Bio-electrochemical removal of nitrate from water and wastewater--a review

Nitrates in different water and wastewater streams raised concerns due to severe impacts on human and animal health. Diverse methods are reported to remove nitrate from water streams which almost fail to entirely treat nitrate, except biological denitrification which is capable of reducing inorganic nitrate compounds to harmless nitrogen gas. Review of numerous studies in biological denitrification of nitrate containing water resources, aquaculture wastewaters and industrial wastewater confirmed the potential of this method and its flexibility towards the remediation of different concentrations of nitrate. The denitrifiers could be fed with organic and inorganic substrates which have different performances and subsequent advantages or disadvantages. Review of heterotrophic and autotrophic denitrifications with different food and energy sources concluded that autotrophic denitrifiers are more effective in denitrification. Autotrophs utilize carbon dioxide and hydrogen as the source of carbon substrate and electron donors, respectively. The application of this method in bio-electro reactors (BERs) has many advantages and is promising. However, this method is not so well established and documented. BERs provide proper environment for simultaneous hydrogen production on cathodes and appropriate consumption by immobilized autotrophs on these cathodes. This survey covers various designs and aspects of BERs and their performances.     

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.     

Role of heterotrophic nitrifiers and aerobic denitrifiers in simultaneous nitrification and denitrification process: a nonconventional nitrogen removal pathway in wastewater treatment

Bacterial species capable of performing both nitrification and denitrification in a single vessel under similar conditions have gained significance in the wastewater treatment scenario considering their unique character of performing the above reactions under heterotrophic and aerobic conditions respectively. Such a novel strategy often referred to as simultaneous nitrification and denitrification (SND) has a tremendous potential in dealing with various wastewaters having low C : N content, considering that the process needs very little or no external carbon source and oxygen supply thus adding to its cost-effective and environmentally friendly nature. Though like other micro-organisms, heterotrophic nitrifiers and aerobic denitrifiers convert inorganic or organic nitrogen-containing substances into harmless dinitrogen gas in the wastewater, their ecophysiological role in the global nitrogen cycle is still not fully understood. Attempts to highlight the role played by the heterotrophic nitrifiers and aerobic denitrifiers in dealing with nitrogen pollution under various environmental operating conditions will help in developing a mechanistic understanding of the SND process to address the issues faced by the traditional methods of aerobic autotrophic nitrification–anaerobic heterotrophic denitrification.     

Effect of sulfide concentration on autotrophic denitrification from nitrate and nitrite in vertical fixed-bed reactors

Autotrophic denitrification coupled with sulfide oxidation represents an interesting alternative for the simultaneous removal of nitrate/nitrite and sulfide from wastewaters. The applicability of such bioprocess is especially advantageous for the post treatment of effluents from anaerobic reactors, since they usually produce sulfides, which can be used as endogenous electron donor for autotrophic denitrification. This study evaluated the effect of sulfide concentration on this bioprocess using nitrate and nitrite as electron acceptors in vertical fixed-bed reactors. The results showed that intermediary sulfur compounds were mainly produced when excess of electron donor was applied, which was more evident when nitrate was used. Visual evidences suggested that elemental sulfur was the intermediary compound produced. There was also evidence that the elemental sulfur previously formed was being used when sulfide was applied in stoichiometric concentration relative to nitrate/nitrite. Nitrite was more readily consumed than nitrate. For both electron acceptors and sulfide concentrations tested, autotrophic denitrification was not affected by residual heterotrophic denitrification via endogenic activity, occurring as a minor additional nitrogen removal process.     

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.     

Denitrifying sulfide removal and carbon methanogenesis in a mesophilic, methanogenic culture

The inhibitory effects of 90-189 mg l⁻¹ of sulfide and 25-75 mg-N l⁻¹ of nitrate on methanogenesis were investigated in a mixed methanogenic culture using butyrate as carbon source. In the initial phase of 90 mg l⁻¹ S²⁻ test, autotrophic denitrification of nitrate occurred with sulfide as the electron donor. Then the sulfate-reducing strains converted the produced sulfur back to sulfide via heterotrophic oxidation pathway. Methanogenesis was not markedly inhibited when 90 mg l⁻¹ of sulfide was dosed alone. When 25-75 mg-N l⁻¹ of nitrate was presented, initiation of methanogenesis was seriously delayed. Nitrogen oxides (NO_x), the intermediates for nitrate reduction via denitrification pathway, inhibited methanogenesis. The 90 mg l⁻¹ of sulfide favored heterotrophic dissimilatory nitrate reduction to ammonia (DNRA) pathway for nitrate reduction. Possible ways of maximizing methane production from an organic carbon-rich wastewater with high levels of sulfide and nitrate were discussed.     

