Denitrifyingphosphorusremoval in a step-feed CAST with alternating anoxic-oxic operational strategy

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污泥龄对AAO工艺反硝化除磷的影响

TN
38016 4816 5613 010 010 411 6410 2713
8712 100
— 5713
TP
710 018
8816
பைடு நூலகம்
MLSS
2 590
1) 数据为 3 次试验的平均值
进水 38416 5314
010 6418 714
SRT = 10 d
出水去除率Π%
6218 8317
018 9815
尽管反硝化除磷现象在 AΠAΠO 工艺并没有被认识到 ,但当研究者在其它一系列工艺系统中发现并确定该现象时 ,AΠAΠO 工艺构造实际上也是有利于 DNPAOs 的富集. 在 AΠAΠO 工艺中 ,可通过适当调整工艺参数使 DNPAOs 的富集程度达到最大 ,采取的措施有 2 个 : ①增加混合液回流比便于在缺氧段为 DNPAOs 提供足够的电子受体 NO3- 2N ; ② 调整污
结果. 由表 1 可见 ,COD 去除几乎不受 SRT 长短的影响 ,去除率达 83 %～88 % ,出水浓度基本达到我国城镇污水处理厂排放标准规定的一级标准[9] ; NH4+ 2N 去除率几乎达到 100 % ,可达到一级排放标准中 A 标准 ; TN 去除率随着 SRT 的延长呈上升趋势 ;但当 SRT 为 8 和 10 d 时 ,出水中出现 NO2- 2N 的积累 ,表明硝化反应的第 2 步受到抑制. 在 SRT 为 8 、10 和 12 d 时 ,出水 TP 浓度均低于 110 mgΠL ,达到一级排放标准中 B 标准 ;但 SRT 为 15 d 时出水磷浓度较高 ,只符合二级排放标准. 综合考虑有机物去除、脱氮和除磷效果 , 以 SRT 为 12 d 时处理效果最佳.

英文文献翻译 - 中文

倒置AAO工艺的生产性试验研究陈宏斌1，唐先春1，董斌1，高廷耀1，Martin Wagner21 中国上海同济大学污染控制和资源化研究实验室2 IWAR Institute, Technical University of Darmstadt, 64287 Germany摘要倒置缺氧/厌氧/好氧工艺（倒置AAO工艺）是上世纪90年代中期开发出来的用于脱氮除磷的污水处理工艺。

本文主要阐述了松江污水处理厂（中国）二期工程倒置AAO工艺处理城市污水的运行效果、运行参数以及影响因素。

近两年的运行结果表明：对CODCr 、BOD5、SS、NH3-N和TN具有较好的处理效率，对TN 和NH3-N的处理效率分别达到了0.022 kg TN·kgMLSS-1·d-1和 0.026 kg NH3-N·kgMLSS-1·d-1。

然而，对PO4-P和TP的去除率却并不高。

因此，我们提出了一个除磷效率更高的改进措施。

运行结果表明：倒置AAO工艺不仅适用于新建的污水处理厂的脱氮除磷，同样也适用于现有的脱氮除磷效率不高的污水处理厂改造和扩建。

关键词：硝化反硝化除磷倒置AAO工艺城市污水引言氮和磷是城市污水中导致受纳水体富营养化的主要因素。

生物硝化、反硝化和除磷工艺是经济的可行的控制排放水水质的方法。

生物脱氮除磷技术主要有：AAO工艺系列，氧化沟工艺和序批式活性污泥工艺（SBR法）。

不同菌种脱氮除磷的新陈代谢过程是需要在缺氧，厌氧和好氧条件下进行的。

在AAO工艺和氧化沟工艺中，可通过搅拌、曝气和污泥回流等方法实现上述条件。

SBR工艺是一个以时间顺序实现缺氧，厌氧，好氧的AAO工艺。

在中国，AAO工艺广泛应用于许多污水处理设施。

传统的AAO工艺有很多优点，同时也有两个缺点：1）需要两个回流系统，其总回流比不少于300％；2）从二沉池抽走的剩余污泥有部分没有完全经过厌氧，缺氧和好氧阶段，这可能会减少总磷（TP）的去除率。

