Monitoring the denitrification of wastewater containing high concentrations of nitrate with methanol in a sulfur-packed reactor

Biological denitrification of high nitrate-containing wastewater was examined in a sulfur-packed column using a smaller amount of methanol than required stoichiometrically for heterotrophic denitrification. In the absence of methanol, the observed nitrate removal efficiency was only about 40%, and remained at 400 mg NO(3)(-)-N/l, which was due to an alkalinity deficiency of the pH buffer and of CO(2) as a carbon source. Complete denitrification was achieved by adding approximately 1.4 g methanol/g nitrate-nitrogen (NO(3)(-)-N) to a sulfur-packed reactor. As the methanol concentration increased, the overall nitrate removal efficiency increased. As influent methanol concentrations increased from 285 to 570, 855, and 1,140 mg/l, the value of Delta mg alkalinity as CaCO(3) consumed/Delta mg NO(3)(-)-N removed increased from -1.94 to -0.84, 0.24, and 0.96, and Delta mg SO(4)(2-) produced/Delta mg NO(3)(-)-N removed decreased from 4.42 to 3.57, 2.58, and 1.26, respectively. These results imply the co-occurrence of simultaneous autotrophic and heterotrophic denitrification. Sulfur-utilizing autotrophic denitrification in the presence of a small amount of methanol is very effective at decreasing both sulfate production and alkalinity consumption. Most of methanol added was removed completely in the effluent. A small amount of nitrite accumulated in the mixotrophic column, which was less than 20 mg NO(2)(-) -N/l, while under heterotrophic denitrification conditions, nitrite accumulated steadily and increased to 60 mg NO(2)(-) -N/l with increasing column height.     

Nitrogen fertilizer rate and crop management effects on nitrate leaching from an agricultural field in central Pennsylvania

Eighteen pan lysimeters were installed at a depth of 1.2 m in a Hagerstown silt loam soil in a corn field in central Pennsylvania in 1988. In 1995, wick lysimeters were also installed at 1.2 m depth in the same access pits. Treatments have included N fertilizer rates, use of manure, crop rotation (continuous corn, corn-soybean, alfalfa-corn), and tillage (chisel plow-disk, no-till). The leachate data were used to evaluate a number of nitrate leaching models. Some of the highlights of the 11 years of results include the following: 1) growing corn without organic N inputs at the economic optimum N rate (EON) resulted in NO3--N concentrations of 15 to 20 mg l(-1) in leachate; 2) use of manure or previous alfalfa crop as partial source of N also resulted in 15 to 20 mg l(-1) of NO3--N in leachate below corn at EON; 3) NO3--N concentration in leachate below alfalfa was approximately 4 mg l(-1); 4) NO3--N concentration in leachate below soybeans following corn was influenced by fertilizer N rate applied to corn; 5) the mass of NO3--N leached below corn at the EON rate averaged 90 kg N ha(-1) (approx. 40% of fertilizer N applied at EON); 6) wick lysimeters collected approximately 100% of leachate vs. 40-50% collected by pan lysimeters. Coefficients of variation of the collected leachate volumes for both lysimeter types were similar; 7) tillage did not markedly affect nitrate leaching losses; 8) tested leaching models could accurately predict leachate volumes and could be calibrated to match nitrate leaching losses in calibration years, but only one model (SOILN) accurately predicted nitrate leaching losses in the majority of validation treatment years. Apparent problems with tested models: there was difficulty estimating sizes of organic N pools and their transformation rates, and the models either did not include a macropore flow component or did not handle macropore flow well.     

Influence of reactive media composition and chemical oxygen demand as methanol on autotrophic sulfur denitrification

Sulfur-utilizing autotrophic denitrification relies on an inorganic carbon source to reduce the nitrate by producing sulfuric acid as an end product and can be used for the treatment of wastewaters containing high levels of nitrates. In this study, sulfur-denitrifying bacteria were used in anoxic batch tests with sulfur as the electron donor and nitrate as the electron acceptor. Various medium components were tested under different conditions. Sulfur denitrification can drop the medium pH by producing acid, thus stopping the process half way. To control this mechanism, a 2:1 ratio of sulfur to oyster shell powder was used. Oyster shell powder addition to a sulfurdenitrifying reactor completely removed the nitrate. Using 50, 100, and 200 g of sulfur particles, reaction rate constants of 5.33, 6.29, and 7.96 mg(1/2)/l(1/2)·h were obtained, respectively; and using 200 g of sulfur particles showed the highest nitrate removal rates. For different sulfur particle sizes ranging from small (0.85-2.0 mm), medium (2.0-4.0 mm), and large (4.0-4.75 mm), reaction rate constants of 31.56, 10.88, and 6.23 mg(1/2)/l(1/2)·h were calculated. The fastest nitrate removal rate was observed for the smallest particle size. Addition of chemical oxygen demand (COD), methanol as the external carbon source, with the autotrophic denitrification in sufficiently alkaline conditions, created a balance between heterotrophic denitrification (which raises the pH) and sulfur-utilizing autotrophic denitrification, which lowers the pH.     

