Evidence of anoxic methane oxidation coupled to denitrification

Denitrification using methane as sole electron donor under anoxic condition was investigated. Sludge produced by a denitrifying reactor using acetate as electron donor was put in contact with methane at partial pressures from 1.8 to 35.7kPa. Nitrate depletion and gaseous nitrogen production were measured. The denitrification rate was independent of the methane partial pressure when superior or equal to 8.8kPa. The nitrate depletion was asymptotic. A denitrification rate of 0.25g NO(3)(-)-Ng(-1) VSSd(-1) was observed at the onset of culturing, followed by a slower and lineal denitrification rate of 4.9x10(-3)g NO(3)(-)-Ng(-1) VSSd(-1). Abiotic nitrate removal or the availability of another carbon source were discarded from control experiments made in the absence of methane or using sterilized inoculum.     

Enumeration of acetate-consuming bacteria by microautoradiography under oxygen and nitrate respiring conditions in activated sludge

Microautoradiography was used to enumerate bacteria able to take up radiolabelled acetate in activated sludge using oxygen or nitrate as electron acceptors. In each of three wastewater treatment plants (WWTP) with nitrification and denitrification (N-removal), the number of bacteria consuming acetate under aerobic and anoxic conditions was identical in contrast to the acetate removal rates. The rates were clearly lower under anoxic conditions suggesting that the specific activity of the cells and not the number of active cells was reduced under anoxic conditions. The fraction of bacteria able to consume acetate varied in three WWTPs between 47% and 93% of the total number of bacteria as determined by DAPI. In a WWTP without N-removal only 20% of the bacteria were able to consume acetate under aerobic conditions and very few of these were able to do it under anoxic conditions. The cell specific acetate removal rates in all WWTPs were found to be 3.0-13.2 x 10(-15) mol cell(-1) h(-1) under aerobic conditions and between 1.9 and 9.1 x 10(-15) mol cell(-1) h(-1) under anoxic conditions.     

Denitrification with methane as external carbon source

Methane is a potentially inexpensive, widely available electron donor for biological denitrification of wastewater, landfill leachate or drinking water. Although no known methanotroph is able to denitrify, various consortia of microorganisms using methane as the sole carbon source carry out denitrification both aerobically and anaerobically. Aerobic methane-oxidation coupled to denitrification (AME-D) is accomplished by aerobic methanotrophs oxidizing methane and releasing soluble organics that are used by coexisting denitrifiers as electron donors for denitrification. This process has been observed in several laboratory studies. Anaerobic methane oxidation coupled to denitrification (ANME-D) was recently discovered and was found to be mediated by an association of an archaeon and bacteria. Methane oxidizing consortia of microorganisms have also been studied for simultaneous nitrification and denitrification (SND) of wastewater. This review focuses on the AME-D process, but also encompasses methane oxidation coupled to SND as well as ANME-D.     

Phosphate uptake under anoxic conditions and fixed-film nitrification in nutrient removal activated sludge system

Simultaneous enhanced biological phosphate uptake and biological denitrification under anoxic conditions were investigated in a modified lab-scale nutrient removal activated sludge system. The aim of the experiments was to find whether poly-P bacteria are capable of taking up phosphate under anoxic conditions by utilising nitrate as an electron acceptor. The phosphate uptake in anoxic conditions was compared to that in aerobic environment in batch tests. The results of the long-term operation of continuous-flow lab-scale system as well as the results of batch tests showed that the anoxic phosphate uptake with simultaneous denitrification after preceding anaerobic substrate uptake could significantly reduce the extent of competition for organic substrate between poly-P bacteria and denitrifiers. A side-stream nitrification in fixed-film reactor enabled to reduce the losses of organic carbon by aerobic oxidation and to stabilise the slow-growing population of nitrifiers in the system.     

Mixed carbon sources for nitrate reduction in activated sludge-identification of bacteria and process activity studies

Mixtures of methanol and acetate as carbon source were investigated in order to determine their capacity to enhance denitrification and for analysis of the microbial composition and carbon degradation activity in activated sludge from wastewater treatment plants. Laboratory batch reactors at 20 degrees C were used for nitrate uptake rate (NUR) measurements in order to investigate the anoxic activity, while single and mixed carbon substrates were added to activated sludge. Microautoradiography (MAR) in combination with fluorescence in situ hybridisation (FISH) were applied for microbial analysis during exposure to different carbon sources. The NUR increased with additions of a mixture of acetate and methanol compared with additions of a single carbon source. MAR-FISH measurements demonstrated that the probe-defined group of Azoarcus was the main group of bacteria utilising acetate and the only active group utilising methanol under anoxic conditions. The present study indicated an improved denitrification potential by additions of a mixed carbon source compared with commonly used single-carbon additions. It is also established that Azoarcus bacteria are involved in the degradation of both acetate and methanol in the anoxic activated sludge.     

