Novel application of oxygen-transferring membranes to improve anaerobic wastewater treatment

Anaerobic biological wastewater treatment has numerous advantages over conventional aerobic processes; anaerobic biotechnologies, however, still have a reputation for low-quality effluents and operational instabilities. In this study, anaerobic bioreactors were augmented with an oxygen-transferring membrane to improve treatment performance. Two anaerobic bioreactors were fed a synthetic high-strength wastewater (chemical oxygen demand, or COD, of 11,000 mg l(-1)) and concurrently operated until biomass concentrations and effluent quality stabilized. Membrane aeration was then initiated in one of these bioreactors, leading to substantially improved COD removal efficiency (> 95%) compared to the unaerated control bioreactor (approximately 65%). The membrane-augmented anaerobic bioreactor required substantially less base addition to maintain circumneutral pH and exhibited 75% lower volatile fatty acid concentrations compared to the unaerated control bioreactor. The membrane-aerated bioreactor, however, failed to improve nitrogenous removal efficiency and produced 80% less biogas than the control bioreactor. A third membrane-augmented anaerobic bioreactor was operated to investigate the impact of start-up procedure on nitrogenous pollutant removal. In this bioreactor, excellent COD (>90%) and nitrogenous (>95%) pollutant removal efficiencies were observed at an intermediate COD concentration (5,500 mg l(-1)). Once the organic content of the influent wastewater was increased to full strength (COD = 11,000 mg l(-1)), however, nitrogenous pollutant removal stopped. This research demonstrates that partial aeration of anaerobic bioreactors using oxygen-transferring membranes is a novel approach to improve treatment performance. Additional research, however, is needed to optimize membrane surface area versus the organic loading rate to achieve the desired effluent quality.     

Bioleaching of sulfidic tailing samples with a novel, vacuum-positive pressure driven bioreactor

This study presents a design for a novel bioreactor that uses alternating vacuum and positive pressure cycles to transfer acidic leach solution in and out of contact with finely ground sulfidic mine tailings. These tailings constitute an environmental problem that needs experimental data to support the development of management and control strategies. A conventional stirred tank bioreactor was used as a reference system. Both bioreactors were inoculated with mixed cultures of acidophilic iron and sulfur oxidizers. The rate of the bioleaching of tailings was 0.50 +/- 0.14 g Fe/L . day in the stirred tank bioreactor and 0.17 +/- 0.05 g Fe/L . day in the novel bioreactor. Microbial populations were identified in the two-bioreactor systems by analysis of 16S rRNA genes involving amplification, denaturing gradient gel electrophoresis (DGGE), cloning, and sequencing. The inoculum contained sulfur-oxidizing Acidithiobacillus caldus and Acidithiobacillus thiooxidans, iron oxidizers from the genera Leptospirillum and Ferroplasma, and a chemoorganotrophic Alicyclobacillus sp. During bioleaching of the tailings, the microbial populations in both bioreactors were similar to the inoculum culture, except that At. thiooxidans outgrew At. caldus. Sequences consistent with a Sulfobacillus sp. were amplified from both bioreactor samples although this bacterium was initially below the level of detection in the inoculum. After prolonged operation, Ferroplasma acidiphilum and an uncultured bacterium related to the CFB group were also detected in the novel bioreactor, whereas Sulfobacillus sp. was no longer detected. The novel bioreactor has potential uses in other areas of environmental biotechnology that involves periodic contact of liquids with solid substrates.     

Temporal dynamics and degradation activity of an bacterial inoculum for treating waste metal-working fluid

In order for established bioreactors to be effective for treating chemically mixed wastes such as metal working fluids (MWF) it is essential that they harbour microbial populations that can maintain sufficient active biomass and degrade each of the chemical constituents present. In this study we investigated the effectiveness of a bacterial consortium composed of four species (Clavibacter michiganensis, Methylobacterium mesophilicum, Rhodococcus erythropolis and Pseudomonas putida), assembled on the basis of their apparent ubiquity in waste MWF, degradation ability and tolerance to fluctuating chemistry of the waste. The temporal dynamics of the inoculum and its effects on the fate of individual chemical components of the waste were studied, by regular sampling, over 400 h. Using a complementary approach of culture with chemotaxonomic (FAME) analysis and applying group specific probes (FISH), the inoculum was found to represent a significant component of the community in bioreactors with and without presence of indigenous MWF populations. In addition, the reduction in the COD by the consortium was approximately 85% of the total pollution load, and 30-40% more effectively than any other treatment (indigenous MWF community alone or activated sludge). Furthermore, all the chemical constituents, including the biocide (a formaldehyde release agent) demonstrated > 60% reduction. Many chemical components of the MWF proved to be recalcitrant in the other treatments. The results of this study confirm that assemblage of an inoculum, based on a comprehensive knowledge of the indigenous microbial community, in the target habitat, is a highly effective way of selecting microbial populations for bioaugmentation of bioreactors.     

