53#,a strain of efficient alginate degrading bacteria,is isolated from rotted kelp using sodium alginate as the only carbon resource. Strain 53# is identified as Paenibacillus agaridevorans based on physiological characteristics and 16S rRNA sequencing. The optimaltion yield condition of strain 53# is identified at ${\rm{pH = 8}}$,$T = 25^\circ {\rm{C}}$,NaCl 15 g/L,and sodium alginate 15 g/L by orthogonal experiment and analysis,and the highest enzyme activity is ${\rm{390}}{\rm{.53}} \pm {\rm{17}}{\rm{.32\;U/mL}}$. Strain 53# has the advantages such as being cultivated easily,producing enzyme fast and having high enzyme activity. It can achieve a high efficiency for saccharification of alginate and thus has potential value to be utilized in the production of bioethanol from brown algae.以海藻酸钠为唯一碳源,从天然腐烂海带中筛选得到一株高效褐藻胶降解菌株53#,经形态学观察和16S rRNA鉴定,确定为类芽孢杆菌Paenibacillus agaridevorans。采用正交试验方法,以pH、温度、NaCl浓度和海藻酸钠初始浓度为影响因素,对该菌株的产酶条件进行优化,得到53#菌的 最佳产酶条件: ${\rm{pH = 8}}$,25℃,NaCl浓度15 g/L,海藻酸钠初始浓度15 g/L。在最佳产酶条件下,褐藻胶裂解酶最大酶活可达${\rm{390}}{\rm{.53}} \pm {\rm{17}}{\rm{.32\;U/mL}}$。筛选得到的类芽孢杆菌Paenibacillus agaridevorans 53#具有易于培养、产酶速度快和酶活力高等优点,能够实现褐藻胶的高效糖化,在褐藻生产生物乙醇领域具有潜在利用价值。

This paper presents the effect of NaCl on aerobic denitrification by a novel aerobic denitrifier strain Achromobacter sp. GAD-3. Results indicated that the aerobic denitrification process was inhibited by NaCl concentrations ae<yen>20 g L-1, leading to lower nitrate removal rates (1.67 +/- 4.0 mg L-1 h(-1)), higher nitrite accumulation (50.2 +/- 87.4 mg L-1), and increasing N2O emission ratios (13 +/- 72 mg L-1/mg L-1). Poor performance of aerobic denitrification at high salinity was attributed to the suppression of active microbial biomass and electron donating capacity of strain GAD-3. Further studies on the corresponding inhibition of the denitrifying gene expression by higher salinities revealed the significant sensitivity order of nosZ (for N2O reductase) > cnorB (for NO reductase) ae nirS (for cytochrome cd(1) nitrite reductase) > napA (for periplasmic nitrate reductase), accompanied with a time-lapse expression between nosZ and cnorB based on reverse transcription and real-time quantitative polymerase chain reaction (RT-qPCR) analysis. The insights into the effect of NaCl on aerobic denitrification are of great significance to upgrade wastewater treatment plants (WWTPs) containing varying levels of salinity.

Sulfamethoxazole (SMX), as a common sulfonamide antibiotic, was reported to affect conventional anaerobic denitrification. This study presented effects of SMX on aerobic denitrification by an aerobic denitrifier strain Pseudomonas stutzeri PCN-1. Results demonstrated serious inhibition of N2O reduction as SMX reached 4 mu g/L, leading to higher N2O emission ratio (251-fold). Increase of SMX (similar to 8 mu g/L) would induce highest nitrite accumulation (95.3 mg/L) without reduction, and severe inhibition of nitrate reduction resulted in lower nitrate removal rate (0.15 mg/L/h) as SMX reached 20 mu g/L. Furthermore, corresponding inhibition of SMX on denitrifying genes expression (nosZ > nirS > cnorB > napA) was found with a time-lapse expression between nosZ and cnorB. Meanwhile, the decline in electron transport activity and active microbial biomass of strain PCN-1 was revealed. The insight into mechanism of SMX influence on aerobic denitrifier is of particular significance to upgrade nitrogen removal process in antibiotics-containing wastewater treatment plant. (C) 2017 Elsevier Ltd. All rights reserved.

