Sulfide oxidation under chemolithoautotrophic denitrifying conditions

Chemolithoautotrophic denitrifying microorganisms oxidize reduced inorganic sulfur compounds coupled to the reduction of nitrate as an electron acceptor. These denitrifiers can be applied to the removal of nitrogen and/or sulfur contamination from wastewater, groundwater, and gaseous streams. This study investigated the physiology and kinetics of chemolithotrophic denitrification by an enrichment culture utilizing hydrogen sulfide, elemental sulfur, or thiosulfate as electron donor. Complete oxidation of sulfide to sulfate was observed when nitrate was supplemented at concentrations equal or exceeding the stoichiometric requirement. In contrast, sulfide was only partially oxidized to elemental sulfur when nitrate concentrations were limiting. Sulfide was found to inhibit chemolithotrophic sulfoxidation, decreasing rates by approximately 21-fold when the sulfide concentration increased from 2.5 to 10.0 mM, respectively. Addition of low levels of acetate (0.5 mM) enhanced denitrification and sulfate formation, suggesting that acetate was utilized as a carbon source by chemolithotrophic denitrifiers. The results of this study indicate the potential of chemolithotrophic denitrification for the removal of hydrogen sulfide. The sulfide/nitrate ratio can be used to control the fate of sulfide oxidation to either elemental sulfur or sulfate.     

Sulfur-driven autotrophic denitrification: diversity, biochemistry, and engineering applications

Sulfur-driven autotrophic denitrification refers to the chemolithotrophic process coupling denitrification with the oxidation of reduced inorganic sulfur compounds. Ever since 1904, when Thiobacillus denitrificans was isolated, autotrophic denitrifiers and their uncultured close relatives have been continuously identified from highly diverse ecosystems including hydrothermal vents, deep sea redox transition zones, sediments, soils, inland soda lakes, etc. Currently, 14 valid described species within α-, β-, γ-, and ε-Proteobacteria have been identified as capable of autotrophic denitrification. Autotrophic denitrification is also widely applied in environmental engineering for the removal of sulfide and nitrate from different water environments. This review summarizes recent researches on autotrophic denitrification, highlighting its diversity, metabolic traits, and engineering applications.     

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.     

Biological nitrate removal in industrial wastewater treatment: which electron donor we can choose

Biological denitrification was reviewed regarding its potential application to treating nitrate in industrial wastewater. Although heterotrophic denitrification is an efficient and well-developed process, some carbon content in wastewater is essential to maintain bacterial activity. Because of the high operating cost of heterotrophic denitrification caused by the required addition of a carbon source and potential “carbon breakthrough”, the study of autotrophic denitrification has attracted the interest of numerous researchers. Many advances in autotrophic processes have been made in the application of novel concepts and reaction schemes. While the main advantage of autotrophic bacteria rests on the reduction of operating costs by the replacement of an external carbon source with a cheaper electron donor, further decrease in cost requires additional refinement of these processes, including further improvement of reactor structure and optimization of reaction conditions. In the long term, new concepts are required for a compact wastewater treatment process. This review addresses the state of the art of each electron donor candidate for its potential application to the treatment of industrial wastewater containing nitrate.     

Autotrophic denitrification via a novel membrane-attached biofilm reactor

AIMS: A laboratory-scale autotrophic membrane-attached biofilm reactor was developed to remove nitrate from drinking water. METHODS AND RESULTS: Hydrogen and carbon dioxide flowed together into the lumem side of a gas-permeable silicone tube. The gases diffused through the membrane wall to feed Alcaligenes eutrophus that formed a biofilm on the surface of the silicone tube for autotrophic denitrification. Hydrogen provided the energy source, and carbon dioxide, besides serving as the carbon source, was employed to neutralize the alkalinity from denitrification. The optimal carbon dioxide concentration in the silicone tube was between 20% and 50%. CONCLUSION: This study has demonstrated that a gas-permeable silicone tube is a convenient and efficient method to feed A. eutrophus for autotrophic denitrification. Supplying a suitable amount of carbon dioxide together with hydrogen into the silicone tube solved the problem that alkalinity formation caused during denitrification. The pH of the bioreactor was maintained at about 7 to avoid nitrite accumulation, and then the nitrogen removal rate was increased. A high specific nitrogen removal rate (1.6-5.4 g Nm(2)d(-1-1) of surface area of silicone tube) was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: In addition to combining the advantages of the hydrogenotrophic denitrification process and a membrane feeding substrate bioreactor (MFSB), this bioreactor achieved a high nitrogen removal rate and is simple to operate. It therefore is highly promising in drinking-water treatment.     