A two-stage biological treatment system for ammonium-nitrate-laden wastewater

A two-stage non-conventional bench scale biological treatment system was investigated for the treatment of the wastewater laden with ammonium nitrate. The first stage which consisted of a fixed film anoxic reactor effected denitrification of nitrate ion, while the second stage consisting of a pond effected ammonia removal. Dissimilatory denitrification requires external carbon source as an electron donor. Methanol was used as electron donor in this system. The system exhibited substantial nitrate and ammonia removal. The influent nitrate-N concentration which was on average 193.87 ± 12.68 mg/l was reduced to 5.86 ± 4.86 mg/l in the denitrification unit. There was only a marginal reduction of ammonia in the denitrification unit and most of the ammonia-N was removed in the pond. The ammonia-N was reduced from an average value of 104.87 ± 3.49 mg/l at denitrification unit inlet to 33.37 ± 8.12 mg/l at the pond outlet. There was no corresponding increase in the nitrite or nitrate concentration in proportion to ammonia reduction in the pond. The average nitrate concentration in the pond outlet was 2.4 ± 0.93 mg/l. Microbiological investigation of the system revealed the presence of significant populations of denitrifying organisms in the first stage, and denitrifying, nitrifying and algal populations in the second stage. The system also sustained wastewater of pH as low as 3.87 and appears to be very promising for larger scale industrial wastewater treatment.     

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.     

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.     

Autotrophic nitrite removal in the cathode of microbial fuel cells

Nitrification to nitrite (nitritation process) followed by reduction to dinitrogen gas decreases the energy demand and the carbon requirements of the overall process of nitrogen removal. This work studies autotrophic nitrite removal in the cathode of microbial fuel cells (MFCs). Special attention was paid to determining whether nitrite is used as the electron acceptor by exoelectrogenic bacteria (biologic reaction) or by graphite electrodes (abiotic reaction). The results demonstrated that, after a nitrate pulse at the cathode, nitrite was initially accumulated; subsequently, nitrite was removed. Nitrite and nitrate can be used interchangeably as an electron acceptor by exoelectrogenic bacteria for nitrogen reduction from wastewater while producing bioelectricity. However, if oxygen is present in the cathode chamber, nitrite is oxidised via biological or electrochemical processes. The identification of a dominant bacterial member similar to Oligotropha carboxidovorans confirms that autotrophic denitrification is the main metabolism mechanism in the cathode of an MFC.     

Characterization of microbial community and kinetics for spent sulfidic caustic applied autotrophic denitrification

Spent sulfidic caustic was applied to sulfur utilizing autotrophic denitrification as the simultaneous source of electron donor and alkalinity. The two experiment set-up of upflow anoxic hybrid growth reactor (UAHGR) and upflow anoxic suspended growth reactor (UASGR) was adopted and nitrate removals were similar in both reactors. Approximately 90% of the initial nitrate was denitrified at nitrate loading rate of 0.15∼0.40 kgNO₃ ⁻/m³·d. The experimental stoichiometric ratio of sulfate production to nitrate removal was ranged from 1.5 to 2.1 mgSO₄ ²⁻/mgNO₃ ⁻. During the operation period, denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction (PCR)-amplified 16S rDNA fragments for the sludge sample of both reactors showed the change of microbial communities. Thiobacillus denitrificans-like microorganism occupied 28.5% (18 clones) of the 63 clones by cloning the PCR products from the sludge sample of UAHGR. Acidovorax avenae, which can reduce nitrate to nitrogen gas while oxidizing phenol (heterotrophic denitrifier), was also found in 7 clones (11.1%). Although an organic carbon source was not added to the medium, a microorganism (Kaistella koreensis) capable of oxidizing organic compounds was found in 7 clones (11.1%). Therefore, the microbial community of spent sulfidic caustic applied autotrophic denitrification process well corresponds to the substrate components of spent sulfidic caustic. Through the batch cultivation of microorganisms in UAHGR, the microbial kinetic coefficients of spent sulfidic caustic applied autotrophic denitrification were estimated to be μ _max = 0.097 h⁻¹, k _d = 0.0021 h⁻¹, K _s = 200 mgNO₃ ⁻/L, and Y = 0.31 mgMLVSS/mgNO₃ ⁻.     

