investigated by comparing the performance of a thermophilic (55 °C) versus a mesophilic (30 °C) UASB reactor. When only selenate (50 μM) was fed to the UASB reactors (pH 7.3; hydraulic retention time 8 h) with excess electron donor (lactate at 1.38 mM corresponding to an organic loading rate of 0.5 g COD L−1 d−1), the thermophilic UASB reactor achieved a higher total Se removal efficiency (94.4 ± 2.4%) than the mesophilic UASB reactor (82.0 ± 3.8%). When 5000 μM nitrate was further added to the influent, total Se removal was again better under thermophilic (70.1 ± 6.6%) when compared to mesophilic (43.6 ± 8.8%) conditions. The higher total effluent Se concentration in the mesophilic UASB reactor was due to the higher concentrations of biogenic elemental Se nanoparticles (BioSeNPs). The shape of the BioSeNPs observed in both UASB reactors was different: nanospheres and nanorods, respectively, in the mesophilic and thermophilic UASB reactors. Microbial community analysis showed the presence of selenate respirers as well as denitrifying microorganisms.

Comparative Mesophilic and Thermophilic Anaerobic Digestion of Palm Oil Mill Effluent Using Upflow Anaerobic Sludge Blanket

The effects of organic loading rate and operating temperature on the microbial diversity and performances of upflow anaerobic sludge blanket (UASB) reactors treating palm oil mill effluent (POME) were investigated. The following two UASB reactors were run in parallel for comparison: (1) under a mesophilic condition (37 degrees C) and (2) under a mesophilic condition in transition to a thermophilic condition (57 degrees C). A polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis showed that the microbial population profiles significantly changed with the organic loading rate (OLR) and the temperature transition from the mesophilic to the thermophilic condition. Significant biomass washout was observed for the mesophilic UASB when operating at a high organic loading rate (OLR) of 9.5 g chemical oxygen demand (COD)/L.d. In contrast, the thermophilic UASB can be operated at this OLR and at a temperature of 57 degrees C with satisfactory COD removal and biogas production. The PCR-based DGGE analysis suggested that the thermophilic temperature of 57 degrees C was suitable for a number of hydrolytic, acidogenic, and acetogenic bacteria.

An online-monitored thermophilic hydrogen production UASB reactor for long-term stable operation

In this study, the H 2 production and chemical oxygen demand (COD) removal performances of a thermophilic upflow anaerobic sludge blanket (UASB) reactor with online monitoring system were investigated. The online monitoring system enabled a rapid monitoring and timely control of the process. As a consequence, high operating stability was achieved despite of the varied hydraulic retention time (HRT) during 310-day operation. The COD efficiency remained at above 98%, and the hydrogen yield fluctuated slightly within the range of 2.42–3.06 mol H 2 mol −1 sucrose. Thermophilic H 2-producing granules were successfully cultivated in this reactor, which showed better physical and microbial properties than floc sludge and higher H 2 production rate than mesophilic granules. An analysis of the microbial growth kinetics further demonstrated a possibly higher synthesis and metabolism activity of microbes in the thermophilic granule state.

Exploration of water-recycled cassava bioethanol production integrated with anaerobic digestion treatment

Due to the limited success of discharge target-hitting treatment in coping with environmental pollution from the alcohol industry, our attention was directed towards the distillery spent (DS) wash recycle in a cleaner bioethanol production by integrating anaerobic digestion treatment with conventional fermentation. An anaerobic digestion effluent from an alcohol waste water treatment plant is applicable for single use in ethanol fermentation. With further experimental estimate, the recycle of DS treated by a sole thermophilic up flow anaerobic sludge blanket (UASB) treatment was adverse to ethanol fermentation, resulting in a gradual increase of the residual total sugar from 1.2% at batch 1 to 8.0% at batch 5 after 48 h. With a combination of the thermophilic and mesophilic UASB treatment, the thirteenbatch fermentation recycling its DS achieved ~10.5% of ethanol production and > 90% of starch utilization after 48 h, which was comparable to that using tap water. This revealed a potential of the anaerobic digestion treatment in water-saving and emission reduction for bioethanol industry. Key words : Anaerobic digestion, cassava, cleaner bioethanol production, distillery spent wash, thermophilic and mesophilic up flow anaerobic sludge blanket, recycle and reuse.

Estimation of Biogas Production Rate in a Thermophilic UASB Reactor Using Artificial Neural Networks

Biogas production rate was modeled and estimated in a thermophilic upflow anaerobic sludge blanket digester. Data set covers a time period of both steady-state conditions and an abnormal operation condition, i.e., organic loading shocks. Multilayer neural networks topology was used as the modeling tool. Half of the experimental data were used for the training of the model and the remaining half were used for the testing stage. Model results were evaluated from the point of view of both steady conditions and abnormal conditions. It was seen from the time series trends of the estimated data that biogas production rates at steady state operation conditions were closely estimated by the model while the results for organic loading shocks were sufficiently followed. Artificial neural network models gave encouraging estimation results for the online control of thermophilic reactors.