Kinetic competition between phosphorus release and denitrification on sludge under anoxic condition

The kinetic behaviors of simultaneous phosphorus release and denitrification on sludge were investigated under anoxic condition. A phosphorus enriched sludge produced from Anaerobic-Anoxic-Oxic (AnAO) process under various SRT (5, 10 and 15 days) operation conditions was carried out in a series of batch tests. Experimental results indicated that the available organic substrate determined the kinetic behaviors of phosphorus release/uptake and denitrification. The simultaneous phosphorus release and denitrification demonstrated a kinetic competition under anoxic conditions in the presence of an available organic substrate. When the substrate was abundant, sludge was under “releasable-phosphorus-limited” condition; phosphorus release rate decreased slightly by nitrate inhibition. However, nitrate significantly inhibited phosphorus release when sludge was under “initial-substrate-limited” condition. Moreover, the sludge's phosphorus contents (as created by different SRT processes) dominated the kinetics of competition between phosphorus release and denitrification. The sludge with a high phosphorus content had a higher phosphorus release rate in accordance with a lower denitrification rate. Additionally, the substrate sequestrated rate markedly increased under the condition of simultaneous phosphorus release and denitrification. Finally, a preliminary metabolism model of denitrifying phosphorus removal bacteria was proposed, and found to be capable of adequately accounting for simultaneous phosphorus release and denitrification under anoxic conditions.     

Anoxic growth of phosphate-accumulating organisms (PAOs) in biological nutrient removal activated sludge systems

In this paper, research on the growth performance of phosphate-accumulating organisms (PAOs) was conducted based on literature and experimental investigations on biological nutrient removal (BNR) activated sludge (BNRAS) systems. The research aims at presenting the occurrence of denitrifying PAOs (DPAOs), abstracting information on the kinetics and stoichiometry of PAOs under anoxic conditions and determining the conditions that stimulate the PAO growth under anoxic conditions. The research results indicate that the PAOs are capable of utilizing nitrate as electron acceptor instead of oxygen in BNRAS systems, particularly in external nitrification BNRAS (ENBNRAS) systems. However, the growth yield of PAOs under anoxic conditions should be reduced to about 70% of that under aerobic conditions, and further the stoichiometric coefficient for anoxic P uptake per PHB COD utilized should be reduced to about 80% of that under aerobic conditions as the DPAOs show a significantly lower BEPR performance and use the influent RBCOD less "efficiently" compared with aerobic PAOs (APAOs). The research results also indicate that the major factor influencing the occurrence of DPAOs and associated anoxic P uptake is the nitrate load into the anoxic reactor, i.e. the nitrate load should be large enough or exceeds the denitrification potential of ordinary heterotrophic organisms (OHOs), i.e. non-PAO organisms in the anoxic reactor to stimulate DPAOs in the system as the specific denitrification rate of OHOs (K'2 OHO) is significantly larger than that of PAOs (K'2 PAO). In terms of this competition, if the nitrate load into the main anoxic reactor is less than the denitrification potential of OHOs, then the OHOs will outcompete PAOs for using the limited nitrate, while if the nitrate load in the main anoxic reactor exceeds the denitrification potential of OHOs, then the PAOs would have opportunities to use the "excess" nitrate and so develop in the system. The other factors that influence DPAOs include the system aerobic mass fraction, sequence of reactors and frequency of sludge alternation between the aerobic and anoxic states. Although it does appear that these factors above may significantly influence the fraction of DPAOs (etaG), the quantitative relationship between these factors and etaG is not known, and the experimental observations indicate that this will be system-specific, and require calibration for each situation.     

Applying a novel autohydrogenotrophic hollow-fiber membrane biofilm reactor for denitrification of drinking water

We conducted a series of pseudo-steady-state experiments on a novel hollow-fiber membrane biofilm reactor used for denitrification of oligotrophic waters, such as drinking water. We applied a range of nitrate loadings and hydrogen pressures to establish under what conditions the system could attain three goodness-of-performance criteria: partial nitrate removal, minimization of hydrogen wasting, and low nitrite accumulation. The hollow-fiber membrane biofilm reactor could meet drinking-water standards for nitrate and nitrite while minimizing the amount of hydrogen wasted in the effluent when it was operated under hydrogen-limited conditions. For example, the system could achieve partial nitrate removals between 39% and 92%, effluent nitrate between 0.4 and 9.1 mg N/l, effluent nitrite less than 1 mg N/l, and effluent hydrogen below 0.1 mg H2/l. High fluxes of nitrate and hydrogen made it possible to have a short liquid retention time (42 min), compared with 1-13 h in other studies with hydrogen used as the electron donor for denitrification. The fluxes and concentrations for hydrogen, nitrate, and nitrite obtained in this study can be used as practical guidelines for system design.     