电芬顿法的机理研究及其在重金属水污染处理中的应用潜力

重金属的处理中同样能够达到良好的效果。已有相关研究使用传统芬顿法对重金属污染物进行了高效
理论上相同时间内电解池内将生成相同摩尔数的 Fe2+ 和 H2 O2 ,从而使芬顿反应正常的进行。电芬顿铁氧化-H2O2 法的主要反应机理如图 4 所示。
的处理 [14-16] ,Fu 等 [17] 通过芬顿 -化学沉淀法,在投加量为 2 g / L Fe0 ,35 mmol / L H2 O2 ,初始 pH 2. 5,沉淀 pH 11 的情况下,Ni 去除率达到 98. 4% 以上。 Wang 等[18] 采用芬顿氧化 - 铁氧体循环工艺, 在 Fe2+
图 1 电芬顿 -铁还原法反应机理 [13]
1. 2 电芬顿-铁氧化法该方法在一个通过盐桥分隔的双极反应器中进
行,H2 O2 由外部投加提供,Fe2+ 通过铁电极在阳极氧化产生,在这个方法中,溶液可以保持一个较低的 PH 值,减少了氢氧化铁沉淀的生成量, 但又存在着 Fe ( OH) 2 , Fe( OH) 3 的絮凝作用,从而对污染物起到强络合吸附。电芬顿 -铁氧化法主要反应机理如图 2 所示。
环境工程
2020 年第 38 卷增刊
5
电芬顿法的机理研究及其在重金属水污染处理中的应用潜力
柴友正1 秦普丰2∗ 吴志斌3 高骞3 李天佑4
( 1. 湖南省灌溉水源水质污染净化工程技术研究中心,长沙 410128;2. 湖南农业大学资源环境学院,长沙 410128)
0 引言
有吸引力的技术,因为该方法没有毒性,没有残留并
近年来,随着对废水污染的日益重视,各类处理且操作简单[6] 。芬顿法的主要步骤是氧化、中和、絮

污水处理化工毕业设计论文中英文外文资料文献翻译[管理资料]

附件1：外文资料翻译译文城市污水常温处理中的新型改良EGSB（膨胀颗粒污泥床）反应器的发展近年来，厌氧处理技术已经成为一项有吸引力的可持续发展的污水处理技术，因为它耗能少而且产气量少。

特别的，流式厌氧污泥床(UASB)和常规膨胀颗粒污泥床(EGSB)在城市污水处理中得到了广泛应运。

通常，EGSB比UASB 更能有效去除化学需氧量（COD），更能有效抵抗有机负荷率(OLR)、温度和pH 的变化。

然而，由于较高的上升流速和较多的甲烷气泡，使膨胀颗粒污泥床（EGSB）中的三相分离器中的水的流速很高，这就导致了大量生物质的流失,最终废水中的COD浓度就升高了。

所以，有时候不能满足城市污水处理厂或生物处理系统排放的标准，并导致生物处理系统崩溃。

因此,对与EGSB系统来说，城市污水处理中的关键问题是如何控制在高上升流速下的生物量流失。

在本文中,提出一种改进型的EGSB反应器模型，它结合了EGSB 和UASB 两者的优势。

在相同环境下通过比较，试验性地研究EGSB m和EGSB c两种反应器。

在东区污水处理厂中有一个初级出水沉降池。

在对膨胀颗粒污泥床（EGSB m）中水动力特征分析时，进行了停留时间分布(RTD)的实验和Polvmerase连锁反应实验，并且应用变性梯度凝胶电泳(PCR-DGGE)技术来探索颗粒污泥中微生物的多样性。

