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.     

Effect of exogenous carbon sources on removal of inorganic nutrient by the nitrification-denitrification process

A bench scale study was undertaken to examine the effects of exogenous carbon substrates on the removal of inorganic nitrogen and phosphorus by a simple nitrification-denitrification process. Each plastic tank reactor was subjected to a 7-h aeration followed by a 5-h anoxic stage. Methanol, glucose and sodium acetate, at the concentrations equivalent to theoretical COD values of 100 and 200 mg O₂ l⁻¹ were used as external carbon sources and were added to the reactors prior to the anoxic stage. Effects of these additions on biological phosphate release were also investigated. The results showed that 94% of NH₄⁺-N was removed at the end of the aeration period. During the anoxic stage, reduction of nitrate to nitrogen gas was recorded and the denitrification process was significantly enhanced by the addition of organic carbon substrates. At the end of the anoxic stage, over 90% reduction was achieved in the tanks with exogenous carbon substrates while only 47% of NO₃⁻-N was removed in the control reactors. Among the three substrates, sodium acetate was the most efficient and effective source, followed by methanol and glucose. Addition of sodium acetate not only increased the amount of nitrate reduction but also enhanced the rate of N removal especially when a high dosage of sodium acetate was used. With respect to phosphorus removal, 88% ortho-P was removed after the aerobic stage. Throughout the anoxic stage, P concentration was maintained at about 2 mg l⁻¹ in both control and methanol treated reactors. However, significant increase in effluent P content was recorded in both sodium acetate and glucose treatments indicating that phosphorus was released from the bacterial cells during the anoxic stage. The amount of P-release in these two treatments was related to the concentrations of the carbon substrate used.     

利用储碳活性污泥强化反硝化脱氮研究

为提高脱氮效果,在SBR进水的缺氧操作结束后取出部分储碳污泥并加以保存,待好氧末返投储碳污泥作为碳源来强化缺氧反硝化反应.结果表明:对TN的去除率可达98%左右,远高于以缺氧/好氧方式运行的SBR;在好氧段的污泥浓度约为3 000 mg/L的情况下,选择缺氧搅拌段的污泥浓度为5 000 mg/L左右能使TN基本全部被去除.SBR经改造后,能有效实现取、返储碳污泥,提高了脱氮效果.     

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.     

Effects of carbon source on denitrification efficiency and microbial community structure in a saline wastewater treatment process

Two different denitrifying reactors were monitored in order to evaluate the effects of carbon source on denitrification efficiency and microbial community structure under various saline conditions. Nitrogen removal performances were determined when salinity concentrations increase gradually in acetate- or methanol-fed denitrifying reactor. As a result, acetate-fed process attained high nitrate removal at 0-10% NaCl, while methanol was proven beneficial electron donors at 0-3% NaCl. A parallel analysis of T-RFLP and cloning in the acetate-fed sludge showed that a specialized microbial population (i.e. the genera Halomonas and Marinobacter) adapted to a high saline environment. Meanwhile, there were no major changes of bacterial populations in the methanol-fed reactor at 4% NaCl, although the relative abundances of the genera Azoarcus and Methylophaga increased when salinity concentration was at 1-3% NaCl, indicating that methanol-utilizing populations in activated sludge was unable to adapt to a high saline environments (>4% NaCl).     

Ozonation reduces sludge production and improves denitrification

The effectiveness of partial ozonation of return activated sludge for enhancing denitrification and waste sludge minimization were examined. A pair of nitrifying sequencing batch reactors was operated in either aerobic or alternating anoxic/aerobic conditions, with one control and one ozonated reactor in each set. The amount of solids produced decreased with the ozone dose. Biomass in the anoxic/aerobic reactor was easier to destroy (up to 25% of the initial excess sludge) than in the aerobic (10%) one, generating approximately twice as much soluble COD by cell lysis. Denitrification rate improved up to 60% due to additional carbon released by ozonation. Nitrification rates deteriorated much more in the aerobic than in the alternating reactor, possibly as a result of direct destruction of nitrifying autotrophs as well as competition created by growth of heterotrophs receiving the additional COD. Overall, ozonation provided the expected benefits in denitrification and had less impact on nitrification in the alternating reactors.     