好氧反硝化菌脱氮特性研究进展

好氧反硝化菌的发现,是对传统反硝化理论的丰富与突破. 由于其在脱氮方面的独特优势,已成为目前废水生物脱氮领域研究的热点. 好氧反硝化菌能够在有氧条件下,利用有机碳源生长的同时将含氮化合物反硝化生成N2等气态氮化物,多数还能同时进行异养硝化作用,将铵态氮直接转化为含氮气体. 本文从电子理论、反硝化酶系等方面对目前已分离出的一些好氧反硝化菌的脱氮特性及其脱氮机理进行探讨,分析了溶解氧、碳源类型及C/N等环境条件对其脱氮作用的影响,介绍了好氧反硝化菌的筛选方法及应用现状,对其应用前景和发展方向进行了展望.     

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.     

Recent developments on biological nutrient removal processes for wastewater treatment

The need for preserving the environment is tightening regulations limiting the discharge of contaminants into water bodies. Nowadays most of the effort is done on the removal of more specific contaminants such as nutrients (N and P) and sulfurous compounds since they are becoming of great concern due to its impact on the quality of water bodies. There have been two recent discoveries of microbial conversions of nitrogenous compounds. One consisting on the capability of ammonia oxidizers of denitrify under certain conditions resulting in a new one-step method for the removal of N-compounds. The second has been named the ANAMMOX process, wherein ammonium is oxidized to dinitrogen gas with nitrite as the electron acceptor. Other developments consist of operational strategies aiming at obtaining the highest efficiency at removing nitrogen at lowest cost. One strategy consists of the partial nitrification to nitrite (only successful in the SHARON process) and subsequently either the heterotrophic denitrification of nitrites or its autotrophic reduction by ANAMMOX microorganisms. Another strategy consists of the coexistence of nitrifiers and denitrifiers in the same reactor by implementing high frequency oscillations on the oxygen level.The recent developments on biological phosphorous removal are based on the capacity of some denitrifying microorganisms to store ortho-phosphate intracellular as poly-phosphate in the presence of nitrate. These microorganisms store substrate (PHB) anaerobically which is further oxidized when nitrate is present. By extracting excess sludge from the anoxic phase, phosphate is removed from the system. Removing phosphate using nitrate instead of oxygen has the advantage of saving energy (oxygen input) and using less organic carbon.The microbial conversions of sulfurous compounds involve the metabolism of several different specific groups of bacteria such as sulfate reducing bacteria, sulfur and sulfide oxidizing bacteria, and phototrophic sulfur bacteria. Some of these microorganisms can simultaneously use nitrate, what has been reported as autotrophic denitrification by sulfur and sulfide oxidizing microorganisms. More recently, the anaerobic treatment of an industrial wastewater rich in organic matter, nitrogen and sulfate, reported a singular evolution of N and S compounds that initially was hypothesized as SURAMOX (SUlfate Reduction and AMmonia OXidation). The process could not have been verified nor reproduced and further investigations on the proposed SURAMOX mechanism have given no additional insights to those initial observations.     

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.     

Microbial community of granules in expanded granular sludge bed reactor for simultaneous biological removal of sulfate, nitrate and lactate

This study studied the cultivation of granules from an expanded granular sludge bed reactor that simultaneously transforms sulfates, nitrates, and oxygen to elementary sulfur, nitrogen gas, and carbon dioxides, respectively. The living cells accumulate at the granule outer layers, as revealed by the multicolor staining and confocal laser scanning microscope technique. The microbial community comprises sulfate-reducing bacteria (SRB, Desulfomicrobium sp.), heterotrophic (Pseudomonas aeruginosa and Sulfurospirillum sp.), and autotrophic denitrifiers (Sulfurovum sp. and Paracoccus denitrificans) whose population dynamics at different sulfate and nitrate loading rates are monitored with the single-strand conformation polymorphism and denaturing gradient gel electrophoresis technique. The Desulfomicrobium sp. presents one of the dominating strains following reactor startup. At high sulfate and nitrate loading rates, the heterotrophic denitrifiers overcompete autotrophic denitrifiers to reduce SRB activities. Conversely, suddenly reducing nitrate loading rates completely removes the heterotrophic denitrifier Sulfurospirillum sp. from the granules and activates the autotrophic denitrifiers. The physical fixation of different groups of functional strains in granules fine-tunes the strains' activities, and hence the reactor performance.     

