Subsurface Biofilm Barriers for the Containment and Remediation of Contaminated Groundwater

An engineered microbial biofilm barrier capable of reducing aquifer hydraulic conductivity while simultaneously biodegrading nitrate has been developed and tested at a field-relevant scale. The 22-month demonstration project was conducted at the MSE Technology Applications Inc. test facility in Butte, Montana, which consisted of a 130 ft wide, 180 ft long, 21 ft deep, polyvinylchloride (PVC)-lined test cell, with an initial hydraulic conductivity of 4.2 × 10^-2 cm/s. A flow field was established across the test cell by injecting water upgradient while simultaneously pumping from an effluent well located approximately 82 ft down gradient. A 30 ft wide biofilm barrier was developed along the centerline of the test cell by injecting a starved bacterial inoculum of Pseudomonas fluorescens strain CPC211a, followed by injection of a growth nutrient mixture composed of molasses, nitrate, and other additives. A 99% reduction of average hydraulic conductivity across the barrier was accomplished after three months of weekly or bi-weekly injections of growth nutrient. Reduced hydraulic conductivity was maintained by additional nutrient injections at intervals ranging from three to ten months. After the barrier was in place, a sustained concentration of 100 mg/l nitrate nitrogen, along with a 100 mg/l concentration of conservative (chloride) tracer, was added to the test cell influent over a six-month period. At the test cell effluent the concentration of chloride increased to about 80 mg/l while the effluent nitrate concentration varied between 0.0 and 6.4 mg/l.     

Influence of hydrodynamics and nutrients on biofilm structure

Hydrodynamic conditions control two interlinked parameters; mass transfer and drag, and will, therefore, significantly influence many of the processes involved in biofilm development. The goal of this research was to determine the effect of flow velocity and nutrients on biofilm structure. Biofilms were grown in square glass capillary flow cells under laminar and turbulent flows. Biofilms were observed microscopically under flow conditions using image analysis. Mixed species bacterial biofilms were grown with glucose (40 mg/l) as the limiting nutrient. Biofilms grown under laminar conditions were patchy and consisted of roughly circular cell clusters separated by interstitial voids. Biofilms in the turbulent flow cell were also patchy but these biofilms consisted of patches of ripples and elongated 'streamers' which oscillated in the flow. To assess the influence of changing nutrient conditions on biofilm structure the glucose concentration was increased from 40 to 400 mg/l on an established 21 day old biofilm growing in turbulent flow. The cell clusters grew rapidly and the thickness of the biofilm increased from 30 μ to 130 μ within 17 h. The ripples disappeared after 10 hours. After 5 d the glucose concentration was reduced back to 40 mg/l. There was a loss of biomass and patches of ripples were re-established within a further 2 d.     

Biodegradation of acetonitrile by adapted biofilm in a membrane-aerated biofilm reactor

A membrane-aerated biofilm reactor (MABR) was developed to degrade acetonitrile (ACN) in aqueous solutions. The reactor was seeded with an adapted activated sludge consortium as the inoculum and operated under step increases in ACN loading rate through increasing ACN concentrations in the influent. Initially, the MABR started at a moderate selection pressure, with a hydraulic retention time of 16 h, a recirculation rate of 8 cm/s and a starting ACN concentration of 250 mg/l to boost the growth of the biofilm mass on the membrane and to avoid its loss by hydraulic washout. The step increase in the influent ACN concentration was implemented once ACN concentration in the effluent showed almost complete removal in each stage. The specific ACN degradation rate achieved the highest at the loading rate of 101.1 mg ACN/g-VSS h (VSS, volatile suspended solids) and then declined with the further increases in the influent ACN concentration, attributed to the substrate inhibition effect. The adapted membrane-aerated biofilm was capable of completely removing ACN at the removal capacity of up to 21.1 g ACN/m² day, and generated negligible amount of suspended sludge in the effluent. Batch incubation experiments also demonstrated that the ACN-degrading biofilm can degrade other organonitriles, such as acrylonitrile and benzonitrile as well. Denaturing gradient gel electrophoresis studies showed that the ACN-degrading biofilms contained a stable microbial population with a low diversity of sequence of community 16S rRNA gene fragments. Specific oxygen utilization rates were found to increase with the increases in the biofilm thickness, suggesting that the biofilm formation process can enhance the metabolic degradation efficiency towards ACN in the MABR. The study contributes to a better understanding in microbial adaptation in a MABR for biodegradation of ACN. It also highlights the potential benefits in using MABRs for biodegradation of organonitrile contaminants in industrial wastewater.     