常温厌氧颗粒污泥取自中国无锡市的一家污水处理厂，该厂主要利用全比例内循环生物反应器处理酸性废水。

黑色的颗粒污泥有规则的形状（φ= - 2毫米）和良好的沉降性能。

污泥中含有悬浮固体（TSS）（VSS）59克/升。

在EGSB m 和EGSB c两种反应器中，最初的接种污泥量占有效总量的65%。

污水样本取自上海东区城市污水处理厂的一个初级沉淀池中。

其中包括60%生活污水和40%的工业废水。

污水的主要指标如表1。

表1 污水的主要指标工业生产中EGSBm和EGSBc反应器的原理图如图1。

两个反应器都是有机玻璃制成的,容量为300 升,采用连续流动模式。

厌氧氨氧化

Journal of Hazardous Materials 250–251 (2013) 431–438Contents lists available at SciVerse ScienceDirectJournal of HazardousMaterialsj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /j h a z m atDenitriﬁcation performance and microbial diversity in a packed-bed bioreactor using biodegradable polymer as carbon source and bioﬁlm supportZhiqiang Shen a ,b ,Yuexi Zhou a ,Jun Hu b ,Jianlong Wang b ,c ,∗aChinese Research Academy of Environmental Sciences,Beijing 100012,PR ChinabLaboratory of Environmental Technology,INET,Tsinghua University,Beijing 100084,PR China cBeijing Key Laboratory of Fine Ceramics,Tsinghua University,Beijing 100084,PR Chinah i g h l i g h t sStarch/PCL (SPCL)blend was prepared and used for biological denitriﬁcation.The microbial community of attached bioﬁlm was analyzsed by metagenomic method. The vast majority of species bioﬁlm (99.71%)belonged to six major phyla. Proteobacteria were the most abundant phylum (85.50%).Diaphorobacter and Acidovorax was 52.75%of identiﬁed denitrifying bacteria.a r t i c l ei n f oArticle history:Received 15October 2012Received in revised form 10February 2013Accepted 13February 2013Available online 20 February 2013Keywords:Nitrate BioﬁlmMicrobial community DenitriﬁcationSolid carbon sourcea b s t r a c tA novel kind of biodegradable polymer,i.e.,starch/polycaprolactone (SPCL)was prepared and used as carbon source and bioﬁlm support for biological denitriﬁcation in a packed-bed bioreactor.The deni-triﬁcation performances and microbial diversity of bioﬁlm under different operating conditions were investigated.The results showed that the average denitriﬁcation rate was 0.64±0.06kg N/(m 3d),and NH 3–N formation (below 1mg/L)was observed during denitriﬁcation.The nitrate removal efﬁciency at 15◦C was only 55.06%of that at 25◦C.An initial excess release of DOC could be caused by rapid biodegra-dation of starch in the surfaces of SPCL granules,then it decreased to 10.08mg/L.The vast majority of species on SPCL bioﬁlm sample (99.71%)belonged to six major phyla:Proteobacteria,Bacteroidetes,Chloroﬂexi,Firmicutes,Spirochaetes and Actinobacteria.Proteobacteria were the most abundant phylum (85.50%)and mainly consisted of ␤-proteobacteria (82.39%).Diaphorobacter and Acidovorax constituted 52.75%of the identiﬁed genera which were denitrifying bacteria.© 2013 Elsevier B.V. All rights reserved.1.IntroductionThe “solid-phase denitriﬁcation”is a new type of heterotrophic biological denitriﬁcation in which insoluble biodegradable poly-mers were used as bioﬁlm carrier and carbon source simulta-neously [1,2].Solid substrates were used as alternatives to liquid carbon sources,which are accessible only by microbial enzymatic attack,so it can avoid the risk of overdosing in liquid carbon sources supported denitriﬁcation system [1].In the past few years,solid substrates have been investigated as a carbon source in the biolog-ical denitriﬁcation of drinking water [3,4],groundwater [5],landﬁll∗Corresponding author at:Laboratory of Environmental Technology,INET,Tsinghua University,Beijing 100084,China.Tel.:+861062784843;fax:+861062771150.E-mail addresses:wangjl@ ,wangjl@ (J.Wang).leachate [6,7]and recirculated aquaculture system [1,8].Water body is susceptible to the over use of fertilizers and pesticides in agriculture ﬁelds,then the simultaneous removal of nitrate and pesticides has been studied using solid substrates as carbon sources and bioﬁlm carrier [9–11].Furthermore,some researchers have also studied the feasibility of using biodegradable compounds as carbon sources and bioﬁlm carrier for nitrogen removal in simul-taneous nitriﬁcation and denitriﬁcation (SND)system [12–14].There are two kinds of solid carbon sources which have been investigated for denitriﬁcation,synthetic polymers and natural materials especially the cellulose-rich monly,syn-thetic polymers are thermoplastic which are easy processed to various shapes to ﬁt the demand of