Soil environmental conditions rather than denitrifier abundance and diversity drive potential denitrification after changes in land uses

Land‐use practices aiming at increasing agro‐ecosystem sustainability, e.g. no‐till systems and use of temporary grasslands, have been developed in cropping areas, but their environmental benefits could be counterbalanced by increased N₂O emissions produced, in particular during denitrification. Modelling denitrification in this context is thus of major importance. However, to what extent can changes in denitrification be predicted by representing the denitrifying community as a black box, i.e. without an adequate representation of the biological characteristics (abundance and composition) of this community, remains unclear. We analysed the effect of changes in land uses on denitrifiers for two different agricultural systems: (i) crop/grassland conversion and (ii) cessation/application of tillage. We surveyed potential denitrification (PD), the abundance and genetic structure of denitrifiers (nitrite reducers), and soil environmental conditions. N₂O emissions were also measured during periods of several days on control plots. Time‐integrated N₂O emissions and PD were well correlated among all control plots. Changes in PD were partly due to changes in denitrifier abundance but were not related to changes in the structure of the denitrifier community. Using multiple regression analysis, we showed that changes in PD were more related to changes in soil environmental conditions than in denitrifier abundance. Soil organic carbon explained 81% of the variance observed for PD at the crop/temporary grassland site, whereas soil organic carbon, water‐filled pore space and nitrate explained 92% of PD variance at the till/no‐till site, without any residual effect of denitrifier abundance. Soil environmental conditions influenced PD by modifying the specific activity of denitrifiers, and to a lesser extent by promoting a build‐up of denitrifiers. Our results show that an accurate simulation of carbon, oxygen and nitrate availability to denitrifiers is more important than an accurate simulation of denitrifier abundance and community structure to adequately understand and predict changes in PD in response to land‐use changes.     

Ecophysiology of abundant denitrifying bacteria in activated sludge

The abundance of potential denitrifiers in full-scale wastewater treatment plants with biological nitrogen and phosphorus removal was investigated by FISH and various oligonucleotide probes. The potential denitrifiers were characterized as probe-defined populations that were able to consume radiolabelled substrate with oxygen, nitrate and nitrite as electron acceptor as determined by microautoradiography. The most abundant potential denitrifiers were related to the genera Aquaspirillum, Azoarcus, Thauera and Rhodocyclus, all within the Betaproteobacteria. They made up 20-49% of all bacteria in most of the 17 nitrogen removal plants investigated and were hardly present in four plants without denitrification. The ecophysiology of Aquaspirillum, Azoarcus and Thauera-related bacteria was consistent within each probe-defined group in the plants investigated. These three groups showed distinct physiological differences, with the Aquaspirillum-related bacteria appearing as the most specialized one, consuming only amino acids among the substrates tested, and Thauera as the most versatile consuming some volatile fatty acids, ethanol and amino acids. The coexistence of Aquaspirillum, Azoarcus and Thauera-related bacteria in a range of treatment plants with differences in wastewater, design and operation suggest that the populations ensure a functional stability of the plants by occupying different ecological niches related to the carbon transformation.     

好氧反硝化菌的研究进展

综述了好氧反硝化菌的种类和特性、好氧反硝化菌的反硝化作用机制和影响因素.好氧反硝化菌主要包括假单胞菌属(Pseudomonas)、产碱杆菌属(Alcaligenes)、副球菌属(Para-coccus)和芽孢杆菌属(Bacillus)等,属好氧或兼性好氧异养微生物.好氧反硝化菌能在好氧条件下进行反硝化,其主要产物是N2O,并可将铵态氮直接转化成气态产物.催化好氧反硝化菌反硝化作用的硝酸盐还原酶是周质酶而不是膜结合酶.溶解氧和C/N往往是影响好氧反硝化菌反硝化作用的主要因素.介绍了间歇曝气法、选择性培养基法等好氧反硝化菌的主要分离筛选方法.概述了好氧反硝化菌在水产养殖、废水生物处理、降解有机污染物以及对土壤氮素损失的影响方面的研究进展.     

A comparative study of methanol as a supplementary carbon source for enhancing denitrification in primary and secondary anoxic zones