Activity of Nitrifying Biofilms Constructed on Low-Density Polyester Enhances Bioremediation of a Coastal Wastewater Effluent

Summary Nitrifying biofilms were constructed on low density polyester Dacron for the bioremediation of nitrogen from wastewater effluent of a municipal treatment plant. Dacron disks were inoculated with wastewater sludge enriched for 15 days for either ammonia- or nitrite-oxidizing bacteria (AOB or NOB, respectively) and packed into glass bioreactors. Wastewater effluent containing high levels of ammonia, nitrite, and phosphate was collected and fed to inoculated and uninoculated bioreactors. Both inoculated bioreactors showed stable nitrification efficiencies, removing 96 and 76% of the ammonia and 12 and 35% of the nitrite for AOB- and NOB-inoculated bioreactors, respectively. Efficiencies of phosphate removal were similar in both inoculated and uninoculated bioreactors, indicating that nitrifiers were not required for this process. AOB-inoculated bioreactors accumulated nitrite mid-way through the experiment and had low rates of conversion to nitrate, suggesting slow nitrite oxidizer growth. DGGE and sequence analysis of AOB 16S rRNA genes showed enrichment of Nitrosomonas spp. in both inoculated bioreactors, and a dominance of Nitrosospira spp. in non-inoculated bioreactors. This study describes an inexpensive and efficient technology for removing ammonia and nitrite from wastewater effluents of municipal treatment plants before its release to the environment.     

Long-Term Stability of Mercury-Reducing Microbial Biofilm Communities Analyzed by 16S-23S rDNA Interspacer Region Polymorphism

The composition of mercury-reducing communities in two bioreactors retaining Hg(II) from chloralkali electrolysis wastewater for 485 days was analyzed based on effluent community DNA. Packed bed bioreactors with lava chips as carrier of the biofilm were inoculated with nine Hg(II)-resistant isolates that belonged to the alpha and gamma subdivisions of the proteobacteria. A rapid DNA-fingerprinting method was applied, using the intergenic spacer region (ISR) of the 16S-23S rDNA for analysis of the community composition. This allowed discrimination of the inoculum strains down to subspecies level. A merA specific PCR permitted the discrimination of the community's merA genes. During the 485 days of operation, the bioreactors were exposed to various physical stresses (mixing, gas bubbles, temperature increase up to 41°C, increased flow velocity) and repeated high mercury inflow concentrations, resulting in reduced bioreactor performance and decreased culturable cell numbers in the reactor effluent. Nevertheless, the composition of the microbial community remained rather stable throughout the investigated time period. Of the inoculum strains, two could be detected throughout, whereas three were sometimes present with varying periods of nondetection. Two inoculum strains were only detected within the first month. Two strains of gamma-proteobacteria that were able to reduce ionic mercury invaded the bioreactor community. They did not outcompete established strains and had no negative effect on the Hg(II)-retention activity of the bioreactors. The community comprised diverse merA genes. The abundance of merA genes matched the abundance of their respective strains as confirmed by ISR community analysis. The continuously high selection pressure for mercury resistance maintained a stable and highly active mercury-reducing microbial community within the bioreactors.     

Treatment of urban wastewater in a membrane bioreactor at high organic loading rates.

Membrane bioreactors can replace the activated sludge process and the final clarification step in municipal wastewater treatment. The combination of bioreactor and crossflow microfiltration allows for a high chemical oxygen demand (COD) reduction of synthetic wastewater. From biomass, grown at high production rates in the aerobic bioreactor, energy rich biogas can be obtained in a subsequent anaerobic bioreactor. In this paper, experimental data from a laboratory scale membrane bioreactor are presented. The degradation of synthetic wastewater at short hydraulic retention times down to 1.5 h has been studied. The organic loading rate (OLR) has been varied in the range of 6-13 kg m(-3) per day. At steady state a high quality filtrate could be obtained at different operating conditions. At biomass concentrations of 10-22 g l(-1), COD reduction was above 95%.     