different denitriﬁcation process.In the “solid-phase denitriﬁcation”system,synthetic polymers including polyhydroxyalkanoates (PHAs)[2]and polycaprolactone (PCL)[1,8,15]were prepared to granules for denitriﬁcation,mean-while,PCL also can be prepared to plates for use [16].Comparing with the expensive synthetic polymers,natural materials including0304-3894/$–see front matter © 2013 Elsevier B.V. All rights reserved./10.1016/j.jhazmat.2013.02.026432Z.Shen et al./Journal of Hazardous Materials250–251 (2013) 431–438 wheat straw[10,17],cotton[18],waste newspaper[19],pine bark[6],crab-shell chitin[5]were much cheaper but may bring ammo-nia[5],high DOC and color problems in efﬂuent[3].Therefore,thekey issue of“solid-phase denitriﬁcation”is to develop new solidsubstrates with low denitriﬁcation cost and without deteriorationof efﬂuent water quality.Blending with some cheap organic materials is a mostpotential method to lower the price of products especiallyin biodegradable plastics productionﬁparing withother biodegradable thermoplastic polymers,starch is an abun-dant renewable polysaccharide with better biodegradability andlow cost.Aliphatic polyesters are biodegradable thermoplasticpolymers with good processability,thermal stability,excellentmechanical properties,good water resistance,and dimensional sta-bility[20].So it is a potential way to blend starch with aliphaticpolyesters for production of biodegradable plastics.Aliphaticpolyesters such as PCL[21–23],poly(butylene succinate)(PBS)[24],poly(hydroxybutyrate-covalerate)(PHBV)[25]and poly(lacticacid)(PLA)[26,27]were widely adopted to blend with starch forbiodegradable plastics production or medical application.Recently,we investigated the feasibility of using cross-linkedstarch/polycaprolactone blends as solid carbon source and bioﬁlmcarrier for denitriﬁcation[28].The main objective of this study was:(1)to evaluate the denitriﬁcation ability of starch/polycaprolactone(SPCL)blends serving both as carbon source and bioﬁlm carrier;(2)to assess the effect of operating conditions(i.e.nitrate loading rates,temperature and initial pH)on denitriﬁcation under continuous-mode;(3)to analyze the microbial community structure of bioﬁlmattached on SPCL.2.Materials and methods2.1.MaterialsThe polycaprolactone(PCL)used in this study has a molecularweight of60,000g/mol(Dalton).The cornstarch used in this studyis technical grade.Starch/PCL(SPCL)blends were prepared by twin-screw extruder.The main characters of SPCL are listed as follows:Starch,55.44%;PCL,30.00%;Additives(plasticizer and couplingagent),14.56%;Calculated surface area,1833.33m2/m3.2.2.Experiment apparatusContinuous experiments were carried out in a laboratory-scalepacked-bed set-up(Fig.1)using50mm inner diameter by500mmheight cylindrical Plexiglas.A Plexiglas mesh disc(48mm diam-eter,2mm pore size)was placed at the lower end of the columnas support for the packing material.SPCL granules were usedasFig.1.Schematic display of the experimental set-up.carbon source and carrier for bioﬁlm growth,and packing the col-umn up to a height of250mm(273g).2.3.Experiment proceduresSPCL granules were used as bioﬁlm carrier and electron donor to support biological denitriﬁcation.Synthetic water(adding NaNO3 and KH2PO4to tap water,and N:P(w/w)=5:1)seeded with acti-vated sludge which collected from a local municipal wastewater treatment plant(with theﬁnal concentration of800mg/L MLSS) was pumped into the bottom of column at aﬂow rate of4.1mL/min (HRT=2h).Unless otherwise indicated,temperature was25±1◦C, and pH and DO were not controlled.After a stable denitriﬁcation performance obtained,ﬂow rate was then increased stepwise to study the effect of nitrate loading rates(lasted184d).Then,deni-triﬁcation performance at low temperature(15±1◦C)was studied (lasted20d).The effect of pH on denitriﬁcation was investigated using1M hydrochloric acid and sodium hydroxide to control the pH of synthetic water to4.5,6,9and10.5(lasted74d).Samples were taken from the efﬂuent to monitor NO3–N,NO2–N,NH3–N, pH and DOC.2.4.Analytical methodsSamples were taken andﬁltered through0.45␮m membrane before analysis.NO3–N was determined by UV-spectrophotometer (Shimadzu UV-3100)at220nm and275nm,and NO2–N and NH3–N were