Evaluation of a microbial method to reduce hydrogen sulfide levels in a porous rock biofilm

Summary The efficacy of nitrate addition, with and without inoculation with a sulfide-resistant strain ofThiobacillus denitrificans (strain F), in reducing sulfide levels in an experimental system using cores and subsurface formation water from a gas storage facility was examined. The addition of nitrate (40 mM) alone to the formation water injected into core systems operated at hydraulic retention times of 3.2 and 16.7 h resulted in lower effluent sulfide concentrations, from an influent concentration of about 170–190 M to an effluent concentration of 110 and 3 M, respectively. A reduction in effluent nitrate concentrations in both core systems indicated the presence of indigenous nitrate-using populations. After strain F was inoculated into the core system operated at the shorter retention time, the effluent sulfide concentration decreased from 110 to 16–25 M. The effluent sulfate concentration increased, and the effluent nitrate concentration decreased concomitant with the presence of high concentrations of denitrifying thiobacilli in the inoculated core system. The denitrifying thiobacilli detected after inoculation were presumed to be strain F since these organisms were not detected in this core system before inoculation, or in any of the samples from the uninoculated core system. These data suggest that the efficacy of the nitrate treatment may depend on the residence times of the liquids in the core system, and that inoculation with strain F was required to reduce sulfide levels to <20 M in the core system operated at a short hydraulic retention time.     

Packed cage rotating biological contactor system for treatment of cyanide wastewater

The aim of this work was to study the efficiency of the packed cage rotating biological contactor (RBC) system with synthetic wastewater (SWW) containing 800 mg/l BOD(5) with various cyanide residue concentrations and hydraulic loading time. The results showed that cyanide had a negative effect to both the system's efficiency and bio-film quality. An increase in cyanide concentration led to a decrease in bio-film growth and the consequent reduction in the removal efficiency of the system. Also, the effluent suspended solids (SS) of the system was increased with increasing cyanide concentrations because the bio-film detached from the media due to the toxicity of the cyanide residue. The system showed the highest COD, BOD(5), TKN and cyanide removal efficiencies of 94.0 +/- 1.6%, 94.8 +/- 0.9%, 59.1 +/- 2.8% and 95.5 +/- 0.6%, respectively, with SWW containing 5 mg/l cyanide under HRT of 8 days, while they were only 88.8 +/- 0.7%, 89.5 +/- 0.5%, 40.3 +/- 1.1% and 93.60 +/- 0.09%, respectively, with SWW containing 40 mg/l cyanide. In addition, the effluent ammonia, nitrite and nitrate were increased with increases in cyanide concentration or loading. However, the system with SWW containing the highest cyanide concentration of 40 mg/l showed almost constant COD and BOD(5) removal efficiencies of 89% and 90%, even when the system was controlled under the lowest HRT of 8 h.     

Flow cell hydrodynamics and their effects on E. coli biofilm formation under different nutrient conditions and turbulent flow

Biofilm formation is a major factor in the growth and spread of both desirable and undesirable bacteria as well as in fouling and corrosion. In order to simulate biofilm formation in industrial settings a flow cell system coupled to a recirculating tank was used to study the effect of a high (550 mg glucose l?1) and a low (150 mg glucose l?1) nutrient concentration on the relative growth of planktonic and attached biofilm cells of Escherichia coli JM109(DE3). Biofilms were obtained under turbulent flow (a Reynolds number of 6000) and the hydrodynamic conditions of the flow cell were simulated by using computational fluid dynamics. Under these conditions, the flow cell was subjected to wall shear stresses of 0.6 Pa and an average flow velocity of 0.4 m s?1 was reached. The system was validated by studying flow development on the flow cell and the applicability of chemostat model assumptions. Full development of the flow was assessed by analysis of velocity profiles and by monitoring the maximum and average wall shear stresses. The validity of the chemostat model assumptions was performed through residence time analysis and identification of biofilm forming areas. These latter results were obtained through wall shear stress analysis of the system and also by assessment of the free energy of interaction between E. coli and the surfaces. The results show that when the system was fed with a high nutrient concentration, planktonic cell growth was favored. Additionally, the results confirm that biofilms adapt their architecture in order to cope with the hydrodynamic conditions and nutrient availability. These results suggest that until a certain thickness was reached nutrient availability dictated biofilm architecture but when that critical thickness was exceeded mechanical resistance to shear stress (ie biofilm cohesion) became more important.     