Treatment of turkey processing wastewater with sand filtration

This research investigated the feasibility of coarse/fine sand filtration for removing organic materials from turkey processing wastewater. Sand filtration was tested with three organic and hydraulic loadings. Six two-layer sand bioreactors were in three groups, each with 5 cm layer of pea gravel at the bottom to support layers of fine sand (46 cm) and coarse sand (15 cm) to a height of 66 cm. The bioreactors were inoculated with a mixture of 20% (vol/vol) of wastewater lagoon sludge, 40% (vol/vol) of turkey processing wastewater, and 40% (vol/vol) of BOD(5) dilution water before starting the column operation with turkey processing wastewater. The wastewater contained 1270+/-730 mg COD/L and was applied to each sand bioreactor at hydraulic loading rates of 94% during 80 days of column operation at low and medium hydraulic loading rates (132 L/m(2)/day). The removal at the highest hydraulic loading rate (264 L/m(2)/day) declined after the appearance of a black zone in the top layer of fine sand on day 30 for one reactor and day 50 for the other. The sand filtration in this study represents a feasible treatment for turkey processing wastewater and its efficiency and the life span of the process are associated with the extent of hydraulic loading of the sand bioreactors.     

Study of microbial community structures in UASB sludge treating municipal wastewater by denaturing gradient gel electrophoresis of 16S rDNA

The structures of microbial communities in lab-scale upflow anaerobic sludge blanket (UASB) reactors for treating municipal wastewater with different ratios of COD soluble/COD total were studied using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes.The microbial structure of the inoculum sludge obtained from a full-scale UASB reactor of treating potato processing wastewater was compared with the structures of sludges collected from three lab-scale UASB reactors after eight months feeding with raw municipal wastewater, with CEPS (chemically enhanced primary sedimentation) pretreated municipal wastewater, and with a synthetic municipal sewage, respectively. Computer-aided numerical analysis of the DGGE fingerprints showed that the bacterial community underwent major changes. The sludges for treating raw and CEPS pretreated wastewater had very similar bacterial and archaeal communities (82%and 96% similarity) but were different from that for treating the synthetic sewage. Hence, despite similar % COD in the particulate form in the synthetic and the real wastewater, the two wastewaters were selected for different microbial communities. Prominent DGGE bands of Bacteria and Archaea were purified and sequenced. The 16S rRNA gene sequences of the dominant archaeal bands found in the inoculum, and UASB sludge fed with raw sewage, CEPS pretreated wastewater, and synthetic sewage were closely associated with Methanosaeta concilii. In the UASB sludge fed with synthetic sewage, another dominant band associated with an uncultured archaeon 39-2 was found together with M. concilii.     

Nitrification in hybrid bioreactors treating simulated domestic wastewater

Aim

To provide deeper insights into nitrification process within aerobic bioreactors containing supplemental physical support media (hybrid bioreactors).

Methods and Results

Three bench‐scale hybrid bioreactors with different media size and one control bioreactor were operated to assess how biofilm integrity influences microbial community conditions and bioreactor performance. The systems were operated initially at a 5‐day hydraulic retention time (HRT), and all reactors displayed efficient nitrification and chemical oxygen demand (COD) removal (>95%). However, when HRT was reduced to 2·5 days, COD removal rates remained high, but nitrification efficiencies declined in all reactors after 19 days. To explain reduced performance, nitrifying bacterial communities (ammonia‐oxidizing bacteria, AOB; nitrite‐oxidizing bacteria, NOB) were examined in the liquid phase and also on the beads using qPCR, FISH and DGGE. Overall, the presence of the beads in a reactor promoted bacterial abundances and diversity, but as bead size was increased, biofilms with active coupled AOB–NOB activity were less apparent, resulting in incomplete nitrification.

Conclusions

Hybrid bioreactors have potential to sustain effective nitrification at low HRTs, but support media size and configuration type must be optimized to ensure coupled AOB and NOB activity in nitrification.

Significance and Impact of the Study

This study shows that AOB and NOB coupling must be accomplished to minimize nitrification failure.     