assayed by hydrochloric acid naphthyl ethylenedi-amine spectrophotometry method and hypochlorite-salicylic acid spectrophotometry method,respectively[29].Dissolved organic carbon(DOC)was detected using a TOC analyzer(HACH,IL530TOC-TN).Samples were neither acidiﬁed nor sparged during analysis. The pH value was measured with pH meter.The morphology of the samples was examined using a SEM(Fei Quanta200).Bioﬁlm was taken from the column reactor after being operated for280d.DNA from bioﬁlm was extracted directly using glass beads to mechanical lysis of SPCL granulars(with bioﬁlm).The volumetric denitriﬁcation rate R vd in kg/(m3d) NO3–N+NO2–N(total soluble oxidized nitrogen species)of the reactor is given by the Eq.(1):R vd=0.024×Q D×(C in−C ef)V(1) where C in is the inﬂuent NO3–N+NO2–N concentrations(mg/L)and C ef is the efﬂuent NO3–N+NO2–N concentrations.Q D is theﬂow rate(L/h)and V is the reactor volume(L).Nitrate removal efﬁciency(N re)is deﬁned by the equation(2): N re=100×(NO3−N in−NO3−N ef−NO2−N ef−NH3−N ef)NO3−N in(2) where NO3–N in is the inﬂuent NO3–N concentration,NO3–N ef, NO2–N ef and NH3–N ef are the efﬂuent NO3–N,NO2–N and NH3–N concentrations,respectively(negligible accumulation of gaseous-N by-products and organic-N).2.5.Pyrosequencing2.5.1.DNA extraction and puriﬁcationGenomic DNA was extracted using E.Z.N.A.Soil DNA Kit (OMEGA).2.5.2.PCR ampliﬁcationFor each sample,we ampliﬁed V1-V3region of bacterial16S rRNA genes using a broadly conserved primer set(27F and533R). The forward primer(5 -GCC TTG CCA GCC CGC TCA GAG AGT TTGZ.Shen et al./Journal of Hazardous Materials250–251 (2013) 431–438433ATC CTG GCT CAG-3 )contained the454Life Sciences primer B sequence and the broadly conserved bacterial primer27F.The reverse primer(5 -GCC TCC CTC GCG CCA TCA GNN NNN NNN NNT TAC CGC GGC TGC TGG CAC-3 )contained the454Life Sci-ences primer A sequence,a unique10-nt barcode used to tag each PCR product(designated by NNNNNNNNNN),and the broad-range bacterial primer533R.PCR reactions were carried out in tripli-cate20-␮L reactions with0.4␮M forward and reverse primers, 1-␮L template DNA,250nM dNTP and1×FastPfu Buffer.All dilu-tions were carried out using certiﬁed DNA-free PCR water.Thermal cycling consisted of initial denaturation at95◦C for2min followed by25cycles of denaturation at95◦C for30s,annealing at55◦C for 30s,and extension at72◦C for30s,with aﬁnal extension of5min at72◦C.Replicate amplicons were pooled and visualized on2.0% agarose gels using SYBR Safe DNA gel stain in0.5×TBE.Amplicons were puriﬁed using AxyPrep TM DNA Gel Extraction Kit(AXYGEN) according to the manufacturer’s instructions.2.5.3.Amplicon,quantitation,pooling and pyrosequencingAmplicon DNA concentrations were measured using the Quant-iT PicoGreen dsDNA reagent and kit(Invitrogen).DNA samples were diluted in30␮L1X TE,an equal volume2X PicoGreen working solution was added in a total reaction volume of60␮L in minicell cuvette.Fluorescence was measured on a Turner Biosystems TBS-380Fluorometer using the465-485/515-575-nm excitation/emissionﬁlter pair.Following quantitation,cleaned amplicons were combined in equimolar ratios into a single tube.Pyrosequencing was carried out on a454Life Sciences Genome Sequencer FLX Titanium instrument(Roche)by Shanghai Majorbio Bio-pharm Biotechnology Co.Ltd.(Shanghai,China),the sequenc-ing data was analyzed by Mothur[30].3.Results and discussion3.1.Effect of nitrate loading rates on denitriﬁcationThe effect of nitrate loading rates on denitriﬁcation was studied varying the nitrate loading rates between0.60and1.20kg/(m3d) through changing the inﬂuent nitrate concentration and theﬂow rate(Figs.2and3,Table1).As the formation of bioﬁlm on the surface of SPCL,nitrate removal efﬁciency increased gradually and reached98.42%at the7th d,and then it became stable during the period of days8–25(Fig.2).The lag time(=period of adaptation of denitrifying microorganisms)of PCL was16days in recirculated aquaculture systems[1].The lag time of SPCL was only7d,indi-cating that PCL blending with starch can signiﬁcantly shorten the lag time in solid denitriﬁcation system.When the nitrate loading rate increased from0.60to1.20kg/(m3d)(at the25th d),the efﬂu-ent nitrate and nitrite concentrations