Effects of heavy metals on aerobic denitrification have been poorly understood compared with their impacts on anaerobic denitrification. This paper presented effects of four heavy metals (Cd(II), Cu(II), Ni(II), and Zn(II)) on aerobic denitrification by a novel aerobic denitrifying strain Pseudomonas stutzeri PCN-1. Results indicated that aerobic denitrifying activity decreased with increasing heavy metal concentrations due to their corresponding inhibition on the denitrifying gene expression characterized by a time lapse between the expression of the nosZ gene and that of the cnorB gene by PCN-1, which led to lower nitrate removal rate (1.67 similar to 6.67 mg L-1 h(-1)), higher nitrite accumulation (47.3 similar to 99.8 mg L-1), and higher N2O emission ratios (5 similar to 283 mg L-1/mg L-1). Specially, promotion of the nosZ gene expression by increasing Cu(II) concentrations (0 similar to 0.05 mg L-1) was found, and the absence of Cu resulted in massive N2O emission due to poor synthesis of N2O reductase. The inhibition effect for both aerobic denitrifying activity and denitrifying gene expression was as follows from strongest to least: Cd(II) (0.5 similar to 2.5 mg L-1) > Cu(II) (0.5 similar to 5 mg L-1) > Ni(II) (2 similar to 10 mg L-1) > Zn(II) (25 similar to 50 mg L-1). Furthermore, sensitivity of denitrifying gene to heavy metals was similar in order of nosZ > nirS ae cnorB > napA. This study is of significance in understanding the potential application of aerobic denitrifying bacteria in practical wastewater treatment.

Anaerobic denitrification has been proved to be negatively affected by ZnO nanomaterials (NPs), but little is known about how ZnO NPs affects aerobic denitrification. In this study, inhibition of ZnO NPs to an aerobic denitrifier, Pseudomonas stutzeri PCN-1, was firstly reported. The results showed total nitrogen removal efficiency was decreased from 100% to 1.70% with the increase of ZnO NPs from 1 to 128 mg/L. The presence of ZnO NPs caused significant inhibition of gene expressions and catalytic activities of nitrate reductase and nitrite reductase, which finally led to delayed nitrate reduction and high nitrite accumulation. Further studies revealed that the deposition of nanoparticles on the bacterial surface caused by electrostatic forces and the generation of reactive oxygen species (ROS) were responsible for the cytotoxicity of ZnO NPs, where ROS played a more important role. These results were of significance to evaluating the potential ecological toxicity and risks of nanomaterials. (C) 2017 Elsevier Ltd. All rights reserved.

The microbial community diversity in anaerobic-, anoxic- and oxic-biological zones of a conventional Carrousel oxidation ditch system for domestic wastewater treatment was systematically investigated. The monitored results of the activated sludge sampled from six full-scale WWTPs indicated that Proteobacteria, Chloroflexi, Bacteroidetes, Actinobacteria, Verrucomicrobia, Acidobacteria and Nitrospirae were dominant phyla, and Nitrospira was the most abundant and ubiquitous genus across the three biological zones. The anaerobic-, anoxic-and oxic-zones shared approximately similar percentages across the 50 most abundant genera, and three genera (i.e. uncultured bacterium PeM15, Methanosaeta and Bellilinea) presented statistically significantly differential abundance in the anoxic-zone. Illumina high-throughput sequences related to ammonium oxidizer organisms and denitrifiers with top50 abundance in all samples were Nitrospira, uncultured Nitrosomonadaceae, Dechloromonas, Thauera, Denitratisoma, Rhodocyclaceae (norank) and Comamonadaceae (norank). Moreover, environmental variables such as water temperature, water volume, influent ammonium nitrogen, influent chemical oxygen demand (COD) and effluent COD exhibited significant correlation to the microbial community according to the Monte Carlo permutation test analysis (p < 0.05). The abundance of Nitrospira, uncultured Nitrosomonadaceae and Denitratisoma presented strong positive correlations with the influent/effluent concentration of COD and ammonium nitrogen, while Dechloromonas, Thauera, Rhodocyclaceae (norank) and Comamonadaceae (norank) showed positive correlations with water volume and temperature. The established relationship between microbial community and environmental variables in different biologically functional zones of the six representative WWTPs at different geographical locations made the present work of potential use for evaluation of practical wastewater treatment processes.

Although efficient aerobic denitrification has received increasing attention, few studies have been made on simultaneous denitrification and phosphorus removal (SDPR) under aerobic condition. In this study, SDPR by an efficient aerobic denitrifier, Agrobacterium sp. LAD9, was firstly demonstrated. High nitrate and phosphorus removal rates of 7.50 and 1.02 mg L-1 h(-1) were achieved in wide range of O-2 concentration from 5.92 to 20.02 mg L-1. The N2O production would be inhibited as O-2 concentration exceeded 11.06 mg L-1, while the phosphorus removal efficiency would be generally improved with increasing O-2 concentration. N-15 mass spectrometry revealed that nitrogen removal accorded with the typical aerobic denitrification pathway, while P-31 nuclear magnetic resonance spectroscopy (P-31 NMR) indicated the fate of phosphorus to cells, extracellular polymeric substances (EPS), and polyphosphate (poly-P) of the denitrifier. EPS acted as a reservoir of phosphorus and the transformation of poly-P was dynamic and depended on initial orthophosphate (ortho-P) content. The aerobic SDPR would greatly simplify the conventional wastewater treatment processes which required separated considerations of nitrogen and phosphorus removal.