A comparative study on the use of methanol as a supplementary carbon source to enhance denitrification in primary and secondary anoxic zones is reported. Three lab-scale sequencing batch reactors (SBR) were operated to achieve nitrogen and carbon removal from domestic wastewater. Methanol was added to the primary anoxic period of the first SBR, and to the secondary anoxic period of the second SBR. No methanol was added to the third SBR, which served as a control. The extent of improvement on the denitrification performance was found to be dependent on the reactor configuration. Addition to the secondary anoxic period is more effective when very low effluent nitrate levels are to be achieved and hence requires a relatively large amount of methanol. Adding a small amount of methanol to the secondary anoxic period may cause nitrite accumulation, which does not improve overall nitrogen removal. In the latter case, methanol should be added to the primary anoxic period. The addition of methanol can also improve biological phosphorus removal by creating anaerobic conditions and increasing the availability of organic carbon in wastewater for polyphosphate accumulating organisms. This potentially provides a cost-effective approach to phosphorus removal from wastewater with a low carbon content. New fluorescence in situ hybridisation (FISH) probes targeting methanol-utilising denitrifiers were designed using stable isotope probing. Microbial structure analysis of the sludges using the new and existing FISH probes clearly showed that the addition of methanol stimulated the growth of specific methanol-utilizing denitrifiers, which improved the capability of sludge to use methanol and ethanol for denitrification, but reduced its capability to use wastewater COD for denitrification. Unlike acetate, long-term application of methanol has no negative impact on the settling properties of the sludge.     

基于响应面法对一株好氧反硝化菌脱氮效能优化

【目的】水体富营养化是当今我国水环境面临的重大水域环境问题,氮素超标排放是主要的引发因素之一。好氧反硝化菌构建同步硝化反硝化工艺比传统脱氮工艺优势更大。获得高效的好氧反硝化菌株并通过生长因子优化使脱氮效率达到最高。【方法】经过序批式生物反应器(Sequencing batch reactor,SBR)的定向驯化,筛选获得高效好氧反硝化菌株,采用响应面法优化好氧反硝化过程影响总氮去除效率的关键因子(碳氮、溶解氧、pH、温度)。【结果】从运行稳定的SBR反应器中定向筛选高效好氧反硝化菌株Pseudomonas T13,采用响应面法对碳氮比、pH和溶解氧关键因子综合优化获得在18 h内最高硝酸盐去除率95%,总氮去除率90%。该菌株的高效反硝化效果的适宜温度范围为25?30 °C;最适pH为中性偏碱;适宜的COD/NO3?-N为4:1以上;最佳溶解氧浓度在2.5 mg/L。【结论】从长期稳定运行的SBR反应器中筛选获得一株高效好氧反硝化菌Pseudomonas T13,硝酸盐还原酶比例占脱氮酶基因的30%以上,通过运行条件优化获得硝氮去除率达到90%以上,对强化废水脱氮工艺具有良好应用价值。     

好氧反硝化细菌碳氮代谢特点及途径的研究进展

好氧反硝化作用的发现打破了反硝化只能在严格厌氧条件下进行的传统认知,为生物脱氮提供了一条新的途径,已成为近些年的研究热点。碳源可为好氧反硝化过程提供能量和电子供体,其代谢难易程度直接影响着好氧反硝化细菌的脱氮效率,因此有必要明确碳源在好氧反硝化脱氮过程中的代谢机理。基于此,本文阐述了好氧反硝化细菌的种类及其对硝态氮与亚硝态氮的代谢途径;系统分析了不同好氧反硝化细菌对碳氮源代谢的差异与代谢机理;综合分析了碳代谢差异对好氧反硝化脱氮过程的影响,并对未来的研究方向进行了展望,旨在深入理解好氧反硝化细菌同时去除碳氮的机理,为提高废水生物脱氮除碳效率提供理论依据。     

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.     

Enhancing mainstream nitrogen removal by employing nitrate/nitrite-dependent anaerobic methane oxidation processes

Due to serious eutrophication in water bodies, nitrogen removal has become a critical stage for wastewater treatment plants (WWTPs) over past decades. Conventional biological nitrogen removal processes are based on nitrification and denitrification (N/DN), and are suffering from several major drawbacks, including substantial aeration consumption, high fugitive greenhouse gas emissions, a requirement for external carbon sources, excessive sludge production and low energy recovery efficiency, and thus unable to satisfy the escalating public needs. Recently, the discovery of anaerobic ammonium oxidation (anammox) bacteria has promoted an update of conventional N/DN-based processes to autotrophic nitrogen removal. However, the application of anammox to treat domestic wastewater has been hindered mainly by unsatisfactory effluent quality with nitrogen removal efficiency below 80%. The discovery of nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) during the last decade has provided new opportunities to remove this barrier and to achieve a robust system with high-level nitrogen removal from municipal wastewater, by utilizing methane as an alternative carbon source. In the present review, opportunities and challenges for nitrate/nitrite-dependent anaerobic methane oxidation are discussed. Particularly, the prospective technologies driven by the cooperation of anammox and n-DAMO microorganisms are put forward based on previous experimental and modeling studies. Finally, a novel WWTP system acting as an energy exporter is delineated.