increased signiﬁcantly,and a very sharp decline of nitrate removal rate was observed simul-taneously(phase2).These changes may be due to the high nitrate loading rates which exceeded the denitriﬁcation capability of the system.Keeping a constantﬂow rate(8.2mL/min)and decreasing inﬂuent nitrate concentration to25mg/L at the60th d,the efﬂuent nitrate and nitrite concentrations decreased sharply and ca.90% nitrate removal rate reached(phase3).Further increasing nitrate loading rate to0.72kg/(m3d)at the100th d,a littleﬂuctuationofFig.2.Denitriﬁcation performances of SPCL at different nitrate loading rates. denitriﬁcation performance was observed(phase4).Nitrite is an intermediate of nitrate reduction.In this study,nitrite concentra-tion was below1mg/L when high nitrate removal rate reached.The formation of NH3–N(below ca.1mg/L)was observed,possibly due to the dissimilatory nitrate reduction to ammonia(DNRA)process, which was also observed in anaerobic sediments by Kelso et al.[31]. Honda and Osawa[16]also found that a0.1mg/L NH3–N increased in denitriﬁcation system using PCL as substrate.Table1The volumetric denitriﬁcation rate R vd at different nitrate loading rates.Phase Operating date(d)Flow rate(mL/min)Inﬂuent NO3–N(mg/L)HRT(h)NO3–N loading rate(kg/(m3d))R vd(kg/(m3d)) 10–25 4.15020.600.53±0.09a226–598.2501 1.200.54±0.22360–1008.22510.600.59±0.03 4101–18416.4150.50.720.64±0.06a Mean±standard deviation.434Z.Shen et al./Journal of Hazardous Materials 250–251 (2013) 431–4380.60.8 1.0 1.20.60.81.01.2N i t r a t e r e m o v e d [k g /(m 3·d )]Nitrate load [kg/(m 3·d)]Fig.3.Average nitrate removal rates under different loading rates.The variations of DOC and pH in efﬂuent are also depicted inFig.2.During the start-up period,a quick release of DOC occurred,and reached maximum 61.30mg/L at the 14th d.The high DOC might allow rapid microbial growth and the fast colonization on the substrate therefore high removal of nitrate was observed.DOC will accumulate when the amount of released dissolved organic carbon exceeded the need of microbes for both growth and denitriﬁcation.During the start-up period,once microbes stick and proliferate to SPCL granules,they secrete enzymes to biodegrade starch or PCL and use them as carbon source.Since starch is more biosuscep-tible than PCL in starch/PCL blends [21,32],so the accumulated of DOC may be mainly derived from the biodegradation of starch (on the surface of SPCL granules)during the start-up period.HRT may be an important factor affecting the release and accumulation of DOC.At the 24th d,the ﬂow rate was changed to 8.2mL/min (HRT changed from 2h to 1h),the average DOC in the efﬂuent decreased to 19.60mg/L (phases 2and 3).Further increasing the ﬂow rate to 16.4mL/min (HRT =0.5h),the average DOC decreased to 10.08mg/L (phase 4).The previous work also found that HRT has an important inﬂuence on DOC accumulation using cross-linked starch/PCL blends as solid carbon and bioﬁlm carrier for denitriﬁ-cation [28].Aslan and Türkman [3]also found that DOC decreased with increasing ﬂow rate in ﬁxed-bed denitriﬁcation system using wheat straw as substrate.Shear force has signiﬁcant inﬂuences on the structure of the bioﬁlm and mass transfer.A higher shear force may result in a thin-ner and denser bioﬁlm [33],but it has a dual effect on the behaviors of mass transfer in bioﬁlm,i.e.high turbulence would facilitate sub-strate diffusion in bioﬁlms;however,shearforce-enhanced bioﬁlm density in turn reduces the diffusivity of substrate in bioﬁlms.The observed diffusivity of substrate would be a net result of these two phenomena [34].Celmer et al.