Study of microbial community structures in UASB sludge treating municipal wastewater by denaturing gradient gel electrophoresis of 16S rDNA

The structures of microbial communities in lab-scale upflow anaerobic sludge blanket (UASB) reactors for treating municipal wastewater with different ratios of COD soluble/ COD total were studied using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes. The microbial structure of the inoculum sludge obtained from a full-scale UASB reactor of treating potato processing wastewater was compared with the structures of sludges collected from three lab-scale UASB reactors after eight months feeding with raw municipal wastewater, with CEPS (chemically enhanced primary sedimentation) pretreated municipal wastewater, and with a synthetic municipal sewage, respectively. Computer-aided numerical analysis of the DGGE fingerprints showed that the bacterial community underwent major changes. The sludges for treating raw and CEPS pretreated wastewater had very similar bacterial and archaeal communities (82% and 96% similarity) but were different from that for treating the synthetic sewage. Hence, despite     

Lemna minor tolerance to metal‐working fluid residues: implications for rhizoremediation

For the first time in the literature, duckweed (Lemna minor) tolerance (alone or in combination with a consortium of bacteria) to spent metal‐working fluid (MWF) was assessed, together with its capacity to reduce the chemical oxygen demand (COD) of this residue. In a preliminary study, L. minor response to pre‐treated MWF residue (ptMWF) and vacuum‐distilled MWF water (MWFw) was tested. Plants were able to grow in both residues at different COD levels tested (up to 2300 mg·l^?1), showing few toxicity symptoms (mainly growth inhibition). Plant response to MWFw was more regular and dose responsive than when exposed to ptMWF. Moreover, COD reduction was less significant in ptMWF. Thus, based on these preliminary results, a second study was conducted using MWFw to test the effectiveness of inoculation with a bacterial consortium isolated from a membrane bioreactor fed with the same residue. After 5 days of exposure, COD in solutions containing inoculated plants was significantly lower than in non‐inoculated ones. Moreover, inoculation reduced β+γ‐tocopherol levels in MWFw‐exposed plants, suggesting pollutant imposed stress was reduced. We therefore conclude from that L. minor is highly tolerant to spent MWF residues and that this species can be very useful, together with the appropriate bacterial consortium, in reducing COD of this residue under local legislation limits and thus minimise its potential environmental impact. Interestingly, the lipophilic antioxidant tocopherol (especially the sum of β+γ isomers) proved to be an effective plant biomarker of pollution.     

Bioaugmentation strategies for remediating mixed chemical effluents

Operationally exhausted metal working fluids are chemically mixed, produced in large quantities (400 000 tonnes year in the U.K.), and potentially environmentally toxic. It is essential to develop more reliable and economical approaches for their disposal. We investigated the effectiveness of a defined bacterial consortium, constructed specifically for treating metal-working fluid (MWF), and contrasted its performance to that of undefined inocula from activated sludge. Construction of the consortium was based on knowledge of the diversity of bacterial communities that naturally colonize MWF and determination of their catabolic abilities and tolerance to the chemical constituents. Chemical analysis of the inoculated MWF bioreactor revealed that, after 100 h at 28 degrees C, the defined inoculum reduced the pollution load by over 80% from an initial chemical oxygen demand of approximately 48 000 mg L(-)(1). The inocula performance was approximately 50% more effective than that of the undefined microbial community from the activated sludge. Furthermore, the performance of the constructed consortium was more reproducible than that of an undefined community, an essential feature for bioaugmentation treatment of industrial wastes.     

Biodegradation of Trichloroethylene in Continuous-Recycle Expanded-Bed Bioreactors

Experimental bioreactors operated as recirculated closed systems were inoculated with bacterial cultures that utilized methane, propane, and tryptone-yeast extract as aerobic carbon and energy sources and degraded trichloroethylene (TCE). Up to 95% removal of TCE was observed after 5 days of incubation. Uninoculated bioreactors inhibited with 0.5% Formalin and 0.2% sodium azide retained greater than 95% of their TCE after 20 days. Each bioreactor consisted of an expanded-bed column through which the liquid phase was recirculated and a gas recharge column which allowed direct headspace sampling. Pulses of TCE (20 mg/liter) were added to bioreactors, and gas chromatography was used to monitor TCE, propane, methane, and carbon dioxide. Pulsed feeding of methane and propane with air resulted in 1 mol of TCE degraded per 55 mol of substrate utilized. Perturbation studies revealed that pH shifts from 7.2 to 7.5 decreased TCE degradation by 85%. The bioreactors recovered to baseline activities within 1 day after the pH returned to neutrality.     