Characteristics of the bioreactor landfill system using an anaerobic-aerobic process for nitrogen removal

A sequential upflow anaerobic sludge blanket (UASB) and air-lift loop sludge blanket (ALSB) treatment was introduced into leachate recirculation to remove organic matter and ammonia from leachate in a lab-scale bioreactor landfill. The results showed that the sequential anaerobic-aerobic process might remove above 90% of COD and near to 100% of NH4+ -N from leachate under the optimum organic loading rate (OLR). The total COD removal efficiency was over 98% as the OLR increased to 6.8-7.7 g/l d, but the effluent COD concentration increased to 2.9-4.8 g/l in the UASB reactor, which inhibited the activity of nitrifying bacteria in the subsequent ALSB reactor. The NO3- -N concentration in recycled leachate reached 270 mg/l after treatment by the sequential anaerobic-aerobic process, but the landfill reactor could efficiently denitrify the nitrate. After 56 days operation, the leachate TN and NH4+ -N concentrations decreased to less than 200 mg/l in the bioreactor landfill system. The COD concentration was about 200 mg/l with less than 8 mg/l BOD in recycled leachate at the late stage. In addition, it was found that nitrate in recycled leachate had a negative effect on waste decomposition.     

Inactivation of ANAMMOX communities under concurrent operation of anaerobic ammonium oxidation (ANAMMOX) and denitrification

A concurrent operation of anaerobic ammonium oxidation (ANAMMOX) and denitrification was investigated in a well known UASB reactor seeding with both ANAMMOX and anaerobic granular sludges. ANAMMOX activity was confirmed by hydroxylamine test and the hybridization of biomass using the gene probes of Amx 820 and EUB 338 mixed. Denitrification was observed through the reductions of both COD and nitrate-nitrite concentrations under anaerobic/anoxic conditions. By providing a stoichiometric ratio of nitrite to ammonium nitrogen with addition nitrate nitrogen, a gradual reduction of ANAMMOX activity was found with an increase of COD concentration in a range of 100-400 mg l(-1). This is equivalent to the COD to N ratio of 0.9-2.0. The COD concentration was found to be a control variable for process selection between ANAMMOX reaction and denitrification. A reduction of COD and nitrite-nitrate concentrations in all reactors confirmed the undergone concurrent denitrification which thrives when sufficient organic matter is available. COD concentration over 300 mg l(-1) was found to inactivate or eradicate ANAMMOX communities.     

Denitrification of wastewater containing high nitrate and calcium concentrations

The removal of nitrate from rinse wastewater generated in the stainless steel manufacturing process by denitrification in a sequential batch reactor (SBR) was studied. Two different inocula from wastewater treatment plants were tested. The use of an inoculum previously acclimated to high nitrate concentrations led to complete denitrification in 6h (denitrification rate: 22.8mg NO(3)(-)-N/gVSSh), using methanol as carbon source for a COD/N ratio of 4 and for a content of calcium in the wastewater of 150mg/L. Higher calcium concentrations led to a decrease in the biomass growth rate and in the denitrification rate. The optimum COD/N ratio was found to be 3.4, achieving 98% nitrate removal in 7h at a maximum rate of 30.4mg NO(3)(-)-N/gVSSh and very low residual COD in the effluent.     