[35]found that high shear force proved to be effective in improving denitriﬁcation rate by reducing the thickness of the bioﬁparing phases 1with 3(Table 1),under a constant nitrate loading rate (0.60kg/(m 3d))the average denitriﬁcation rate improved since ﬂow rate increased from 4.1to 8.2mL/min.Thus,the need of carbon source for biological denitri-ﬁcation increased,which might be a main result of the decrease of DOC.DOC values did not exceed 5–7mg/L when using PCL as solid carbon source in recirculated aquaculture systems [1].In this study,DOC was higher than net PCL supported denitriﬁcation sys-tem,which probably due to the addition of starch and a high starch content (starch:PCL (w/w)=1.848)in the blends,meanwhile the difference of PCL may be another reason though it maybe play a less important role in the high DOC problem.The values of pH decreased slightly from a range of 6.89–7.87(inﬂuent)to a range of 6.47–7.48(efﬂuent)in the period of days 26–184.The decrease of pH values probably be due to organic acids produced from carbon source by microbial metabolism,and neu-tralization alkalinity represented by denitriﬁers [15].When PCL immersed in enzyme lipase solution,the quantity of acid liberated was coincided with its biodegradability [36].After immersed into lysozyme solution,the pH of media containing pure PCL scaffolds was lower than the initial pH due to the acidic degradation products of the PCL component [37].Increasing nitrate loading rate properly,the average denitriﬁca-tion rate increased (phases 3and 4)while over-loading of nitrate (phase 2)could not improve the denitriﬁcation performance (Fig.3and Table 1).Under the same nitrate loading rate,increasing the ﬂow rate led to a higher average denitriﬁcation rate (phases 1and 3).3.2.Denitriﬁcation performance at low temperatureAt the 185th d,the temperature was decreased to 15◦C (except temperature,other operating parameters equaled to phase 4)to study the denitriﬁcation performance at low pared to phase 4at 25◦C (Fig.2),the efﬂuent average nitrate concentrations increased signiﬁcantly and reached 7.03±0.36mg/L,nitrate removal efﬁciency was 47.50%.Though nitrate removal was inhibited at low temperature,nitrite accu-mulation was low (below 0.6mg/L).The average denitriﬁcation rate was 0.34±0.01kg/(m 3d),indicating that temperature was an important parameter for denitriﬁcation performance.A very sharp decline of denitriﬁcation rate was also observed in wheat straw or cotton supported denitriﬁcation system when tempera-ture decreased [9,18].As denitriﬁcation rate decreased,the demand of carbon source for biological denitriﬁcation decreased.Meanwhile,the amount of released dissolved organic carbon should be reduced since the enzymatic degradation process of starch/PCL blends affected by temperature.PCL showed a slight biodegradability under aquatic conditions at the mesophilic temperature [38],but the biodegrad-ability of PCL was 92%in the diluted sludge at the thermophilic temperature [39].At 15◦C,average DOC was 4.59mg/L,which was only 45.54%of the value in phase 4at 25◦C.pH values slightly decreased from a range of 6.90–7.57(inﬂuent)to a range of 6.72–7.05(efﬂuent).3.3.Effect of pH on denitriﬁcation performanceThe denitriﬁcation performance at different initial pH (4.5–10.5)was studied (except initial pH,other operating parameters equaled to phase 4).Compared with uncontrolled initial pH condition (phase 4,Fig.2),increase of efﬂuent nitrate and decrease of nitrate removal rate were observed at both acid and basic condition,indi-cating that when pH was beyond the optimal range,denitriﬁcation enzymatic activity was inhibited.It was similar to the optimal pH range reported for denitriﬁcation [40,41].At acidic pH (4.5and 6),the average denitriﬁcation rates at pH of 4.5and 6were 0.46±0.05and 0.51±0.04kg/(m 3d),respectively.These values were signiﬁcantly lower than the value in phase 4(average pH ca.7.23),which was 0.64±0.06kg/(m 3d)(Table 1).In addition,the efﬂuent nitrite and DOC concentrations at pH of 4.5were higher than the values at pH of 6.It was interesting to note that the average denitriﬁcation rate increased from 0.35±0.03to 0.52±0.02kg/(m 3d)when pH increased from 9to 10.5,which might be due to the higher DOC release and accumulation at pH of 10.5than 9.At basic condition,more organic acid products were produced to neutralize the alkalinity,which conﬁrmed by pH change.These excess release of dissolved organic carbon should beZ.Shen et al./Journal of Hazardous Materials250–251 (2013) 431–438435Fig.4.Photograph of fresh SPCL(a);SEM images of bioﬁlm attached on SPCL(b);fresh SPCL(c);and used SPCL(d).exceeded the need of microbes for denitriﬁcation,so a higher DOC accumulation was observed at basic condition than acidic or neu-tral condition.Efﬂuent pH tended to be neutral both at acidic and basic initial