Anaerobic treatment of wastewater from a food-manufacturing plant with a low concentration of organic matter and regeneration of usable pure water

Wastewater from a food-manufacturing plant with a low concentration of organic matter below 100 mg/l TOC was first treated at 37°C in an anaerobic fluidized-bed reactor (AFBR) or in an upflow anaerobic sludge blanket (UASB). The TOC removal efficiency in both reactors decreased from 85% to 65% as the influent TOC concentration decreased from 100 to 35 mg/l at a hydraulic retention time (HRT) of 6 h. Treatment at an HRT of 4 h resulted in an effluent TOC concentration of 11 to 15 mg/l. The concentration of suspended solids in the effluent could be reduced to 20 mg/l, which corresponded to 7% of that of the influent. The effluent from both reactors was then treated anaerobically in a fixed-bed reactor system. The TOC concentration and optical density (OD) of the effluent from the aerobic treatment were reduced to 5 mg/l and 0.005, respectively, at an HRT of 2 h. When anaerobically or aerobically treated effluent was passed over an activated carbon column, the effluent TOC concentration was reduced to 2 to 3 mg/l. The conductivity of 1.3 mS/cm in raw wastewater, which was not removed through the above treatments, was reduced to 0.001 mS/cm on an ion-exchange resin column. An effluent quality corresponding to that of ultra-pure water for industrial use was finally attained by the treatment in this multi-step system.     

Characterization and biological treatment of ceramic industry wastewater

Ceramic industry wastewaters not only contain high suspended and total solids but also significant amounts of dissolved organics resulting in high BOD or COD loads. Suspended solids can be removed from the wastewater by chemical precipitation. However, dissolved BOD/COD compounds can only be removed by biological or chemical oxidation. Effluent wastewater from chemical sedimentation stage of EGE CERAMIC industry was characterized and subjected to biological treatment in a laboratory scale activated sludge unit. Experiments were conducted at different hydraulic and solids retention times. The best results were obtained with Š_c=20 h of hydraulic and Š_c=20 days of solids retention times (sludge age) resulting in effluent COD concentration of 40 mg/l from a feed wastewater of 720 mg/l COD content. The suspended solids content of the activated sludge effluent was approximately 52 mg/l.     

Comparative study for the effects of variable nutrient conditions on the biodegradation of microcystin-LR and concurrent dynamics in microcystin-degrading gene abundance

Microcystin-LR (MCLR) degradation capability of biofilm was investigated with and without additional nutrients (nitrate, ammonium, peptone and glucose) at concentrations of 100 and 1000 mg L(-1). The MCLR-degradation was stimulated with nitrate and inhibited with other nutrients, except for that glucose of low concentration had no obvious effect. Both stimulatory and inhibitory effects enhanced with increasing concentration of corresponding nutrient. Quantitative polymerase chain reaction (qPCR) indicated that enhanced inhibition in biodegradation correlated to increased inhibition in functional gene (mlrA) abundance, as nutrient concentration increased. Stimulated biodegradation under low nitrate concentration may result from more rapid increase in mlrA gene abundance. These suggested that MCLR-degradation largely depended upon responsible bacterial population, which was affected by population of other bacteria in biofilm according to 16S rDNA-targeting qPCR. However, inhibited mlrA gene abundance implied that the stimulated biodegradation under high nitrate concentration might be involved in the mechanisms not related to MCLRDB population.     

Biofilm formation at warming temperature: acceleration of microbial colonization and microbial interactive effects

River biofilms that grow on wet benthic surface are mainly composed of bacteria, algae, cyanobacteria and protozoa embedded in a polysaccharide matrix. The effects of increased river water temperature on biofilm formation were investigated. A laboratory experiment was designed employing two temperatures (11.1-13.2°C, night-day; 14.7-16.0°C, night-day) and two nutrient levels (0.054 mg P l(-1), 0.75 mg N l(-1); 0.54 mg P l(-1), 7.5 mg N l(-1)). Biofilm formation at the higher temperature was faster, while the biomass of the mature biofilm was mainly determined by nutrient availability. The specific response of the three microbial groups that colonized the substrata (algae, bacteria and ciliates) was modulated by interactions between them. The greater bacterial growth rate and earlier bacterial colonization at the higher temperature and higher nutrient status was not translated into the accrual of higher bacterial biomass. This may result from ciliates grazing on the bacteria, as shown by an earlier increase in peritrichia at higher temperatures, and especially at high nutrient conditions. Temperature and ciliate grazing might determine the growth of a distinctive bacterial community under warming conditions. Warmer conditions also produced a thicker biofilm, while functional responses were much less evident (increases in the heterotrophic utilization of polysaccharides and peptides, but no increase in primary production and respiration). Increasing the temperature of river water might lead to faster biofilm recolonization after disturbances, with a distinct biofilm community structure that might affect the trophic web. Warming effects would be expected to be more relevant under eutrophic conditions.     