Nitrite reduction by a mixed culture under conditions relevant to shortcut biological nitrogen removal

Dissimilative reduction of nitrite by nitrite-acclimated cellswas investigated in a batch reactor under various environmental conditions that can beencountered in shortcut biological nitrogen removal (SBNR: ammonia to nitrite andnitrite to nitrogen gas). The maximum specific nitrite reduction rate was as much as 4.3 times faster than the rate of nitrate reduction when individually tested, but the reaction was inhibited in the presence of nitrate when the initial nitrate concentration was greater than approximately 25 mg-N/l or the initialNO ₃ ^- N/NO ₂ ^- N ratio was larger than 0.5. Nitrite reduction was also inhibited by nitrite itself when theconcentration was higher than that to which the cells had been acclimated. Therefore, it was desirable to avoid excessively high nitrite and nitrate concentrations in a denitrification reactor. Nitrite reduction, however, was not affected by an alkaline pH (in the range of 7–9) or a high concentration of FA (in the range of 16–39 mg/l), which can be common in SBNR processes. The chemical oxygen demand (COD) requirement for nitrite reduction was approximately 22–38% lower than that for nitrate reduction, demonstrating that the SBNR process can be economical. The specific consumption,measured as the ratio of COD consumed to nitrogen removed, was affected by the availability of COD and the physiological state of the cells. The ratio increased when the cells grew rapidly and were storing carbon and electrons.     

Optimization of Nitrate and Manganese Removal by Bacterium Pseudomonas sp. H117 in Mixotrophic Condition

Previous study has revealed that Pseudomonas sp. H117 could exhibit excellent performance on autotrophic and heterotrophic denitrification in polluted groundwater. However, a novel character of simultaneous denitrification and manganese removal by the bacteria remained to be further explored. In this study, we investigated optimum conditions of nitrate and Mn(II) removal by the strain H117 in mixotrophic condition. Different factors (temperature, initial pH, nitrate concentration, and Mn(II) concentration) were investigated and optimized by response surface methodology (RSM), demonstrating that the highest nitrate removal ratio (100%) in the mixotrophic condition occurred at the temperature of 30.20?°C, pH of 6.90, and Mn(II) concentration of 61.81?mg/l. Meanwhile, the optimal Mn(II) removal (73.34%) conditions were at the temperature of 29.33?°C, pH of 7.22, and nitrate concentration of 20.74?mg/l. Furthermore, microbial development pattern, cellular metabolites, and bioprecipitation were characterized by the excitation emission matrix (EEM), meteorological chromatography analysis, and scanning electron microscopy (SEM) methods, respectively. These results demonstrated that strain H117 can have good adaptability to the environment, thus exhibiting an efficient ability for bioremediation of groundwater polluted by nitrate and Mn(II).     

Effects of external carbon source and empty bed contact time on simultaneous heterotrophic and sulfur-utilizing autotrophic denitrification

The effects of external carbon source (both type and concentration) and empty bed contact time (EBCT) on denitrification efficiency during simultaneous heterotrophic and sulfur-utilizing autotrophic denitrification were evaluated. Continuous experiments were conducted with up-flow mode sulfur packed bed reactors (SPBRs) fed with nitrified leachate containing 700–900 mg/l NO₃⁻–N. The fraction of NO₃⁻–N removed by heterotrophic denitrification (HDNR_fraction) for alkalinity production to balance the alkalinity consumption by autotrophic denitrification varied with the type of external carbon source. When methanol or sodium acetate was added at HDNR_fraction values of 60 and 44%, respectively, 100% denitrification was achieved without alkalinity addition. However, glucose required a HDNR_fraction value of 70% for complete denitrification and denitrification was not complete when molasses was used at a HDNR_fraction value of 70%. The EBCT and volumetric loading rate at which 100% denitrification efficiency could be achieved were 6.76 h and 2.84 kg NO₃⁻–N/m³ day, respectively. The maximum nitrogen removal rate was 5.05 kg NO₃⁻–N/m³ day observed with 89% removal efficiency. At short HRT, a clogging problem was observed near the bottom of the SPBR with excess growth of heterotrophic denitrifiers and gas accumulation within the pores of the SPBR. This problem may be eliminated by back-washing or by separating heterotrophic denitrification from sulfur-utilizing denitrification.     