污泥减量工艺:HA-A/A-MCO的好氧脱氮机制分析

针对污泥减量技术存在对氮、磷去除能力低的问题,开发了一种具有强化脱氮除磷功能并可实现污泥减量化的HA-A/A-MCO工艺。在该工艺取得同步脱氮除磷和污泥减量优异效果的条件下,采用其处理校园生活污水,当进水TN平均为47 mg/L时,出水TN为10.9 mg/L,系统的总脱氮率为76.8%,其中好氧脱氮量占总脱氮量的50%,缺氧脱氮量占26%;HA-A/A-MCO系统存在着在好氧条件下具有反硝化能力的菌属,对好氧脱氮有一定贡献,且DO浓度对其反硝化能力没有抑制作用;好氧池中的DO浓度梯度有利于在污泥絮体内形成缺氧环境,从而促进同步硝化反硝化(SND)的发生,但减小污泥絮体尺寸会削弱絮体内部缺氧区域比例、降低SND的脱氮效率。     

Nitrate attenuation in groundwater: a review of biogeochemical controlling processes

Biogeochemical processes controlling nitrate attenuation in aquifers are critically reviewed. An understanding of the fate of nitrate in groundwater is vital for managing risks associated with nitrate pollution, and to safeguard groundwater supplies and groundwater-dependent surface waters. Denitrification is focused upon as the dominant nitrate attenuation process in groundwater. As denitrifying bacteria are essentially ubiquitous in the subsurface, the critical limiting factors are oxygen and electron donor concentration and availability. Variability in other environmental conditions such as nitrate concentration, nutrient availability, pH, temperature, presence of toxins and microbial acclimation appears to be less important, exerting only secondary influences on denitrification rates. Other nitrate depletion mechanisms such as dissimilatory nitrate reduction to ammonium and assimilation of nitrate into microbial biomass are unlikely to be important in most subsurface settings relative to denitrification. Further research is recommended to improve current understanding on the influence of organic carbon, sulphur and iron electron donors, physical restrictions on microbial activity in dual porosity aquifers, influences of environmental condition (e.g. pH in poorly buffered environments and salinity in coastal or salinized soil settings), co-contaminant influences (particularly the contrasting inhibitory and electron donor influences of pesticides) and improved quantification of denitrification rates in the laboratory and field.     

反硝化聚磷的诱导效果试验

在厌氧/缺氧和厌氧/缺氧/好氧两种系统内进行了试验，结果表明：缺氧段都有较为明显的反硝化聚磷效果，某些情况下甚至表现出良好的稳定性。     

Storage of substrate mixtures by activated sludges under dynamic conditions in anoxic or aerobic environments

In spite of the fact that in most activated sludge plants substrate complex mixtures are removed under alternating anoxic and aerobic conditions, most studies on the dynamic response of biomass are limited to feeding a single substrate (acetate or glucose) under a single redox condition (aerobic or anoxic). In this study, the dynamic response of biomass in a sequencing batch reactor is described in terms of substrate removal and related storage as internal polymers, as functions of single or simultaneous feed of several substrates (acetate, glucose, glutamic acid and ethanol) and of anoxic vs. aerobic conditions. Under anoxic conditions, the four substrates were simultaneously removed at a significantly greater nitrate removal rate than when single substrates were present, so showing that the simultaneous removal was partially due to independent metabolic activities. On the other hand, the removal of every substrate was affected (positively or negatively) by the presence of the others, demonstrating that the substrates can be also used by the same metabolism. As an exception, acetate removal was not affected by the presence of other substrates. As for the comparison of aerobic and anoxic conditions, the acetate uptake rate almost doubled moving from anoxic to aerobic conditions, whereas other substrates were only slightly affected. This difference was probably due to the additional presence of aerobic denitrification, which was much more important for acetate. This also confirmed that acetate removal was independent from other substrates. In all cases, storage was the main mechanism of solids formation, so confirming the general importance of such phenomenon under dynamic conditions, independently from feed complexity and redox conditions.     