Effects of cationic polymer on performance of UASB reactors treating low strength wastewater

The effect of cationic polymer additives on biomass granulation and COD removal efficiency had been examined in lab-scale upflow anaerobic sludge blanket (UASB) reactors, treating low strength synthetic wastewater (COD 300-630 mg/l). Under identical conditions, two reactors were operated with and without polymer additives in inoculum under four different organic loading rates (OLRs). The optimum polymer dose was adopted based upon the results of jar test and settling test carried out with inoculum seed sludge. With the use of thick inoculum, SS greater than 110 g/l and VSS/SS ratio less than 0.3, granulation was observed in UASB reactor treating synthetic wastewater as well as actual sewage, when OLR was greater than 1.0 kg COD/m(3) d. Polymer additive with such thick inoculum was observed to deteriorate percentage granules and COD removal efficiency compared to inoculum without polymer additives. At OLR less than 1.0 kg COD/m(3) d, proper granulation could not be achieved in both the reactors inoculated with and without polymer additive. Also, under this low loading, drastic reduction in COD removal efficiency was observed with polymer additives in inoculum. Hence, it is rational to conclude that biomass granulation for treatment of low strength biodegradable wastewater depends on the applied loading rate and selection of thick inoculum sludge.     

Study of microbial community structures in UASB sludge treating municipal wastewater by denaturing gradient gel electrophoresis of 16S rDNA

The structures of microbial communities in lab-scale upflow anaerobic sludge blanket (UASB) reactors for treating municipal wastewater with different ratios of COD_soluble/ COD_total were studied using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes. The microbial structure of the inoculum sludge obtained from a full-scale UASB reactor of treating potato processing wastewater was compared with the structures of sludges collected from three lab-scale UASB reactors after eight months feeding with raw municipal wastewater, with CEPS (chemically enhanced primary sedimentation) pretreated municipal wastewater, and with a synthetic municipal sewage, respectively. Computer-aided numerical analysis of the DGGE fingerprints showed that the bacterial community underwent major changes. The sludges for treating raw and CEPS pretreated wastewater had very similar bacterial and archaeal communities (82% and 96% similarity) but were different from that for treating the synthetic sewage. Hence, despite similar % COD in the particulate form in the synthetic and the real wastewater, the two wastewaters were selected for different microbial communities. Prominent DGGE bands of Bacteria and Archaea were purified and sequenced. The 16S rRNA gene sequences of the dominant archaeal bands found in the inoculum, and UASB sludge fed with raw sewage, CEPS pretreated wastewater, and synthetic sewage were closely associated withMethanosaeta concilii. In the UASB sludge fed with synthetic sewage, another dominant band associated with an uncultured archaeon 39-2 was found together withM. concilii.     

Anaerobic treatment of antibiotic production wastewater and kinetic evaluations

In this study, the anaerobic treatment of high-strength antibiotic production wastewater and the development of a mathematical model for the treatment were attempted. Anaerobic treatability was investigated using synthetic solutions and original wastewater of which the initial chemical oxygen demand (COD) was determined. Initial COD of solutions was increased from 3,000 to 43,000 mg O(2)/liter in an anaerobic bioreactor. The bioreactor pH was maintained at 6.5-7.5. The temperature was kept constant at 37 +/- 1 degrees C. Raw materials and original wastewater containing penicillin antibiotics were obtained from Fako Pharmaceutical Factory (Fako) in Istanbul, Turkey. Anaerobic sludge used for treatment was obtained from Pakmaya Baker's Yeast Producing Factory (Pakmaya) in Izmit, Turkey and the Fako. A mathematical model based on substrate (total COD) concentration was developed assuming that only three consecutive reactions, namely, hydrolysis, acidogenesis and methanogenesis, are significant. From the experimental data, a model that can be used for COD calculation as a function of time was developed using the first- and the second-order kinetic approaches. Making use of the developed model equation, it was proved that the anaerobic treatment of high strength (COD > 25,000 mg O(2)/liter) antibiotic production wastewater fits the second-order kinetics.     