Nitrogen removal from wastewaters at low C/N ratios with ammonium and acetate as electron donors

A denitrifying upflow anaerobic sludge blanket (UASB) reactor was operated at different nitrate loading rates at a C/N ratio of 1.2, with acetate as an electron donor. This resulted in an increase in the accumulation of nitrite. After this, the UASB reactor was supplemented with 100 mg NH4+-Nl(-1) d(-1), while acetate was gradually limited in the medium. This prevented nitrite accumulation at a C/N ratio of 0.6 due to an enhanced nitrite reduction rate achieved in the reactor. An increasing amount of ammonium was consumed when the C/N ratio was lowered in the medium. This suggested that ammonium was used as an alternative electron donor during denitrification, which is supported by nitrogen balances. Nitrite was shown to be toxic for the nitrogen removal process at 200-400 mg NO2--N(l(-1) when the C/N ratio was decreased to 0.4 leading to formation of ammonium. The present study showed that addition of ammonium as an alternative electron donor for denitrification achieved a nitrogen removal process with negligible accumulation of undesirable intermediates.     

Nitrous oxide emissions from denitrifying activated sludge of urban wastewater treatment plants, under anoxia and low oxygenation

Batch experiments were made to better understand the mechanisms of N2O emissions from activated sludge in denitrifying conditions found in urban WWTPs, i.e. under anoxic and low oxygenation conditions. The results showed that in completely anoxic conditions, denitrification, related to a periplasmic nitrate reductase activity, is the major producer of N2O (100% of the N2O production), whereas the nitrate ammonifying activity is not significant. In a gradient of low oxygenation, the highest N2O emissions (49.7+/-3.8 microg N2O-N/g SS/h on average) occurred at a dissolved-oxygen concentration of around 0.3mg O2/L. Below 0.3mg O2/L, heterotrophic denitrification appeared to be the major process responsible for the N2O emission (100% at zero oxygenation). From 0.4 to 1.1mg O2/L, N2O emissions were due to two processes: (i) heterotrophic denitrification that represented about 40% of the N2O production, and (ii) autotrophic nitrifier denitrification that accounted for about 60%. The N2O emissions from activated sludge represented on average 0.4% of reduced NO3(-) in anoxic conditions. The N2O emissions associated with denitrification of entire nitrogen load would amount to 155 T N2O-N/year, if all the Paris wastewater was treated by a process using activated sludge.     

Use of limestone for pH control in autotrophic denitrification: continuous flow experiments in pilot-scale packed bed reactors

The sulfur-utilizing autotrophic denitrification process consumes about 4 g alkalinity (as CaCO(3)) per g NO(3)-N reduced resulting in a decrease of pH. Using limestone as an alkalinity source to control the pH, autotrophic denitrification of synthetic wastewater with varying alkalinity to NO(3)-N ratios was evaluated in pilot-scale packed bed reactors operating in the upflow mode, which contained limestone and sulfur granules in different volumetric ratios. The results demonstrated that limestone supplies effective buffering capacity, if the initial alkalinity of the wastewater is insufficient for complete denitrification. The alkalinity supplied by limestone is a function of hydraulic retention time and the pH, which in turn depends on the extent of biological denitrification and the initial alkalinity to NO(3)-N ratio in the wastewater. The dissolution rate of limestone is inversely proportional to pH for pH values lower than 7.1. It was found that the ratio of influent alkalinity to theoretically required alkalinity in the wastewater should not be lower than 0.5 in order to prevent a decrease in nitrate removal performance. Based on the established chemical-biological interactive relationships, a multilayer approach was proposed to determine the optimum sulfur:limestone ratio for nitrate removal under steady state conditions, taking into account the characteristics of the influent wastewater.     

Characteristics of Autotrophic and Heterotrophic Denitrification by the Strain Pseudomonas sp. H117

Strain H117 was isolated from the Tang Yu reservoir. Based on the phylogenetic characteristics, strain H117, which was identified as Pseudomonas sp. strain H117, had the capability to utilize bicarbonate and sodium acetate as a carbon source under anaerobic conditions. Furthermore, the strain could grow on both autotrophic and heterotrophic media, and could perform both autotrophic and heterotrophic denitrification in the medium. Response surface methodology analysis demonstrated that the maximum degradation ratio of nitrate-occurred under the following conditions in the autotrophic medium: initial pH of 6.00, C/N ratio of 4.68 and temperature of 31.33°C. The maximum degradation ratio of nitrate occurred under the following conditions in the heterotrophic medium: initial pH of 6.16, C/N ratio of 8.23 and temperature of 28.48°C. Finally, the denitrification performance of strain H117 was evaluated under the optimum conditions. These results suggest that strain H117 has potential applications for the bioremediation of polluted groundwater.     