Simultaneous urea hydrolysis, formaldehyde removal and denitrification in a multifed upflow filter under anoxic and anaerobic conditions

A multifed upflow filter (MUF), working under anoxic or anaerobic conditions, coupled with an aerobic biofilm airlift suspension (BAS) reactor was operated in order to treat a wastewater with high formaldehyde (up to 1.5 g L-1) and urea (up to 0.46 g L-1) concentrations. In the MUF, formaldehyde removal, denitrification and urea hydrolysis took place simultaneously. The MUF was operated at 37 degrees C, at a hydraulic retention time (HRT) ranging from 1 to 0.3 d. An organic loading rate (OLR) of 0.5 kg-formaldehyde m-3 d-1 was efficiently eliminated during anaerobic operation and transformed into methane, while a much higher OLR (up to 2 kg-formaldehyde m-3 d-1) was oxidised under anoxic conditions by the nitrite or nitrate from the nitrifying airlift. However, only 80% of urea was hydrolysed to ammonia in an anoxic environment while complete conversion occurred under anaerobic conditions. Moreover, formaldehyde concentrations higher than 50 mg L-1 provoked a loss of efficiency of urea hydrolysis, decreasing to 10% at formaldehyde concentrations above 300 mg L-1. Methane production rate during the anaerobic stage was adversely affected by accumulations of formaldehyde in the reactor causing lower formaldehyde removal efficiency. However, denitrification proceeded properly even at a formaldehyde concentration of 700 mg L-1 in the reactor, although nitrous oxide appears in the off-gas. The COD/N ratios required for complete nitrite and nitrate denitrification with formaldehyde were estimated at 2.1 and 3.5 kg-COD/kg-N, respectively.     

Effects of predation and ORP conditions on the performance of nitrifiers in activated sludge systems

Effects of changing oxidation-reduction potential (ORP) and grazing of protozoa on nitrifiers in activated sludge systems was investigated. This study used sequencing batch reactors which were acclimated under aerobic and alternating anoxic/aerobic conditions, with and without inhibition of protozoa, the predatory microorganisms. The feed used was a synthetic wastewater containing beef and yeast extracts as a carbon source. It was found that the biomass, determined by mixed liquor volatile suspended solids (MLVSS) in the reactors, was significantly affected by predation while ORP (aerobic and alternating anoxic/aerobic conditions) has no impact on the MLVSS regardless of the presence or absence of predation. However, the nitrification rates in the reactors show completely different trends indicating that the ORP of the system has a significant impact on the rates while predation does not. It was found that nitrification rates in alternating reactors were almost double the rates in the aerobic reactors, both with and without predatory inhibition. The decay rate of autotrophic bacteria (b(A)) in aerobic reactors was determined by tracking the decrease of the maximum nitrification rate under both anoxic and aerobic starvation conditions. The b(A) in alternating anoxic/aerobic reactors was also determined under alternating starvation conditions. It was found that, in any case, the alternating anoxic/aerobic autotrophic biomass b(A) was much smaller that the b(A) of aerobic biomass determined under aerobic and anoxic starvation conditions. The alternating anoxic/aerobic b(A) was 62.1% less than the aerobic biomass b(A) under aerobic starvation and 40.2% less for the aerobic biomass starved under anoxic conditions. No statistically significant differences in b(A) were observed between reactors with or without the inhibition of predators.     

Fungal and bacterial mediated denitrification in wetlands: Influence of sediment redox condition

Fungal and bacterial denitrification rates were determined under a range of redox conditions in sediment from a Louisiana swamp forest used for wastewater treatment. Sediment was incubated in microcosms at 6 Eh levels (−200, −100, 0, +100, +250 and +400 mV) ranging from strongly reducing to moderately oxidizing conditions. Denitrification was determined using the substrate-induced respiration (SIR) inhibition and acetylene inhibition methods. Cycloheximide (C₁₅H₂₃NO₄) was used as the fungal inhibitor and streptomycin (C₂₁H₃₉N₇O₁₂) as the bacterial inhibitor. At Eh values of +250 mV and +400 mV, denitrification rates by fungi and bacteria were 34.3-35.1% and 1.46-1.59% of total denitrification, respectively, indicating that fungi were responsible for most of the denitrification under aerobic or weakly reducing conditions. On the other hand, at Eh −200 mV, denitrification rates of fungi and bacteria were 17.6% and 64.9% of total denitrification, respectively, indicating that bacteria were responsible for most of the denitrification under strongly reducing conditions. Results show fungal denitrification was dominant under moderately reducing to weakly oxidizing conditions (Eh > +250 mV), whereas bacterial denitrification was dominant under strongly reducing condition (Eh < −100 mV). At Eh values between −100 to +100 mV, denitrification by fungi and bacteria were 37.9-43.2% and 53.0-51.1% of total denitrification, respectively, indicating that both bacteria and fungi contributed significantly to denitrification under these redox conditions. Because N₂O is an important gaseous denitrification product in sediment, fungal denitrification could be of greater ecological significance under aerobic or moderately reducing conditions contributing to greenhouse gas emission and global warming potential (GWP).     