Denitrification unit biocenosis

It has been found that during heterotrophic denitrification, biocenosis of the upflow anaerobic sludge blanket (UASB) reactor is composed of bacteria, fungi, protozoa and rotifers. Among these groups of organisms close ecological relationships exist. The scheme of these relationships is proposed. Also described are quantity, taxonomy and biochemical features of denitrifying bacteria and fungi occurring in biocenoses during the processes carried out in the presence of methanol, glycerol, acetic and lactic acids.

denitrification, carbon sources for denitrification, denitrifying biocenosis, yeasts and yeast-like microorganisms, ecology of wastewater purification process     

Granulation in high-load denitrifying upflow sludge bed (USB) pulsed reactors

In this work, the effect of the application of a pulse system to anoxic upflow sludge bed (USB) denitrifying reactors for enhancing sludge granulation was studied. In all, three 0.8 L reactors (two operated with flow pulsation, P1 with effluent recycling and P2 without recycling, and one without pulsation and effluent recycling, no pulsation (NP)) were fed with a mixture of NaNO3 and glucose and inoculated with methanogenic granular sludge. The organic loading rate (OLR) and the nitrogen loading rate (NLR) were progressively increased and, at the end of the experiment, extremely high values were obtained (67.5 kgCOD/m3d and 11.25 kgN-NO3-/m3 d). Ammonia and nitrite accumulation in reactor NP were important in the maturation stage, decreasing the denitrification efficiency to 90%, while in reactor P1 only low nitrite values were obtained in the last few days of the experiment. In reactor P2, nitrogen removal was 100% most of the time. Several operational problems (flotation and the subsequent wash out of biomass) appeared in the NP reactor when working at high denitrifying loading rates, while in reactors P1 and P2 there were no notable problems, mainly due to the good characteristics of the sludge developed and the efficient degasification produced by the pulsing flow. The sludge formed in the NP reactor presented a flocculent structure and a total disintegration of the initial methanogenic granules occurred, while a small-sized granular biomass with a high specific density was developed in the pulsed reactors due to the shear stress produced.     

Low temperature removal of nitrate by bacterial denitrification

The efficiency of two denitrifying sludges enriched at 5 and 20°C were compared using methanol as an electron donor. Both sludges were exposed to the same hydraulic and chemical conditions using an influent containing methanol and mineral salts. The low temperature sludge seemed to have several advantages over the sludge selected for at the higher temperature. In the range 0–17°C, the specific denitrification rate was 1.5–4 times the rate for the high temperature sludge, temperatures below 8°C being the most favourable. At 2°C, under nitrate limiting conditions, 98% nitrate reduction was obtained at a hydraulic residence time of 3.5 h, with an effluent concentration of 0.8 mg NO₃---Nl⁻¹. Sedimentation characteristics were always better for the low temperature sludge, and the utilization of methanol equally good as the high temperature sludge. The low temperature sludge appeared to be biochemically and microbiologically stable to temperature changes within the range 0–17°C, the latter temperature being close to the limit for maintaining the psychrophilic characteristics of the sludge. Studies on pure culture isolates of the denitrifying bacteria showed >90% dominance of one bacterial strain in both sludges. Studies of the isolates also showed that the low-temperature sludge consisted predominantly of psychrotrophs/psychrophiles, and not well-adapted mesophiles, which were only present in low concentrations. The dominant strain in both sludges was unable to grow on methanol in pure culture without access to nutrient growth factors. Only a few minor strains were obligate methylotrophs.Low temperature sludges were tested in a 3-stage biological process receiving domestic sewage. Each stage; carbon oxidation, nitrification and denitrification had separate sludge recycle, and methanol was added to the denitrification stage. These sludges were grown and selected for at temperatures 5°C. At 5°C the laboratory scale process gave 90% removal of total nitrogen at hydraulic residence times of 1.5, 9 and 4 h for the two aeration stages and the anaerobic stage respectively. Overall nitrification/denitrification was 95%, while denitrification separately was 98%. The effluent contained 0.4 mg NO₃---Nl⁻¹. The critical step in the process was unquestionably nitrification. Oxidation of ammonium was satisfactory at low temperature, but the reaction was somewhat vulnerable to changes in external conditions. The low temperature denitrifying sludge was originally enriched on synthetic waste but did not appear to change its microbial composition or characteristics by exposure to municipal wastewater.     

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.     

Factors influencing deterioration of denitrification by oxygen entering an anoxic reactor through the surface

The purpose of the paper is to examine the factors that influence the deterioration of denitrification in open anoxic reactors. For this investigation an ASM 1-based simulation model was developed and successfully applied to fit data from batch experiments carried out in lab-scale reactor vessels (uncovered and covered) using both clarified domestic wastewater and synthetic wastewater. Applying the verified model, simulation studies were performed to investigate the effects of available denitrifiable substrate, biomass concentration, oxygen transfer rate, and temperature on deterioration of denitrification in open anoxic reactors. It has been shown that oxygen entering an anoxic reactor through the surface may not just affect denitrification metabolically, but also kinetically, due to increased dissolved oxygen (DO) concentration exerting an inhibitory effect on the denitrification rate. When the exogenous substrate concentration in the reactor vessel is high enough for a high consumption rate, the DO concentration is kept low. The higher the biomass concentration, and thereby the consumption rate of endogenous substrate, the lower the DO concentration during the low-rate denitrification phase. At low substrate removal rates, decreasing temperature will cause the DO concentration in anoxic vessels to increase. The results suggest that assuring removal of available exogenous carbon source at high rate by staging of open anoxic bioreactors may significantly improve denitrification efficiency.     