Feasibility of nitrification/denitrification in a sequencing batch biofilm reactor with liquid circulation applied to post-treatment

An investigation was performed on the biological removal of ammonium nitrogen from synthetic wastewater by the simultaneous nitrification/denitrification (SND) process, using a sequencing batch biofilm reactor (SBBR). System behavior was analyzed as to the effects of sludge type used as inoculum (autotrophic/heterotrophic), wastewater feed strategy (batch/fed-batch) and aeration strategy (continuous/intermittent). The presence of an autotrophic aerobic sludge showed to be essential for nitrification startup, despite publications stating the existence of heterotrophic organisms capable of nitrifying organic and inorganic nitrogen compounds at low dissolved oxygen concentrations. As to feed strategy, batch operation (synthetic wastewater containing 100 mg COD/L and 50 mg N-NH(4)(+)/L) followed by fed-batch (synthetic wastewater with 100 mg COD/L) during a whole cycle seemed to be the most adequate, mainly during the denitrification phase. Regarding aeration strategy, an intermittent mode, with dissolved oxygen concentration of 2.0mg/L in the aeration phase, showed the best results. Under these optimal conditions, 97% of influent ammonium nitrogen (80% of total nitrogen) was removed at a rate of 86.5 mg N-NH(4)(+)/Ld. In the treated effluent only 0.2 mg N-NO(2)(-)/L,4.6 mg N-NO(3)(-)/L and 1.0 mg N-NH(4)(+)/L remained, demonstrating the potential viability of this process in post-treatment of wastewaters containing ammonium nitrogen.     

一株海洋好氧反硝化细菌的鉴定及其好氧反硝化特性

【目的】从处理海洋养殖循环水的生物滤器生物膜中分离到1株具有好氧反硝化活性的细菌(菌株2-8),并进一步研究了该菌的分类地位及反硝化特性。【方法】采用16S rRNA基因序列分析对菌株进行初步鉴定,采用好氧培养技术,探讨了碳源种类、起始pH、NaCl浓度、C/N、温度和摇床转速对菌株2-8好氧反硝化活性的影响。【结果】该菌株的16S rRNA基因序列与Pseudomonas segetis FR1439T(AY770691)的相似性最高,达到99.9%,因此初步鉴定菌株2-8属于假单胞菌属(Pseudomonas sp.2-8)。碳源类型和C/N对其好氧反硝化作用的影响最为显著,以柠檬酸钠为唯一碳源,C/N为15时脱氮效率最高,低C/N导致亚硝酸盐的积累;其好氧反硝化的最适温度和pH分别为30℃和7.5;菌株2-8在摇床转速为160r/min下脱氮效果最好;NaCl浓度对其反硝化活性的影响不明显。【结论】在初始硝酸氮浓度为140mg/L,以柠檬酸钠为唯一碳源、C/N为15、pH为7.5、NaCl浓度为30g/L,30℃以及160r/min摇床培养的条件下,菌株2-8在48h内脱氮率可达92%且无亚硝酸盐积累。     

Combined removal of sulfur compounds and nitrate by autotrophic denitrification in bioaugmented activated sludge system

An autotrophic denitrification process using reduced sulfur compounds (thiosulfate and sulfide) as electron donor in an activated sludge system is proposed as an efficient and cost effective alternative to conventional heterotrophic denitrification for inorganic (or with low C/N ratio) wastewaters and for simultaneous removal of sulfide or thiosulfate and nitrate. A suspended culture of sulfur-utilizing denitrifying bacteria was fast and efficiently established by bio-augmentation of activated sludge with Thiobacillus denitrificans. The stoichiometry of the process and the key factors, i.e. N/S ratio, that enable combined sulfide and nitrogen removal, were determined. An optimum N/S ratio of 1 (100% nitrate removal without nitrite formation and low thiosulfate concentrations in the effluent) has been obtained during reactor operation with thiosulfate at a nitrate loading rate (NLR) of 17.18 mmol N L(-1) d(-1). Complete nitrate and sulfide removal was achieved during reactor operation with sulfide at a NLR of 7.96 mmol N L(-1) d(-1) and at N/S ratio between 0.8 and 0.9, with oxidation of sulfide to sulfate. Complete nitrate removal while working at nitrate limiting conditions could be achieved by sulfide oxidation with low amounts of oxygen present in the influent, which kept the sulfide concentration below inhibitory levels.     