Microbial communities of activated sludge (AS) play a key role in the performance of wastewater treatment processes. However, seasonal variability of microbial population in varying AS-based processes has been poorly correlated with operation of full-scale wastewater treatment systems (WWTSs). In this paper, significant seasonal variability of AS microbial communities in eight WWTSs located in the city of Guangzhou were revealed in terms of 16S rRNA-based Miseq sequencing. Furthermore, variation redundancy analysis (RDA) demonstrated that the microbial community compositions closely correlated with WWTS operation parameters such as temperature, BOD, NH4+-N and TN. Consequently, support vector regression models which reasonably predicted effluent BOD, SS and TN in WWTSs were established based on microbial community compositions. This work provided an alternative tool for rapid assessment on performance of full-scale wastewater treatment plants.

PCN bacteria capable of heterotrophic-aerobic nitrogen removal was successfully applied for bioaugmented treatment of municipal wastewater in a pilot-scale SBR. At an appropriate COD/N ratio of 8, the bioaugmentation system exhibited stable and excellent carbon and nutrients removal, the averaged effluent concentrations of COD, NH4+-N, TN and TP were 20.6, 0.69, 14.1 and 0.40 mg/L, respectively, which could meet the first class requirement of the National Municipal Wastewater Discharge Standards of China (COD < 50 mg/L, TN < 15 mg/L, TP < 0.5 mg/L). Clone library and real-time PCR analysis revealed that the introduced bacteria greatly improved the structure of original microbial community and facilitated their aerobic nutrients removal capacities. The proposed emerging technology was shown to be an alternative technology to establish new wastewater treatment systems and upgrade or retrofit conventional systems from secondary-level to tertiary-level. (C) 2015 Elsevier Ltd. All rights reserved.

Much effort has been made for reducing nitrous oxide (N2O) emission in wastewater treatment processes. This paper presents an interesting way to minimize N2O in aerobic denitrification by strain Pseudomonas stutzeri PCN-1 with help of corn flour as cheaper additional carbon source. Experimental results showed that maximal N2O accumulation by strain PCN-1 was only 0.02% of removed nitrogen if corn flour was used as sole carbon source, which was significantly reduced by 52.07-99.81% comparing with others such as succinate, glucose, acetate and citrate. Sustained release of reducing sugar from starch and continuous expression of nosZ coding for N2O reductase contributed to the special role of corn flour as the ideal carbon source for strain PCN-1. Further experiments in sequencing batch reactors (SBRs) demonstrated similarly efficient nitrogen removal with much less N2O emission due to synergy of the novel strain and activated sludge, which was then confirmed by quantitative PCR analysis. (C) 2015 Elsevier Ltd. All rights reserved.

Nitrous oxide (N2O) emission from wastewater treatment plants (WWTPs) has received increasing attention. This paper presented how N2O emission was significantly reduced in a pilot-scale Carrousel oxidation ditch under reasonable nitrification and denitrification. N2O emission from the reactor was found as low as 0.027% of influent nitrogen, which was much less than that from other processes. Further measurements on spatial variation of N2O emission in the alternative aerobic/anoxic zones with help of a series of batch experiments demonstrated that about 90% of the emission was contributed by nitrifier denitrification (ND). Moreover, the taxonomic analysis based on high through-put 16S rRNA gene sequencing revealed that the high abundance of denitrifying bacteria and nitrite-oxidizing bacteria (NOB) was responsible for low nitrite accumulations and consequent low N2O emissions. However, N2O generation would be greatly increased upon the normal operation being shocked by either ammonia overload or aeration failure of the oxidation ditch system. (C) 2014 Elsevier Ltd. All rights reserved.