Effects of operating parameters on performances of nitrification and denitrification processes

Nitrification and denitrification of synthetic wastewater was studied by using two reactors in series. An activated sludge unit was used for nitrification followed by a downflow biofilter (packed column) for denitrification. A glucose solution was fed to the denitrification column to supply carbon source. Effects of important process variables such as sludge age, hydraulic residence time and feed ammonium concentration on system's performance were investigated. Effluent ammonium-nitrogen (NH4-N) concentration decreased with increasing sludge age and hydraulic residence time and remained constant for sludge age and hydraulic residence times greater than 12 d and 15 h, respectively. Feed ammonium-nitrogen concentration above 200 mg/l resulted in significant levels of NH4-N in the effluent at Š_c = 15 d and Š_H = 12 h in nitrification. Performance of denitrification stage was not satisfactory for feed NO₃-N concentrations above 150 mg N/l resulting in significant effluent NO₃-N levels at hydraulic residence time of Š_H = 6 h.     

Treatment of cyanide-containing wastewater from the food industry in a laboratory-scale fixed-bed methanogenic reactor

During the process of producing cassava starch from Manihot esculenta roots, large amounts of cyanoglycosides were released, which rapidly decayed to CN⁻ following enzymatic hydrolysis. Depending on the varying cyanoglycoside content of the cassava varieties, the cyanide concentration in the wastewater was as high as 200 mg/l. To simulate anaerobic stabilization, a wastewater with a chemical oxygen demand (COD) of about 20 g/l was prepared from cassava roots and was fermented in a fixed-bed methanogenic reactor. The start-up phase for a 99% degradation of low concentrations of cyanide (10 mg/l) required about 6 months. After establishment of the biofilm, a cyanide concentration of up to 150 mg CN⁻/l in the fresh wastewater was degraded during anaerobic treatment at a hydraulic retention time of 3 days. All nitrogen from the degraded cyanide was converted to organic nitrogen by the biomass of the effluent. The cyanide-degrading biocoenosis of the anaerobic reactor could tolerate shock concentrations of cyanide up to 240 mg CN⁻/l for a short time. Up to 5 mmol/l NH₄Cl (i.e. 70 mg N/l = 265 mg NH₄Cl/l) in the fresh wastewater did not affect cyanide degradation. The bleaching agent sulphite, however, had a negative effect on COD and cyanide removal. For anaerobic treatment, the maximum COD space loading was 12 g l⁻¹ day⁻¹, equivalent to a hydraulic retention time of 1.8 days. The COD removal efficiency was around 90%. The maximum permanent cyanide space loading was 50 mg CN⁻ l⁻¹ day⁻¹, with tolerable shock loadings up to 75 mg CN⁻ l⁻¹day⁻¹. Under steady-state conditions, the cyanide concentration of the effluent was lower than 0.5 mg/l. Received: 15 August 1997 / Received revision: 10 October 1997 / Accepted: 14 October 1997     

Aerobic treatment of dairy wastewater with sequencing batch reactor systems

Performances of single-stage and two-stage sequencing batch reactor (SBR) systems were investigated for treating dairy wastewater. A single-stage SBR system was tested with 10,000 mg/l chemical oxygen demand (COD) influent at three hydraulic retention times (HRTs) of 1, 2, and 3 days and 20,000 mg/l COD influent at four HRTs of 1, 2, 3, and 4 days. A 1-day HRT was found sufficient for treating 10,000-mg/l COD wastewater, with the removal efficiency of 80.2% COD, 63.4% total solids, 66.2% volatile solids, 75% total Kjeldahl nitrogen, and 38.3% total nitrogen from the liquid effluent. Two-day HRT was believed sufficient for treating 20,000-mg/l COD dairy wastewater if complete ammonia oxidation is not desired. However, 4-day HRT needs to be used for achieving complete ammonia oxidation. A two-stage system consisting of an SBR and a complete-mix biofilm reactor was capable of achieving complete ammonia oxidation and comparable carbon, solids, and nitrogen removal while using at least 1/3 less HRT as compared to the single SBR system.     