Using the combined bioelectrochemical and sulfur autotrophic denitrification system for groundwater denitrification

A combined bioelectrochemical and sulfur autotrophic denitrification system (CBSAD) was evaluated to treat a groundwater with nitrate contamination (20.9-22.0mgNO(3)(-)-N/L). The reactor was operated continuously for several months with groundwater to maximize treatment efficiency under different hydraulic retention times (HRT) and electric currents. The denitrification rate of sulfur autotrophic part followed a half-order kinetics model. Moreover, the removal efficiency of bioelectrochemical part depended on the electric current. The reactor could be operated efficiently at the HRT ranged from 4.2 to 2.1h (corresponding nitrogen volume-loading rates varied from 0.12 to 0.24 kg N/m(3)d; and optimum current ranged from 30 to 1000 mA), and the NO(3)(-)-N removal rate ranged from 95% to 100% without NO(3)(-)-N accumulation. The pH of effluent was satisfactorily adjusted by bioelectrochemical part, and the sulfate concentration of effluent was lower than 250 mg/L, meeting the drinking water standard of China EPA.     

Denitrification capacity in response to increasing nitrate loads and decreasing organic carbon contents in injected leachate of a simulated landfill reactor

Ex situ nitrification followed by denitrification inside the landfill has been recommended to remove ammonia from leachate. The effects of increasing nitrate load and decreasing organic carbon content in the injected leachate on the denitrifying capacity of municipal solid waste (MSW) were investigated. Results showed that MSW possesses a high denitrification capacity. Nitrate reduction could be initiated within 48 h after the first addition of nitrate. Nitrate reduction rate increased with the increasing nitrate loading concentration. When the nitrate loading concentration was increased to 850 mg L^?1, nitrate reduction rate reached up to 35 mg L^?1 h^?1. Nitrite accumulation could be found after the addition of nitrate in each test. However, the maximum nitrite accumulation efficiency declined with increased nitrate load. Organic carbon played an important role in the reduction of nitrate, and both endogenous and exogenous organic materials could act as electron donors.     

Denitrification of a landfill leachate with high nitrate concentration in an anoxic rotating biological contactor

The denitrification performance of a lab-scale anoxic rotating biological contactor (RBC) using landfill leachate with high nitrate concentration was evaluated. Under a carbon to nitrogen ratio (C/N) of 2, the reactor achieved N-NO₃ ⁻ removal efficiencies above 95% for concentrations up to 100 mg N-NO₃ ⁻ l⁻¹. The highest observed denitrification rate was 55 mg N-NO₃ ⁻ l⁻¹ h⁻¹ (15 g N-NO₃ ⁻ m⁻² d⁻¹) at a nitrate concentration of 560 mg N-NO₃ ⁻ l⁻¹. Although the reactor has revealed a very good performance in terms of denitrification, effluent chemical oxygen demand (COD) concentrations were still high for direct discharge. The results obtained in a subsequent experiment at constant nitrate concentration (220 mg N-NO₃ ⁻ l⁻¹) and lower C/N ratios (1.2 and 1.5) evidenced that the organic matter present in the leachate was non-biodegradable. A phosphorus concentration of 10 mg P-PO₄ ³⁻ l⁻¹ promoted autotrophic denitrification, revealing the importance of phosphorus concentration on biological denitrification processes.     

Sulfide enhances methanogenesis in nitrate-containing methanogenic cultures

The effects of sulfide on nitrate reduction and methanogenesis using butyrate as a carbon source were investigated in a mixed mesophilic, methanogenic culture. In the sulfide-free medium, 25-75 mg l⁻¹ nitrate markedly inhibited the efficiencies of acetogenesis and methanogenesis processes. Adding 25 mg-S l⁻¹ increased methane production in nitrate-amended medium. Low sulfide levels shifted the nitrate reduction pathway from denitrification to dissimilatory nitrate reduction to ammonia (DNRA), thereby reducing the amounts of toxic nitric oxide and nitrous oxide produced that inhibit methanogenesis. The dose of 25 mg l⁻¹ sulfide was oxidized completely, during which heterotrophic DNRA predominated. The oxidized forms of sulfide reformed, limiting induction of the heterotrophic denitrification pathway. The actions of heterotrophic and autotrophic DNRA bacteria, denitrifiers, sulfate-reducing bacteria and methanogens mitigate nitrate toxicity during methanogenesis in an anaerobic process.