SBR工艺对低碳量城市污水的反硝化除磷研究

广州地区的城市污水含碳量低，碳、氮、磷浓度比例失调，采用传统工艺处理很难达到理想的脱氮除磷效果，为此采用SBR工艺对其进行处理，考察了该工艺的反硝化除磷效果。结果表明，在厌氧／缺氧／好氧的运行模式下，采用逐步增加缺氧段运行时间的方法可有效提高污泥的反硝化除磷性能；在试验进水水质条件下，反应器厌氧运行30min、缺氧运行3h、好氧运行1h可保证对磷的稳定高效去除，出水TP〈1mg／L；ORP值无法指示缺氧反硝化与吸磷过程，pH值可作为缺氧吸磷结束的指示参数，而ORP和pH值均可作为好氧吸磷结束的控制参数。     

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.     

Methanol-induced biological nutrient removal kinetics in a full-scale sequencing batch reactor

The primary goal of this research was to determine the potential for denitrification and phosphorus removal of a full-scale sequencing batch reactor (SBR), with and without the use of methanol as an external carbon source. The control SBR, without methanol addition, achieved negligible denitrification. Two denitrification rates were observed in the experimental SBR, with methanol addition, an initial fast rate and a slower second rate. The denitrification rate during the first rate period increased with increasing methanol concentration, until a maximum denitrification rate of approximately 19 mg NOx-N/g MLVSS/day was attained. Following the depletion of the methanol, denitrification reactions probably continued by using the available natural carbon in the influent, resulting in a slower, second denitrification rate. Biological phosphorus uptake and release was significant only in the SBR with methanol addition. Methanol was probably not utilized as the carbon source for the enhanced biological phosphorus removal (EBPR) process. However, methanol addition was critical, since it depleted the available nitrates and thus allowed EBPR to take place.     

Biological denitrification in the presence of cyanide

The influence of cyanide presence on the denitrification process using methanol, propanol, sodium acetate, glucose and acetone as the energy and carbon sources were described. It was shown that during denitrification the carbonyl compounds used as the sole energy and carbon sources for denitrifiers can form cyanohydrins with the cyanide ion. The product of cyanohydrin hydrolysis is biodegradable serving as electron donors for denitrifying bacteria. The simultaneous denitrification and cyanide removal was proved.     

沸石SBR/缺氧上升流污泥床实现氧化铁红废水脱氮

采用沸石序批式反应器(ZSBR)与缺氧上升流污泥床反应器(A-USB)组合工艺处理氧化铁红高氨氮废水,探究ZSBR稳定亚硝化特性以及组合工艺的脱氮性能。结果表明,通过游离氨(FA)抑制亚硝酸盐氧化菌(NOB),ZSBR可实现稳定高效的完全亚硝化。在进水NH4+-N浓度约为700 mg/L的情况下,ZSBR的出水NH4+-N基本稳定在30 mg/L以下,亚硝化率(NAR)维持在95%以上,平均亚硝酸盐产率(NPR)最高可达0. 68 kg/(m³·d)。提升外回流比能够有效利用A-USB反硝化产生的碱度并减少ZSBR中碳酸氢钠碱度的投加量。以葡萄糖作为外加碳源进行反硝化试验,ZSBR出水经过A-USB反硝化处理后,总氮去除率(NRE)能够较稳定维持在85%以上,最高总氮去除负荷(NRR)可达5. 10 kg/(m³·d)。高通量测序分析表明,ZSBR样品中AOB(Nitrosomonas)的相对丰度达到了50. 93%,未检测出NOB,而具有反硝化功能的副球菌属、丛毛单胞菌属和假单胞菌属的相对丰度总占比可达7. 05%,进一步验证了组合工艺高效且稳定的脱氮性能。     

Denitrifying phosphorus removal: linking the process performance with the microbial community structure