厌氧氨氧化工艺的应用现状和问题

厌氧氨氧化(Anaerobic ammonium oxidation,ANAMMOX)工艺因其高效低耗的优势,在废水生物脱氮领域具有广阔的应用前景。在过去的20年中,许多基于ANAMMOX反应的工艺得以不断研究和应用。预计到2014年末,全球范围内的ANAMMOX工程将会超过100座。综述了各种形式的ANAMMOX工艺,包括短程硝化-厌氧氨氧化、全程自养脱氮、限氧自养硝化反硝化、反硝化氨氧化、好氧反氨化、同步短程硝化-厌氧氨氧化-反硝化耦合、单级厌氧氨氧化短程硝化脱氮工艺。对一体式和分体式工艺运行条件进行了比较,结合ANAMMOX工艺工程(主要包括移动床生物膜,颗粒污泥和序批式反应器系统)应用现状,总结了工程化应用过程中遇到的问题及其解决对策,在此基础上对今后的研究和应用方向进行了展望。今后的研究重点应集中于运行条件的优化和水质障碍因子的解决,尤其是工艺自动化控制系统的开发和特殊废水对工艺性能影响的研究。     

ORGANIC CARBON SUPPLEMENTATION OF STERILIZED MUNICIPAL WASTEWATER IS ESSENTIAL FOR HETEROTROPHIC GROWTH AND REMOVING AMMONIUM BY THE MICROALGA CHLORELLA VULGARIS1

Heterotrophic growth of the microalga Chlorella vulgaris Beij. in synthetic as well as sterilized municipal wastewater of a nonindustrialized city was measured. The city wastewater contained high levels of ammonium and nitrate, medium levels of phosphate, and low levels of nitrite and organic molecules and could not support heterotrophic growth of C. vulgaris. Evaluation of 11 known carbon sources for this microalga that were added to standard synthetic wastewater containing the same levels of nitrogen and phosphorus as the municipal wastewater revealed that the best carbon sources for heterotrophic growth were Na‐acetate and d ‐glucose. These provided the highest growth rates and the largest removal of ammonium. Growth increased with concentration of the supplement to an optimum at 0.12 M Na‐acetate. This carbon source was consumed completely within 10 d of incubation. Higher concentrations inhibited the growth of C. vulgaris. The microalgal populations under heterotrophic growth conditions were one level of magnitude higher than that under autotrophic growth conditions that served as a comparison. No growth occurred in the dark in the absence of a carbon source. Na‐acetate was superior to d ‐glucose. In municipal wastewater, when Na‐acetate or d ‐glucose was added, C. vulgaris significantly enhanced ammonium removal under heterotrophic conditions, and its capacity was equal to ammonium removal under autotrophic growth conditions. This study showed that sterilized wastewater can be treated by C. vulgaris under heterotrophic conditions if supplemented with the appropriate organic carbon source for the microalgae.     

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.     

Anaerobic ferrous oxidation by heterotrophic denitrifying enriched culture

Heterotrophic denitrifying enriched culture (DEC) from a lab-scale high-rate denitrifying reactor was discovered to perform nitrate-dependent anaerobic ferrous oxidation (NAFO). The DEC was systematically investigated to reveal their denitrification activity, their NAFO activity, and the predominant microbial population. The DEC was capable of heterotrophic denitrification with methanol as the electron donor, and autotrophic denitrification with ferrous salt as the electron donor named NAFO. The conversion ratios of ferrous-Fe and nitrate-N were 87.41 and 98.74 %, and the consumption Fe/N ratio was 2.3:1 (mol/mol). The maximum reaction velocity and half saturation constant of Fe were 412.54 mg/(l h) and 8,276.44 mg/l, and the counterparts of N were 20.87 mg/(l h) and 322.58 mg/l, respectively. The predominant bacteria were Hyphomicrobium, Thauera, and Flavobacterium, and the predominant archaea were Methanomethylovorans, Methanohalophilus, and Methanolobus. The discovery of NAFO by heterotrophic DEC is significant for the development of wastewater treatment and the biogeochemical iron cycle and nitrogen cycle.     