Effect of chloride and condensable tannin in anaerobic degradation of tannery wastewaters

Toxic effect due to chloride and condensable tannin on anaerobic digestion of vegetable tanning wastewater was investigated at different hydraulic retention times viz 24, 48 and 60 hr respectively. The toxicity to anaerobic contact filter was observed at a chloride concentration of 4500 mg/l and tannin concentration of 790 mg/l respectively under synergistic condition. In the case of constant influent tannin concentration of 600 mg/l, the toxicity due to chloride on anaerobic contact filter was observed at 5500 mg/l. The COD removal percent ranged from 50% to 79% at 48 hr and 67% to 91% at 60 hr HRT respectively. In the case of constant influent chloride concentration of 4000 mg/l and at an increasing influent COD concentration, the toxicity due to tannin on anaerobic contact filter was observed at 1180 mg/l. The COD removal percent ranged from 64% to 89% at 48 hr and 78% to 96% at 60 hr HRT respectively. The results showed that at least 60 hr HRT would be desirable to have good COD removal percent.     

Heterotrophic bacterial production on solid fish waste: TAN and nitrate as nitrogen source under practical RAS conditions

The drumfilter effluent from a recirculation aquaculture system (RAS) can be used as substrate for heterotrophic bacteria production. This biomass can be re-used as aquatic feed. RAS effluents are rich in nitrate and low in total ammonia nitrogen (TAN). This might result in 20% lower bacteria yields, because nitrate conversion into bacteria is less energy efficient than TAN conversion. In this study the influence of TAN concentrations (1, 12, 98, 193, 257mgTAN/l) and stable nitrate-N concentrations (174+/-29mg/l) on bacteria yields and nitrogen conversions was investigated in a RAS under practical conditions. The effluent slurry was supplemented with 1.7gC/l sodium acetate, due to carbon deficiency, and was converted continuously in a suspended bacteria growth reactor (hydraulic retention time 6h). TAN utilization did not result in significantly different observed yields than nitrate (0.24-0.32gVSS/gC, p=0.763). However, TAN was preferred compared to nitrate and was converted to nearly 100%, independently of TAN concentrations. TAN and nitrate conversions rates were differing significantly for increasing TAN levels (p<0.000 and p=0.012), and were negatively correlated. It seems, therefore, equally possible to supply the nitrogenous substrate for bacteria conversion as nitrate and not as TAN. The bacteria reactor can, as a result, be integrated into an existing RAS as end of pipe treatment.     

The role of nitrate in osmoregulation of Italian ryegrass

Summary The role of nitrate in osmotic control was studied with Italian ryegrass grown in a nutrient solution in a climate room. Quantum-flux density, osmotic potential of the nutrient solution and availability of nitrate and chloride were varied independently. Plants at high quantum flux density (650 mol m^–2 s^–1) had a lower osmotic potential, a higher carbohydrate concentration and a lower nitrate concentration than plants at low quantum flux density (310 mol m^–2 s^–1), the decrease in nitrate concentration was osmotically equivalent to the increase in carbohydrate concentration. When nitrate in the nutrient solution was partly replaced by chloride, the chloride taken up substituted an equivalent part of the nitrate in the plant. It is concluded that nitrate plays a role in osmoregulation of the plant and compensates for a shortage of other solutes.     

The role of salvinia rotundifolia in scavenging aquatic Pb (II) pollution: a case study