As two obligatory intermediates of denitrification, both NO and N2O had harmful environmental and biological impacts. An aerobic denitrifying bacterial strain PCN-1 was newly isolated and identified as Pseudomonas stutzeri, which was capable of high efficient nitrogen removal under aerobic condition with maximal NO and N2O accumulation as low as 0.003% and 0.33% of removed NO3 -N, respectively. Further experiment taking nitrite as denitrifying substrate indicated similar low NO and N2O emission of 0.006% and 0.29% of reduced NO2 -N, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the coordinate expression of denitrification gene nirS (for cytochrome cd(1) nitrite reductase), cnorB (for NO reductase) and nosZ (for N2O reductase) was the fundamental reason of low NO and N2O accumulation. Activated sludge system bioaugmented by strain PCN-1 demonstrated a significant reduction of NO and N2O emission from wastewater during aerobic denitrification, implied great potential of PCN-1 in practical applications. (C) 2014 Elsevier Ltd. All rights reserved.

The carbon source materials are important influencing factors in the progress of biological removal of nitrogen as the electron donors in denitrification. External carbon source materials are essential for the treatment of wastewater with low C/N ratio. For estimating the proper dosage of external carbon source, back-propagation (BP) neural network and radial basis function (RBF) neural network were introduced to develop a non-linear model between the dosage of external carbon source and influent conditions, using the experiment data from the cyclic activated sludge technology (CAST) on laboratory scale. Results show that both two networks prove to be effective in estimating the dosage of external carbon source; RBF neural network model turns out to be better in training speed and approximation capability, while BP neural network model shows higher prediction accuracy.碳源作为反硝化过程的电子供体,是影响生物脱氮过程的重要因素,低碳氮比污水需外加碳源以保证反硝化反应的顺利进行。为了优化控制碳源投加量,对实验室搭 建的CAST工艺污水处理装置的进水条件和外加碳源量的非线性关系分别进行了基于BP和RBF神经网络的模型研究,并对外加碳源量进行了预测。结果表明, 两种网络模型均能有效预测外加碳源量,RBF神经网络模型在训练速度和逼近能力方面优于BP神经网络模型,但在预测性能方面BP神经网络模型则有更高的预 测精度。

The metabolic pathway of heterotrophic nitrification-aerobic denitrification bacteria was directly determined by the actions of their hydroxylamine oxidase(HAO). Isolating high-purity HAO from this kind of bacteria has become particularly important to explain the mechanism of nitrogen removal. In this study, the separation and purification technic of HAO from a novel heterotrophic nitrification-aerobic denitrification strain Agrobacterium tumefaciens LAD9 was established. Electrophoretic purity of HAO could be sucessfully purified through DEAE Sepharose CL-6B ion-exchange chromatography and Sephacryl S-100 gel filtration from its periplasm. The final purification fold was 5.79 and the yield was 39.71%. SDS-PAGE eclectrophoresis results revealed that the molecular weight of HAO in the strain LAD9 was 18.8 kD. Studies on enzymatic properties showed that the purified enzyme could oxidize hadroxylamine to nitrite and its activity could be enhanced by the addition of Fe~(2+).羟氨氧化酶(Hydroxylamine oxidase,HAO)的作用方式直接决定了异养硝化好氧反硝化细菌的代谢途径,分离得到纯度较高的HAO也就成为研究这类细菌脱氮机制的重要环节。以 异养硝化好氧反硝化细菌Agrobacterium tumefaciens LAD9为代表,建立了该菌株HAO的分离纯化方法:首先采用渗透压休克法提取细胞周质液,然后采用DEAE Sepharose CL-6B离子交换层析和Sephacryl S-100凝胶过滤层析对细胞周质液进行分离纯化。结果表明,经过离子交换层析可得到分子量分别为55.3、35.7和19.2 kD的杂蛋白,进一步经过凝胶过滤层析即可得到电泳纯的HAO,纯化倍数为5.79,产率为39.71%。对其酶学性质的初步研究表明,该菌株HAO的分 子量为18.8 kD,能够将羟胺氧化为亚硝酸盐氮,且Fe~(2+)的加入可显著增强其酶活。

Nitrogen removal in wastewater treatment plants is usually severely inhibited under cold temperature. The present study proposes bioaugmentation using psychrotolerant heterotrophic nitrification-aerobic denitrification consortium to enhance nitrogen removal at low temperature. A functional consortium has been successfully enriched by stepped increase in DO concentration. Using this consortium, the specific removal rates of ammonia and nitrate at 10 degrees C reached as high as 3.1 mg N/(g SS h) and 9.6 mg N/ (g SS h), respectively. PCR-DGGE and clone library analysis both indicated a significant reduction in bacterial diversity during enrichment. Phylogenetic analysis based on nearly full-length 16S rRNA genes showed that Alphaproteobacteria. Deltaproteobacteria and particularly Bacteroidetes declined while Gammaproteobacteria (all clustered into Pseudomonas sp.) and Betaproteobacteria (mainly Rhodoferax ferrireducens) became dominant in the enriched consortium. It is likely that Pseudomonas spp. played a major role in nitrification and denitrification, while R. ferrireducens and its relatives utilized nitrate as both electron acceptor and nitrogen source. Crown Copyright (C) 2012 Published by Elsevier Ltd. All rights reserved.