Diversity, Localization, and Physiological Properties of Filamentous Microbes Belonging to Chloroflexi Subphylum I in Mesophilic and Thermophilic Methanogenic Sludge Granules

We previously reported that the thermophilic filamentous anaerobe Anaerolinea thermophila, which is the first cultured representative of subphylum I of the bacterial phylum Chloroflexi, not only was one of the predominant constituents of thermophilic sludge granules but also was a causative agent of filamentous sludge bulking in a thermophilic (55 degrees C) upflow anaerobic sludge blanket (UASB) reactor in which high-strength organic wastewater was treated (Y. Sekiguchi, H. Takahashi, Y. Kamagata, A. Ohashi, and H. Harada, Appl. Environ. Microbiol. 67:5740-5749, 2001). To further elucidate the ecology and function of Anaerolinea-type filamentous microbes in UASB sludge granules, we surveyed the diversity, distribution, and physiological properties of Chloroflexi subphylum I microbes residing in UASB granules. Five different types of mesophilic and thermophilic UASB sludge were used to analyze the Chloroflexi subphylum I populations. 16S rRNA gene cloning-based analyses using a 16S rRNA gene-targeted Chloroflexi-specific PCR primer set revealed that all clonal sequences were affiliated with the Chloroflexi subphylum I group and that a number of different phylotypes were present in each clone library, suggesting the ubiquity and vast genetic diversity of these populations in UASB sludge granules. Subsequent fluorescence in situ hybridization (FISH) of the three different types of mesophilic sludge granules using a Chloroflexi-specific probe suggested that all probe-reactive cells had a filamentous morphology and were widely distributed within the sludge granules. The FISH observations also indicated that the Chloroflexi subphylum I bacteria were not always the predominant populations within mesophilic sludge granules, in contrast to thermophilic sludge granules. We isolated two mesophilic strains and one thermophilic strain belonging to the Chloroflexi subphylum I group. The physiological properties of these isolates suggested that these populations may contribute to the degradation of carbohydrates and other cellular components, such as amino acids, in the bioreactors.

Bicarbonate dosing: a tool to performance recovery of a thermophilic methanol-fed UASB reactor

The thermophilic-anaerobic treatment of methanol-containing wastewater in an upflow anaerobic sludge blanket (UASB) reactor, was found to be quite sensitive to pH shocks, both acid and alkaline. The results of the recovery experiments of sludge exposed to an alkaline shock, indicated that the addition or deprivation of sodium bicarbonate (NaHCO3) in the medium, plays an important role in the competition of methanogens and (homo)acetogens for methanol. In addition, caution has to be taken when using NaHCO3 for buffering methanol-containing wastewaters, since its introduction in the system will favour (homo)acetogenesis when proper conditions are not established. Based on these results, a recovery strategy for methanogenesis was proposed where bicarbonate is supplied stepwise, and the reactor is operated in a batch mode. This strategy was found to be appropriate, i.e. the results revealed that the recovery of methanogenesis on methanol from a reactor upset or complete failure caused by pH shock is possible, even in systems where (homo)acetogens are outcompeting methanogens. The time and the number of feedings required will depend on the degree of deterioration of the sludge.

Pelotomaculum thermopropionicum gen. nov., sp. nov., an anaerobic, thermophilic, syntrophic propionate-oxidizing bacterium.

An anaerobic, thermophilic, syntrophic propionate-oxidizing bacterium, strain SI(T), isolated previously from granular sludge in a thermophilic upflow anaerobic sludge blanket (UASB) reactor, was characterized. The strain could grow fermentatively on pyruvate and fumarate in pure culture. The strain grew on propionate, ethanol, lactate, 1-butanol, 1-pentanol, 1,3-propanediol, 1-propanol and ethylene glycol in co-culture with the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus strain deltaH(T). The optimum temperature for growth was 55 degrees C and the pH optimum was 7.0. The G+C content of the DNA was 52.8 mol %. Strain SI(T) contained MK-7 and MK-7(H4) as the major quinones and contained iso-C15:0 as the major fatty acid. Based on 16S rDNA sequence analysis, strain SI(T) formed a novel lineage within the gram-positive, spore-forming, sulphate-reducing bacterial group Desulfotomaculum. However, the strain lacked the ability to conduct dissimilatory sulphate reduction. Instead, it could reduce fumarate to succinate with concomitant growth on several organic substances as electron donor. These phenotypic and genetic properties support the formation of a novel species of a new genus, for which the name Pelotomaculum thermopropionicum gen. nov., sp. nov. is proposed. The type strain is strain SI(T) (= DSM 13744T = JCM 10971T).