Bio-surveillance of environmental pollution is increasingly gaining ground, as it is felt that if and when standardized, it might prove to be a long term cost-effective alternative technology. In aquatic media, for monitoring heavy metal pollutants, which are mostly xenobiotic, a number of hydrophytes have been tried by earlier researchers. These hydrophytes have shown to have varying degree of accumulation capacities, which could be preferably utilized in scavenging the toxicants such as Pb(II) among others. The screening criteria of such a suitable scavenger also take into account the ease of harvesting and handling the biomass on a large scale. Here comes the use of a macrophyte, and its potentiality of forming metallo-protein complexes (phytochelatin) to hold back the diffused metal, Pb(II), ions. In the present investigation, the experimental test system was the common tropical aquatic weed, Salvinia rotundifolia, Willd. It has shown a high promise for Pb(II) removal from synthetic as well as industrial (battery producing unit) wastewater. Within a span of 4 days, 50?gm (wet weight) of the plant was capable of removing about 85–95% of Pb(II) from 1.50 litres of both kinds of wastewaters containing 0.65–1.8?ppm of the metal at an optimum pH of 5.5. The uptake potentials were examined under various combinations of pH, plant weight, and metal concentration. It is suggested that the high uptake and recovery of Pb(II) by Salvinia Rotundifolia can be efficiently used as a possible future biotechnological solution for industrial wastewater treatment in a tropical and developing country like India. In the present study, a reduction of biomass weight from 80 to 72.3 (in mg dry wt/g of fresh wt.) was noted, while an increase of tissue conductivity from 161 to 181.5 in micro-mhos/cm was observed. This reflects the potential injurious effect of heavy metals to the cell membrane of the biomass. These changes, when standardized, are likely to serve as a suitable `biomonitoring' device for aquatic metal pollution. In the present case, effluent quality of a battery manufacturing unit, India was evaluated. Even though there is an existing effluent treatment plant (ETP) of the industry, the effluent from the balancing tank (discharge point) was found to contain high concentrations of lead (0.657–1.021?mg/l), BOD (470?mg/l) and COD (680?mg/l) as against State Pollution Control Board's standards of 0.1?mg/l, 30?mg/l, and 250?mg/l respectively. In the present investigation, an attempt has also been made to treat wastewater from the balancing tank of the existing effluent treatment plant (ETP) of the above-stated industrial unit to bring down the concentrations of Pb(II), BOD (5 days), and COD within the permissible limits prescribed by the regulatory board. The treatment was done in a laboratory-scale oxidation pond, cultured with Salvinia rotundifolia, of 85 litres capacity using a hydraulic retention period of about 10 days.     

Colour removal from bagasse-based pulp mill effluent using a white rot fungus

Colour removal of pulp plant effluent was studied using white rot fungus, Trametes (Coriolus) versicolor. The batch experiments were carried out using fungus in the form of mycelial pellets. In the present investigation, the effect of pH, concentrations of glucose (substrate), initial effluent colour and ammonium chloride (nutrient) on colour removal efficiency were studied. It was found that the maximum colour removal efficiency of 82.5% was obtained with an optimal glucose and ammonium chloride concentrations of 15 g/l and 0.5 g/l respectively at a pH of 4.5 without diluting the effluent.     

Membrane bioreactor for drinking water denitrification

The aim of this study is to evaluate the performance of a membrane bioreactor with cell recycle to be used for drinking water denitrification, when operated with a high nitrate load (up to 7.68?kgNO₃ ^?/m³?day) and low hydraulic retention time (down to 0.625?h). Nitrate and nitrite were always completely removed for all the operational conditions used. The effluent's nitrite concentration kept below 0.1?mg NO₂ ^?/l with exception of a short period, during the reactor start-up, when it accumulates. The performance of the membrane bioreactor was also evaluated using a groundwater containing 148?mg NO₃ ^?/l. Nitrate and nitrite concentration in the effluent were below the recommended values for drinking water when the reactor was controlled at pH 7.0. The membrane flux decreases during operation as a consequence of membrane fouling. The flux decrease was more severe during operation with synthetic medium than with contaminated groundwater due to the existence of molecular complexes in the synthetic broth. A backshock technique was used to reduce the surface fouling of the membrane. Combining this technique with the use of a reserve asymmetric structured membrane it was found that the membrane flux remains nearly unchanged.     

Study of a combined sulfur autotrophic with proton-exchange membrane electrodialytic denitrification technology: sulfate control and pH balance

A novel combined system established for nitrate removal from aqueous solution consisted of two parts: sulfur autotrophic denitrification and bio-electrochemical denitrification based on proton-exchange membrane electrodialysis (PEMED). The system was operated at various hydraulic retention times (HRT) and current intensities. Its optimum operation condition was also determined. The combined process had pH adjustment thus generating less nitrite than PEMED process. The denitrification rate of sulfur autotrophic part was dependent on HRT, while shorter HRT could reduce the sulfate generated by the sulfur autotrophic process. The denitrification rate of PEMED process depended on the applied current. For 32 ± 1 mg-N/L nitrate in influent, the optimum operation parameters of combined process were: HRT 2 h; applied current 350 mA. The combined reactor could achieve 95.8% nitrate removal without nitrite accumulation, the pH of effluent kept neutral and the sulfate of effluent was 202.1 mg/L, lower than the drinking water standard in China.