The denitrifier method is widely used as a novel pretreatment method for the determination of nitrogen and oxygen isotope ratios as it can provide quantitative and high-sensitivity measurements. Nevertheless, the method is limited by relatively low measurement accuracy for δ18O. In this study, we analyzed the factors influencing the accuracy of δ18O determination, and then systematically investigated the effects of dissolved oxygen concentrations and nitrate sample sizes on estimates of the δ15N and δ18O of nitrate reference materials. The δ18O contraction ratio was used to represent the relationship between the measured difference and true difference between two reference materials. We obtained the following main results: (1) a gas-liquid ratio of 3:10 (v/v) in ordinary triangular flasks and a shaking speed of 120 r/min produced an optimal range (1.9 to 2.6 mg/L) in the concentration of dissolved oxygen for accurately determining δ18O, and (2) the δ18O contraction ratio decreased as nitrate sample size decreased within a certain range (1.0 to 0.1 μmol). Our results suggested that δ18O contraction is influenced mainly by dissolved oxygen concentrations in pure culture, and provided a model for improving the accuracy of oxygen isotope analysis.
Denitrification ; Nitrates/analysis* ; Oxygen Isotopes/analysis*

Denitrification is an indispensable part of most sewage treatment systems. The biological denitrification process has attracted much attention in the past decades due to the advantages such as cost-effectiveness, process simplicity, and absence of secondary pollution. This review summarized the advances on biological denitrification processes in recent years according to the different physiological characteristics and denitrification mechanisms of denitrification microorganisms. The pros and cons of different biological denitrification processes developed based on nitrifying bacteria, denitrifying bacteria, and anaerobic ammonia-oxidizing bacteria were compared with the aim to identify the best strategy for denitrification in a complex wastewater environment. The rapid development of synthetic biology provides possibilities to develop highly-efficient denitrifying strains based on mechanistic understandings. Combined with the applications of automatic simulation to obtain the optimal denitrification conditions, cost-effective and highly-efficient denitrification processed can be envisioned in the foreseeable future.
Aerobiosis ; Denitrification ; Nitrification ; Nitrogen ; Waste Water

Biological denitrification is the most widely used technology for nitrate removal in wastewater treatment. Conventional denitrification requires long hydraulic retention time, and the nitrate removal efficiency in winter is low due to the low temperature. Therefore, it is expected to develop new approaches to enhance the denitrification process. In this paper, the effect of adding different concentrations of Fe₃O₄ nanoparticles on the denitrification catalyzed by Pseudomonas stutzeri was investigated. The maximum specific degradation rate of nitrate nitrogen improved from 18.0 h⁻¹ to 23.7 h⁻¹ when the concentration of Fe₃O₄ increased from 0 mg/L to 4 000 mg/L. Total proteins and intracellular iron content also increased along with increasing the concentration of Fe₃O₄. RT-qPCR and label-free proteomics analyses showed that the relative expression level of denitrifying genes napA, narJ, nirB, norR, nosZ of P. stutzeri increased by 55.7%, 24.9%, 24.5%, 36.5%, 120% upon addition of Fe₃O₄, and that of denitrifying reductase Nap, Nar, Nir, Nor, Nos increased by 85.0%, 147%, 16.5%, 47.1%, 95.9%, respectively. No significant difference was observed on the relative expression level of denitrifying genes and denitrifying reductases between the bacteria suspended and the bacteria adhered to Fe₃O₄. Interestingly, the relative expression level of electron transfer proteins of bacteria adhered to Fe₃O₄ was higher than that of the bacteria suspended. The results indicated that Fe₃O₄ promoted cell growth and metabolism through direct contact with bacteria, thereby improving the denitrification. These findings may provide theoretical support for the development of enhanced denitrification.
Aerobiosis ; Denitrification ; Nitrates ; Nitrogen ; Pseudomonas stutzeri/genetics*

Heterotrophic nitrification-aerobic denitrification (HN-AD) is an enrichment and breakthrough theory of traditional autotrophic nitrification heterotrophic denitrification. Heterotrophic nitrification-aerobic denitrifiers with the feature of wide distribution, strong adaptability and unique metabolic mechanism have many special advantages, including fast-growing, rapid biodegradability and long lasting activity, which can rapidly remove ammonia nitrogen, nitrate nitrogen (NO₃⁻-N) and nitrite nitrogen (NO₂⁻-N) under aerobic conditions simultaneously. Therefore, HN-AD bacteria show the important potential for denitrification under extreme conditions with high-salt, low-temperature or high-ammonia nitrogen environment, and HN-AD bacteria attract extensive attention in the field of biological denitrification of wastewater. In this review, we first introduce the previously reported HN-AD bacterial species which have denitrification performance in the extreme environments and state their typical metabolic mechanism. Then, we systematically analyze the nitrogen removal characteristics and potential under extreme conditions. We also briefly describe the progress in the application of HN-AD bacterial. Finally, we outlook the application prospects and research directions of HN-AD denitrification technology.
Aerobiosis ; Bacteria ; Denitrification ; Heterotrophic Processes ; Nitrification ; Nitrites ; Nitrogen

Heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria are aerobic microorganisms that can remove nitrogen under high-salt conditions, but their performance in practical applications are not satisfactory. As a compatible solute, trehalose helps microorganisms to cope with high salt stress by participating in the regulation of cellular osmotic pressure, and plays an important role in promoting the nitrogen removal efficiency of microbial populations in the high-salt environment. We investigated the mechanism of exogenous-trehalose-enhanced metabolism of HN-AD community under high-salt stress by starting up a membrane aerobic biofilm reactor (MABR) to enrich HN-AD bacteria, and designed a C₁₅₀ experimental group with 150 μmol/L trehalose addition and a C₀ control group without trehalose. The reactor performance and the community structure showed that NH₄⁺-N, total nitrogen (TN) and chemical oxygen demand (COD) removal efficiency were increased by 29.7%, 28.0% and 29.1%, respectively. The total relative abundance of salt-tolerant HN-AD bacteria (with Acinetobacter and Pseudofulvimonas as the dominant genus) in the C₁₅₀ group reached 66.8%, an 18.2% increase compared with that of the C₀ group. This demonstrated that trehalose addition promoted the enrichment of salt-tolerant HN-AD bacteria in the high-salt environment to enhance the nitrogen removal performance of the system. In-depth metabolomics analysis showed that the exogenous trehalose was utilized by microorganisms to improve proline synthesis to increase resistance to high-salt stress. By regulating the activity of cell proliferation signaling pathways (cGMP-PKG, PI3K-Akt), phospholipid metabolism pathway and aminoacyl-tRNA synthesis pathway, the abundances of phosphoethanolamine, which was one of the glycerophospholipid metabolites, and purine and pyrimidine were up-regulated to stimulate bacterial aggregation and cell proliferation to promote the growth of HN-AD bacteria in the high-salt environment. Meanwhile, the addition of trehalose accelerated the tricarboxylic acid (TCA) cycle, which might provide more electron donors and energy to the carbon and nitrogen metabolisms of HN-AD bacteria and promote the nitrogen removal performance of the system. These results may facilitate using HN-AD bacteria in the treatment of high-salt and high-nitrogen wastewater.
Nitrification ; Denitrification ; Trehalose ; Phosphatidylinositol 3-Kinases/metabolism* ; Heterotrophic Processes ; Salt Stress ; Nitrogen/metabolism* ; Aerobiosis ; Bioreactors/microbiology*

Water eutrophication poses great threats to protection of water environment. Microbial remediation of water eutrophication has shown high efficiency, low consumption and no secondary pollution, thus becoming an important approach for ecological remediation. In recent years, researches on denitrifying phosphate accumulating organisms and their application in wastewater treatment processes have received increasing attention. Different from the traditional nitrogen and phosphorus removal process conducted by denitrifying bacteria and phosphate accumulating organisms, the denitrifying phosphate accumulating organisms can simultaneously remove nitrogen and phosphorus under alternated anaerobic and anoxic/aerobic conditions. It is worth noting that microorganisms capable of simultaneously removing nitrogen and phosphorus absolutely under aerobic conditions have been reported in recent years, but the mechanisms remain unclear. This review summarizes the species and characteristics of denitrifying phosphate accumulating organisms and the microorganisms capable of performing simultaneous nitrification-denitrification and phosphorous removal. Moreover, this review analyzes the relationship between nitrogen removal and phosphorus removal and the underlying mechanisms, discusses the challenges of denitrifying phosphorus removal, and prospects future research directions, with the aim to facilitate process improvement of denitrifying phosphate accumulating organisms.
Phosphorus ; Phosphates ; Wastewater ; Denitrification ; Waste Disposal, Fluid ; Nitrogen ; Bioreactors/microbiology* ; Nitrification ; Sewage

Anaerobic ammonia oxidation (ANAMMOX) process is an efficient and low-cost biological nitrogen removal process. However, it still faces some challenges in mainstream applications due to the limitation of substrate types and nitrate accumulation. In recent years, the combined process of anammox has been widely studied to solve the above problems. In this paper, the combined processes of anammox developed in recent years are reviewed, and discussed from the process principle, advantages and disadvantages, influencing factors, process extensibility and the key bottlenecks existing in the promotion and application, as well as the relevant work of the subject group. Finally, we take an outlook on the development of the combined anaerobic ammonia oxidation process in municipal domestic wastewater treatment.
Ammonium Compounds ; Anaerobiosis ; Bioreactors ; Denitrification ; Nitrogen ; Oxidation-Reduction ; Sewage ; Waste Water