A bacterium capable of phosphorus removal was isolated. Through morphology observation and 16S rRNA gene sequence analysis, the isolate was identified as Salinivibrio sp. (named HG-1). Salinity tolerance and phosphorus removal efficiency under different salinity conditions of the strain were further investigated. The results showed that HG-1 grew well with the salt content varying from 1% to 13% and achieved the highest phosphorus removal efficiency under salt content of 3%. Furthermore, the single-factor and orthogonal experiment results indicated that the optimal phosphorus removal performance was obtained under the conditions with an initial pH of 6.5-7.0, C/N ratio of 9, temperature of 30°C and inoculation ratio of 10%. Under such a condition, the phosphorus removal efficiency could reach 100% in 24 hours. The strain HG-1 can independently complete phosphorus removal process, and thus could provide a novel and promising alternative for biological phosphorus removal under high salinity conditions.筛选出一株能够高效除磷的耐盐菌株HG-1。通过个体形态、菌落特征的观察和16S rRNA基因的序列分析, 初步鉴定为盐弧菌属(Salinivibrio sp.)。对菌株HG-1的耐盐性能及其在不同盐度下对磷酸盐的去除效果进行考察, 结果表明菌株对盐度的耐受范围为1%~13%, 最适盐度为3%。进一步的单因素和正交实验表明, 4个环境因素对菌株HG-1磷酸盐去除率影响的强弱为: pH>碳氮比>温度>接种量, 最优的除磷条件为pH 6.5~7.0, 温度30℃, 接种量10%, 碳氮比9, 在该条件下菌株在24小时内对磷酸盐的去除率可达100%。 将该菌株应用于高盐废水的处理, 可实现磷酸盐的有效去除, 具有良好的实际应用价值, 为高盐条件下生物除磷难题的解决提供了一条新的途径。

Characteristics of ammonium removal by a newly isolated heterotrophic nitrification-aerobic denitrification bacterium Agrobacterium sp. LAD9 were systematically investigated. Succinate and acetate were found to be the most favorable carbon sources for LAD9. Response surface methodology (RSM) analysis demonstrated that maximum removal of ammonium occurred under the conditions with an initial pH of 8.46, C/N ratio of 8.28, temperature of 27.9 degrees C and shaking speed of 150 rpm, where temperature and shaking speed produced the largest effect. Further nitrogen balance analysis revealed that 50.1% of nitrogen was removed as gas products and 40.8% was converted to the biomass. Moreover, the occurrence of aerobic denitrification was evidenced by the utilization of nitrite and nitrate as nitrogen sources, and the successful amplifications of membrane bound nitrate reductase and cytochrome cd(1) nitrite reductase genes from strain LAD9. Thus, the nitrogen removal in strain LAD9 was speculated to comply with the mechanism of heterotrophic nitrification coupled with aerobic denitrification (NH4+-NH2OH-NO2--N2O-N-2), in which also accompanied with the mutual transformation of nitrite and nitrate. The findings can help in applying appropriate controls over operational parameters in systems involving the use of this kind of strain. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.

Three novel strains capable of heterotrophic nitrification-aerobic denitrification were isolated from the landfill leachate treatment system. Based on their phenotypic and phylogenetic characteristics, the isolates were identified as Agrobacterium sp. LAD9, Achromobacter sp. GAD3 and Comamonas sp. GAD4, respectively. Batch tests were carried out to evaluate the growth and the ammonia removal patterns. The maximum growth rates as determined from the growth curve were 0.286, 0.228, and 0.433 h(-1) for LAD9, GAD3 and GAD4, respectively. The maximum aerobic nitrification-denitrification rate was achieved by the strain GAD4 of 0.381 mmol/l h, followed by LAD9 of 0.374 mmol/l h and GAD3 of 0.346 mmol/l h. Moreover, hydroxylamine oxidase and periplasmic nitrate reductase were successfully expressed in all the isolates. The relationship between the enzyme activities and the aerobic nitrification-denitrification rates revealed that hydroxylamine oxidation may be the rate-limiting step in the heterotrophic nitrification-aerobic denitrification process. The study results are of great significance to the wastewater treatment systems where simultaneous removal of carbon and nitrogen is desired.