Identity and potential functions of heterotrophic bacterial isolates from a continuous-upflow fixed-bed reactor for denitrification of drinking water with bacterial polyester as source of carbon and electron donor

A collection of 186 heterotrophic bacteria, isolated directly from a continuous-upflow fixed-bed reactor for the denitrification of drinking water, in which poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) granules acted as biofilm carrier, carbon source and electron donor, was studied with regard to taxonomic affiliation and degradation and denitrification characteristics. Two granule samples were taken from a fully operating reactor for enumeration and isolation of heterotrophic bacteria. One sample was drawn from the lower part of the reactor, near the oxic zone, and the other sample from the upper, anoxic part of the fixed bed. Dominant colonies were isolated and the cultures were identified using fatty acid analysis and 16S rDNA sequencing. Their ability to degrade the polymer and 3-hydroxybutyrate and to denitrify in pure culture was assessed. The results show that high numbers of heterotrophic bacteria were present in the biofilms on the polymer granules, with marked differences in taxonomic composition and potential functions between the lower and upper part of the fixed bed. The majority of the isolates were Gram negative bacteria, and most of them were able to reduce nitrate to nitrite or to denitrify, and to utilize 3-hydroxybutyrate as sole source of carbon. Only two groups, one identified as Acidovorax facilis and the other phylogenetically related to Brevundimonas intermedia, could combine denitrification and utilization of poly(3-hydroxybutyrate) (PHB), and were found only in the upper sample. The other groups occurred either in the lower or upper part, or in both samples. They were assigned to Brevundimonas, Pseudomonas, Agrobacterium, Achromobacter, or Phyllobacterium, or were phylogenetically related to Afipia or Stenotrophomonas.     

Microbial community structure and dynamics in a mixotrophic nitrogen removal process using recycled spent caustic under different loading conditions

A laboratory-scale Bardenpho process was established to investigate the proper nitrogen loading rate (NLR) when modified spent caustic (MSC) is applied as electron donor and alkalinity source for denitrification. MSC injection induced autotrophic nitrogen removal with sulfur as electron donor and heterotrophic denitrification. The nitrogen removal rate (NRR) did not increase proportionally to NLR. Based on the total nitrogen concentration in the effluent observed in the trials with MSC, the NLR in the influent should not exceed 0.15 kg N/m³ d in order to satisfy water quality regulations. Microbial communities in the anoxic reactors were characterized by pyrosequencing of 16S rRNA gene sequences amplified by the polymerase chain reaction of DNA extracted from sludge samples. Microbial diversity was lower as MSC dosage was increased, and the injection of MSC caused an increase in SOB belonging to the genus Thiobacillus which is responsible for denitrification using sulfur.     

Sulfur-driven autotrophic denitrification of nitric oxide for efficient nitrous oxide recovery

Nitrous oxide (N₂O) was previously deemed as a potent greenhouse gas but is actually an untapped energy source, which can accumulate during the microbial denitrification of nitric oxide (NO). Compared with the organic electron donor required in heterotrophic denitrification, elemental sulfur (S⁰) is a promising electron donor alternative due to its cheap cost and low biomass yield in sulfur-driven autotrophic denitrification. However, no effort has been made to test N₂O recovery from sulfur-driven denitrification of NO so far. Therefore, in this study, batch and continuous experiments were carried out to investigate the NO removal performance and N₂O recovery potential via sulfur-driven NO-based denitrification under various Fe(II)EDTA-NO concentrations. Efficient energy recovery was achieved, as up to 35.5%–40.9% of NO was converted to N₂O under various NO concentrations. N₂O recovery from Fe(II)EDTA-NO could be enhanced by the low bioavailability of sulfur and the acid environment caused by sulfur oxidation. The NO reductase (NOR) and N₂O reductase (N₂OR) were inhibited distinctively at relatively low NO levels, leading to efficient N₂O accumulation, but were suppressed irreversibly at NO level beyond 15 mM in continuous experiments. Such results indicated that the regulation of NO at a relatively low level would benefit the system stability and NO removal capacity during long-term system operation. The continuous operation of the sulfur-driven Fe(II)EDTA-NO-based denitrification reduced the overall microbial diversity but enriched several key microbial community. Thauera, Thermomonas, and Arenimonas that are able to carry out sulfur-driven autotrophic denitrification became the dominant organisms with their relative abundance increased from 25.8% to 68.3%, collectively.     

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.