序批式生物膜法除磷机理研究

利用^31P-核磁共振谱图证实了生物除磷的机理，即除磷菌在厌氧条件下分解胞内的聚磷酸盐并释放出正磷酸盐形式的无机磷酸盐，而在好氧或缺氧条件下吸收胞外的无机磷酸盐后转化为聚磷酸盐而贮存于胞内。同时证明了淹没序批式生物膜反应器中磷的去除是由生物完成的。     

Comparison between alternating aerobic-anoxic and conventional activated sludge systems

Conventional activated sludge systems ensure removal of colloidal and dissolved carbonaceous organic matter whereas alternating aerobic-anoxic systems, in addition, satisfy a further reduction in nitrogen content of wastewater. Main difference between them is that the alternating system should also include an anoxic operation mode which satisfies denitrification. In other words conventional systems are operated under aerobic conditions whereas alternating systems require a periodical change from aerobic conditions to anoxic conditions. So the most important problem in alternating systems is to find the appropriate durations for both sequences. In this study a comparison between conventional and alternating systems is considered in terms of nitrogen removal and aeration time by simulation under the same conditions together with an optimization algorithm. The results show that an activated sludge system can be operated as an alternating aerobic-anoxic system so that nitrogen removal is also possible during treatment without any additional investment or operational cost.     

Nitrate contamination of shallow groundwaters in Ontario,Canada

Highly elevated concentrations of NO₃^? have been found in the groundwaters from shallow aquifers at several locations in Ontario. The nitrate is derived either from the fertilizers applied to the agricultural soils, or from industrial point sources, and should be regarded as a major water quality problem. However, the nitrate levels may be reduced by denitrification processes under reducing conditions in the aquifer. The distributions of ammonia, chloride, dissolved oxygen, redox potential, methane, calcium and magnesium are presented and related to the hydrogeochemical changes undergone by the pollutant nitrate during flux along the groundwater flow systems.     

Hydrogen-dependent denitrification of water in an anaerobic submerged membrane bioreactor coupled with a novel hydrogen delivery system

Hydrogen-dependent denitrification has gained significant attention due to its potential economic advantage over heterotrophic denitrification. However, effective hydrogen delivery and biomass retention under anaerobic conditions are significant challenges to implementation of this process. An innovative hydrogenotrophic denitrification system, that addresses these challenges, consisting of an anaerobic submerged membrane bioreactor (MBR) and a novel hydrogen delivery unit, was evaluated for removal of nitrate from a synthetic groundwater feed. The hydrogen delivery unit was designed to release hydrogen-supersaturated water to the reactor and was efficient in hydrogen delivery, providing complete mass transfer. The anaerobic submerged MBR was successful in both reducing nitrate from 25 mg NO(3)-Nl(-1) to below detection and separating biomass from treated water to produce effluent free of suspended solids. Nitrogen gas produced during denitrification was internally recycled to effectively achieve membrane scouring and reactor mixing. The total organic carbon was similar to that of the incoming feed water, averaging approximately 6 mgl(-1).     

A biofilm model for prediction of pollutant transformation in sewers

This study developed a new sewer biofilm model to simulate the pollutant transformation and biofilm variation in sewers under aerobic, anoxic and anaerobic conditions. The biofilm model can describe the activities of heterotrophic, autotrophic, and sulfate-reducing bacteria (SRB) in the biofilm as well as the variations in biofilm thickness, the spatial profiles of SRB population and biofilm density. The model can describe dynamic biofilm growth, multiple biomass evolution and competitions among organic oxidation, denitrification, nitrification, sulfate reduction and sulfide oxidation in a heterogeneous biofilm growing in a sewer. The model has been extensively verified by three different approaches, including direct verification by measurement of the spatial concentration profiles of dissolved oxygen, nitrate, ammonia, and hydrogen sulfide in sewer biofilm. The spatial distribution profile of SRB in sewer biofilm was determined from the fluorescent in situ hybridization (FISH) images taken by a confocal laser scanning microscope (CLSM) and were predicted well by the model.