To investigate the bioelectrochemical enhanced anaerobic ammonia oxidation (anammox) nitrogen removal process, a bioelectrochemical system with coupled anammox cathode was constructed using a dual-chamber microbial electrolysis cell (MEC). Specifically, a dark incubation batch experiment was conducted at 30 ℃ with different influent total nitrogen concentrations under an applied voltage of 0.2 V, and the enhanced denitrification mechanism was investigated by combining various characterization methods such as cyclic voltammetry, electrochemical impedance spectroscopy and high-throughput sequencing methods. The results showed that the total nitrogen removal rates of 96.9%±0.3%, 97.3%±0.4% and 99.0%±0.3% were obtained when the initial total nitrogen concentration was 200, 300 and 400 mg/L, respectively. In addition, the cathode electrode biofilm showed good electrochemical activity. High-throughput sequencing results showed that the applied voltage enriched other denitrifying functional groups, including Denitratisoma, Limnobacter, and ammonia oxidizing bacteria SM1A02 and Anaerolineaceae, Nitrosomonas europaea and Nitrospira, besides the anammox bacteria. These electrochemically active microorganisms comprised of ammonium oxidizing exoelectrogens (AOE) and denitrifying electrotrophs (DNE). Together with anammox bacteria Candidatus Brocadia, they constituted the microbial community structure of denitrification system. Enhanced direct interspecies electron transfer between AOE and DNE was the fundamental reason for the further improvement of the total nitrogen removal rate of the system.
Denitrification ; Wastewater ; Anaerobic Ammonia Oxidation ; Nitrogen ; Oxidation-Reduction ; Bioreactors/microbiology* ; Ammonium Compounds ; Bacteria/genetics* ; Microbiota ; Sewage

Anaerobic ammonium oxidation (ANAMMOX), as its essential advantages of high efficiency and low cost, is a promising novel biological nitrogen elimination process with attractive application prospects. Over the past two decades, many processes based on the ANAMMOX reaction have been continuously studied and applied to practical engineering, with the perspective of reaching 100 full-scale installations in operation worldwide by 2014. Our review summarizes various forms of ANAMMOX processes, including partial nitritation-ANAMMOX, completely autotrophic nitrogen removal over nitrite, oxygen limited autotrophic nitrification and denitrification, denitrifying ammonium oxidation, aerobic deammonification, simultaneous partial nitrification, ANAMMOX and denitrification, single-stage nitrogen removal using ANAMMOX and partial nitritation. We also compare the operating conditions for one-stage and two-stage processes and summarize the obstacles and countermeasures in engineering application of ANAMMOX systems, such as moving bed biofilm reactor, sequencing batch reactor and granular sludge reactor. Finally, we discuss the future research and application direction, which should focus on the optimization of operating conditions and applicability of the process to the actual wastewater, especially on automated control and the impact of special wastewater composition on process performance.
Ammonia ; chemistry ; Bioreactors ; Denitrification ; Nitrification ; Nitrites ; chemistry ; Nitrogen ; chemistry ; Oxygen ; chemistry ; Sewage ; chemistry ; Waste Disposal, Fluid ; methods ; Waste Water ; chemistry

The effects of two different external carbon sources (acetate and ethanol) and electron acceptors (dissolved oxygen, nitrate, and nitrite) were investigated under aerobic and anoxic conditions with non-acclimated process biomass from a full-scale biological nutrient removal-activated sludge system. When acetate was added as an external carbon source, phosphate release was observed even in the presence of electron acceptors. The release rates were 1.7, 7.8, and 3.5 mg P/(g MLVSS·h) (MLVSS: mixed liquor volatile suspended solids), respectively, for dissolved oxygen, nitrate, and nitrite. In the case of ethanol, no phosphate release was observed in the presence of electron acceptors. Results of the experiments with nitrite showed that approximately 25 mg NO₂-N/L of nitrite inhibited anoxic phosphorus uptake regardless of the concentration of the tested external carbon sources. Furthermore, higher denitrification rates were obtained with acetate (1.4 and 0.8 mg N/(g MLVSS·h)) compared to ethanol (1.1 and 0.7 mg N/ (g MLVSS·h)) for both anoxic electron acceptors (nitrate and nitrite).
Biomass ; Bioreactors ; Carbon/chemistry* ; Denitrification ; Electrons ; Nitrates ; Nitrites ; Oxygen ; Phosphates ; Phosphorus/chemistry* ; Sewage ; Waste Disposal, Fluid/methods* ; Wastewater ; Water Pollutants, Chemical ; Water Purification/methods*