Reactive black-5 azo dye treatment in suspended and attach growth sequencing batch bioreactor using different co-substrates

Treatment of textile wastewater is a big challenge because of diverse chemical composition, high chemical strength and color of the wastewater. In the present study, treatment of wastewater containing reactive black-5 azo dye was studied in anaerobic sequencing batch bioreactor (SBBR) using mixed liquor suspended solids (MLSS) from suspended and attach growth bioreactors. MLSS at concentration of 1000 mg/L and reactive black-5 azo dye at 100 mg/L were used. A culture (10⁸–10⁹ CFU/ml) of pre-isolated bacterial strains (Psychrobacter alimentarius KS23 and Staphylococcus equorum KS26)) capable of degrading azo dyes in mineral salt medium was used to accelerate the treatment process in bioreactor. Different combinations of sludge, culture and dye were used for treatment using different co-substrates. About 85% COD removal was achieved by consortium (MLSS + KS23 + KS26) after 24 h in attach growth bioreactor. Similarly, 92% color removal was observed with consortium in attach growth bioreactor compared to 85% color removal in suspended bioreactor. Addition of bacterial culture (20%, v/v) to the bioreactor could enhance the rate of color removal. This study suggests that biotreatment of wastewater containing textile dyes can be achieved more efficiently in the attach growth bioreactor using yeast extract as a co-substrate and MLSS augmented with dye-degrading bacterial strains.     

Perturbation‐independent community development in low‐temperature anaerobic biological wastewater treatment bioreactors

The reproducibility and stability of low‐ temperature anaerobic wastewater treatment systems undergoing transient perturbations was investigated. Three identical anaerobic expanded granular sludge bed‐based bioreactors were used to degrade a volatile fatty acid and glucose‐based wastewater under sub‐ambient (15°C) conditions. The effect of a variety of environmental perturbations on bioreactor performance was assessed by chemical oxygen demand removal. Temporal microbial community development was monitored by denaturation gradient gel electrophoresis (DGGE) of 16S rRNA genes extracted from sludge granules. Methanogenic activity was monitored using specific methanogenic activity assays. Bioreactor performance and microbial population dynamics were each well replicated between both experimental bioreactors and the control bioreactor prior to, and after the implementation of most of the applied perturbations. Gene fingerprinting data indicated that Methanosaeta sp. were the persistent, keystone members of the archaeal community, and likely were pivotal for the physical stability and maintenance of the granular biofilms. Cluster analyses of DGGE data suggested that temporal shifts in microbial community structure were predominantly independent of the applied perturbations. Biotechnol. Bioeng. 2010;105: 79–87. © 2009 Wiley Periodicals, Inc.     

Performance of a pilot-scale submerged membrane bioreactor (MBR) in treating bathing wastewater

Bathing wastewater was treated by a pilot-scale submerged membrane bioreactor (MBR) for more than 60 days. The results showed that the removal rates of main pollutants of wastewater such as COD(Cr), LAS, NH(4)(+)-N and total nitrogen (TN) were above 93%, 99%, 99%, and 90%, respectively. The results of denaturing gel gradient electrophoresis (DGGE) and fluorescent in situ hybridization (FISH) indicated that the bacteria were stable. The abundant nitrobacteria intercepted by the membrane led to the high removal rate of ammonia and TN. FISH and 16S rDNA gene sequence analysis revealed that some specific phylogenetic group of bacteria, the Pseudomonas sp. Ochrobactrum anthropi sp. and Enterobacter sp. probably played a major role in the development of the mature biofilms, which led to the severe irreversible membrane biofouling.     

Scanning electron microscopy of biofilm development in anaerobic fixed-bed reactors: Influence of the inoculum

Summary Scanning electron microscopy was applied to evaluate the influence of inoculum on efficiency of initial biofilm formation and reactor performance. Five anaerobic fixed-bed reactors were inoculated with anaerobic sludges from different sources and operated in parallel under identical conditions with defined wastewater and acetate, propionate and butyrate as constituents In all sludges Methanothrix sp. was the predominant acetotroph. The reactors inoculated with anaerobic sludge adapted to the wastewater achieved the highest space loading with 21.0 g COD/l·d after 58 days. The inoculation with granular sludge from an upflow anaerobic sludge blanket (UASB) reactor resulted in significantly less reactor efficiency. Time course of biofilm formation and biofilm thickness (ranging from 20–200 m) depended on the type of inoculum.