This study investigated the link between the process performance of two denitrifying phosphorus (P) removal systems and their microbial community structure. Two sequencing batch reactors (SBRs) were operated with either acetate or propionate as the sole carbon source, and were gradually acclimatised from anaerobic-aerobic to anaerobic-anoxic conditions. It was found that the propionate SBR was able to sustain denitrifying P removal after acclimatisation, while the enhanced biological phosphorus removal (EBPR) activity in the acetate reactor collapsed after the aerobic phase was eliminated. The results suggested that the anoxic glycogen production rate in the acetate SBR was insufficient to support the anaerobic glycogen demand for acetate uptake. The chemical transformations in each SBR suggested that different types of polyphosphate-accumulating organisms (PAOs) were present in each system, possessing different affinities for nitrate. Microbial characterisation with fluorescence in situ hybridisation (FISH) revealed that Accumulibacter was the dominant organism in each reactor, although different cell morphotypes were observed. A coccus morphotype was predominant in the acetate SBR while the propionate SBR was enriched in a rod morphotype. It is hypothesised that the coccus morphotype corresponds to an Accumulibacter strain that is unable to use nitrate as electron acceptor but is able to use oxygen, and possibly nitrite. The rod morphotype is proposed to be a PAO able to use nitrate, nitrite and oxygen. This hypothesis is in agreement with literature studies focussed on the identity of denitrifying PAOs (DPAOs), as well as a recent metagenomic study on Accumulibacter.     

Anaerobic/oxic/anoxic granular sludge process as an effective nutrient removal process utilizing denitrifying polyphosphate-accumulating organisms

In a biological nutrient removal (BNR) process, the utilization of denitrifying polyphosphate-accumulating organisms (DNPAOs) has many advantages such as effective use of organic carbon substrates and low sludge production. As a suitable process for the utilization of DNPAOs in BNR, an anaerobic/oxic/anoxic granular sludge (AOAGS) process was proposed in this study. In spite of performing aeration for nitrifying bacteria, the AOAGS process can create anaerobic/anoxic conditions suitable for the cultivation of DNPAOs because anoxic zones exist inside the granular sludge in the oxic phase. Thus, DNPAOs can coexist with nitrifying bacteria in a single reactor. In addition, the usability of DNPAOs in the reactor can be improved by adding the anoxic phase after the oxic phase. These characteristics enable the AOAGS process to attain effective removal of both nitrogen and phosphorus. When acetate-based synthetic wastewater (COD: 600 mg/L, NH4-N: 60 mg/L, PO(4)-P: 10 mg/L) was supplied to a laboratory-scale sequencing batch reactor under the operation of anaerobic/oxic/anoxic cycles, granular sludge with a diameter of 500 microm was successfully formed within 1 month. Although the removal of both nitrogen and phosphorus was almost complete at the end of the oxic phase, a short anoxic period subsequent to the oxic phase was necessary for further removal of nitrogen and phosphorus. As a result, effluent concentrations of NH(4)-N, NO(x)-N and PO(4)-P were always lower than 1 mg/L. It was found that penetration depth of oxygen inside the granular sludge was approximately 100 microm by microsensor measurements. In addition, from the microbiological analysis by fluorescence in situ hybridization, existence depth of polyphosphate-accumulating organisms was further than the maximum oxygen penetration depth. The water quality data, oxygen profiles and microbial community structure demonstrated that DNPAOs inside the granular sludge may be responsible for denitrification in the oxic phase, which enables effective nutrient removal in the AOAGS process.     

Denitrification with natural gas and various new growth media

Biological denitrification was investigated in an attached growth reactor system using several growth media, denitrifying cultures and natural gas (95% methane) as a carbon source. In order to establish a baseline of operation, initial experiments were conducted with a bed of 2–3 mm sand and methanol as a carbon source using a methylotrophic denitrifying culture and then the system was compared with natural gas using various methane utilising cultures. Compared to methanol, performance with methane was considerably lower. In order to improve denitrification with methane a plastic medium (Etapak, surface area 200 m²/m³) was placed above the sand bed (which increased the surface area for bacteria growth in the upper part of the bed), and a new methanotrophic mixed culture (NCIMB-code 11085) was introduced to the system. This combination resulted in a 27% higher denitrification efficiency. Experiments were continued by systematically varying the operating conditions to obtain highest denitrification using methane gas and replacing the Etapak media with different plastic media of higher surface area, but keeping the NCIMB culture unchanged. Other media tested were Pall-rings (surface area 319 m²/m³), IP-spacers (surface area 500 m²/m³) and granular activated carbon (GAC-code: Norit PK 1–3). Best results were obtained with IP-spacers which, surprisingly, are designed for use in the concrete industry rather than as a bacterial support medium. These produced nitrate removal efficiencies of up to 93% at 0.6 m/h or 55% at 1.6 m/h water filtration rates. Run times of 10 days or more to a limiting headloss of about 1.0 m,were usually achieved before “bumping” or back-washing to reduce headloss. Effluent turbidities were generally below 1.0 NTU. Tests for bacteria present with GAC media and COD removal with IP spacers were also carried out. Results are discussed with operational conditions and denitrification efficiencies achieved.     

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.