Molecular and conventional analyses of microbial diversity in mesophilic and thermophilic upflow anaerobic sludge blanket granular sludges

The microbial community structure of mesophilic (35 degrees C) and thermophilic (55 degrees C) methanogenic granular sludges was surveyed by using both cultivation-independent molecular approach and conventional cultivation technique in order to address the fundamental questions on the microbial populations, i.e. who are present, where they are located, and what they are doing there. To elucidate the microbial constituents within both sludges, we first constructed 16S ribosomal DNA clone libraries, and partial sequencing of the clones was conducted for phylogenetic analysis. In this experiment, we found a number of unidentifiable clones within the domain Bacteria as well as clones that were closely related with 16S rDNAs of cultured microbes. The unidentifiable clones accounted for approximately 60-70% of the total clones in both mesophilic and thermophilic libraries. 16S rRNA-targeted in situ hybridization combined with confocal laser scanning microscopy was subsequently employed to examine where the uncultured populations were located within sludge granules. Spatial organization of uncultured microbes was visualized in thin-sections of both types of granules using fluorescent oligonucleotide probes, which were designed based on the clone sequences of certain novel clusters. This resulted in the detection of two types of uncultured cells in specific locations inside the granules. Finally, the goal-directed conventional cultivation technique was employed to recover such uncultured anaerobes and uncover their physiology and functions. In this approach, a total of five new species of thermophilic microorganisms were isolated, including several types of syntrophs and a novel sugar-fermenting bacterium. In the previous molecular approaches, all of these isolates were suggested to be significant populations within thermophilic granular sludge, hence obtaining these isolates in pure culture decreased the fraction of unknown clones in the previous thermophilic clone library from 70% to 40%. In conclusion, these approaches successfully revealed biodiversity and spatial organization of microbes of interest in sludge granules, and enlarged the fundamental knowledge of microbial constituents functioning as significant populations in the UASB processes.

高温嫌気性廃水処理汚泥から分離したプロピオン酸酸化共生細菌の生理学的特徴とグラニュール汚泥内空間分布

A propionate-oxidizing microorganism in the methanogenic granular sludge from a thermophilic (55°C) upflow anaerobic sludge blanket (UASB) process was studied by cultivation and in situ hybridization analysis. To isolate the propionate-oxidizing anaerobe, primary enrichment was made with propionate as the sole carbon source under anaerobic conditions at 55°C. After several attempts to purify the propionate-oxidizing microorganism, we finally isolated a thermophilic, syntrophic bacterium that was able to oxidize propionate in syntrophic coculture with Methanothermobacter thermautotrophicus (“Methanobacterium thermoautotrophicum”) strain ΔH. This syntrophic propionate-oxidizing bacterium, designated as strain SI, oxidized propionate as well as ethanol, lactate, 1-butanol, 1-pentanol, 1,3-propanediol, 1-propanol and ethylene glycol in syntrophic coculture. In addition, strain SI fermented pyruvate and fumarate in pure culture. The spatial distribution of strain SI in sludge granules was investigated by fluorescence in situ hybridization (FISH) with a 16S rRNA-targeted oligonucleotide probe specific for strain SI in combination with confocal laser scanning microscopy. Using these methods, strain SI was shown to be present in the middle and inner layers of the thermophilic granules and it was juxtaposed with hydrogenotrophic methanogens. In addition, strain SI-type cells were found to account for 1.1% of the total cells in the sludge by the FISH-direct count method. These results indicate that strain SI may be responsible for propionate oxidation in situ as one of the significant populations in sludge granules.

Removal of Slowly Biodegradable COD in Combined Thermophilic UASB and MBBR Systems

Starch, cellulose and polyvinyl alcohol (PVA) are common substrates of the slowly biodegradable COD (SBCOD) in industrial wastewaters. Removal of the individual and mixed SBCOD substrates was investigated in a combined system of thermophilic upflow anaerobic sludge blanket (TUASB) reactor (55°C) and aerobic moving bed biofilm reactor (MBBR). The removal mechanisms of the three SBCOD substrates were quite different. Starch-COD was almost equally utilized and removed in the two reactors. Cellulose-COD was completely (97-98%) removed from water in the TUASB reactor by microbial entrapment and sedimentation of the cellulose fibers. PVA alone was hardly biodegraded and removed by the combined reactors. However, PVA-COD could be removed to some extent in a binary solution of starch (77%) plus PVA (23%). The PVA macromolecules in the binary solution actually affected the microbial activity in the TUASB reactor resulting accumulation of volatile fatty acids, which shifted the overall COD removal from the TUASB to the MBBR reactor where SBCOD including PVA-COD was removed. Since the three SBCOD substrates were removed by different mechanisms, the combined reactors showed a better and more stable performance than individual reactors.