conditions,which would be a net result of acidity by acidic degradation products and alkalinity derived from denitriﬁ-cation.NH3–N was produced over the pH range of4.5–10.5,but the concentration was below1.00mg/L.3.4.SEM observationSPCL carriers used in this study were cylindrical granules (Fig.4a),and bioﬁlm attached on their surfaces comprised predo-minately of rod bacteria from SEM observation(Fig.4b).The fresh SPCL displayed an irregular surface(Fig.4c),which will favor the attachment of bacterial cells on the surfaces.Generally,without deformed starch granules is homogeneously dispersed throughout the PCL/starch blends as droplet-like particles[22].The starch parti-cles present in SPCL showed a thermoplastic nature,indicating that it underwent signiﬁcant paring the SEM images of the fresh SPCL with the used one,the later one(Fig.4d)showed that its surface covered with pits and pores,the result of biodegradation was visible.3.5.Microbial community of bioﬁlmIn SPCL bioﬁlm sample,most abundant sequences were assigned to the node of bacteria or to its descendants,and a few sequences of eukaryotic organisms were also received. In bacteria,the vast majority of sequences(99.71%)belonged to one of the six major phyla:Proteobacteria,Bacteroidetes,Chloroﬂexi,Firmicutes,Spirochaetes and Actinobacteria(Fig.5), and Proteobacteria was the most abundant phylum(85.50%) which was mainly␤-proteobacteria(82.39%).␤-proteobacteria were reported to be abundant in activated sludge of denitrifying reactors[42].ProteobacteriaBacteroidetesChloroflexiFirm icutesSpirochaetesActinobacteria 0153045607590Relativeabundance(%)Fig.5.Relative abundance of the main phyla identiﬁed on SPCL bioﬁlm sample.Only phyla with a relative abundance greater than1%were shown.These six predomi-nant phyla together account for>99.71%of sequences identiﬁed.Total numbers of sequences was N=9623.436Z.Shen et al./Journal of Hazardous Materials 250–251 (2013) 431–438Di a p ho ro b a c t e r A c i d o v o r a x De c h l o r o m o n a s A l i c y c l i p h i l u s R o s e if l e x u s P r e v o t e l l ac e a e u n c u l t u r ed T re p o n e m a C l o a c i b a c t e r i u m P e c t i n a t u s S t e n o t r o p h o m o n a s C e l l u l o m o n a s D e s u lf o v i b r i o A z o s p i r a F l a v o b a c t e r i u m A n a e r o a r c u s 05101520253035R e l a t i v e a b u n d a n c e (%)Fig.6.Relative abundance of the main genera identiﬁed on SPCL bioﬁlm sample.Only genera with a relative abundance greater than 1%are shown.These 15pre-dominant genera together account for >93.79%of genera identiﬁed.At the genus level,sequences from SPCL represented 58differ-ent genera,but 53.42%of the sequences were not related to the known bacteria.Diaphorobacter and Acidovorax constituted 52.75%of the identiﬁed genera in SPCL bioﬁlm sample (Fig.6).Diaphorobac-ter was reported to be denitrifying bacteria [43].Both nitrate and nitrite reductase activities were presented in eight strains of Diaphorobacter isolated [44].However,the nitrate reduction rate was 1.5times more than the nitrite reduction in Diaphorobac-ter sp.,but a nitrite accumulation was also received especially at high nitrate concentration,suggesting that Diaphorobacter possi-bly transfers electron sequentially in the denitriﬁcation system from nitrate to dinitrogen formation [45].Acidovorax species are commonly observed in wastewater treatment reactors and have been shown to be able to metabolize several different carbon sources [46].For example,Acidovorax avenae subsp.avenae LMG 17238can be successfully used ethanol,methanol,sodium acetate,glucose and poly(␧-caprolactone)as carbon source for denitriﬁca-tion [47].Coates et al.[48]reported two Dechloromonas strains,RCB and JJ that can completely mineralize various mono-aromatic compounds including benzene to CO 2in the absence of O 2with nitrate as the electron acceptor.Hong et al.[49]reported that Alicycliphilus was one of the abundant genus in the denitrifying bioreactor.Alicycliphilus denitriﬁcans was the main denitriﬁer that could use nitrate,nitrite,and oxygen as electron acceptors as reported by Mechichi et al.[50].Stenotrophomonas was isolated directly from a continuous up-ﬂow ﬁxed-bed denitriﬁcation reac-tor using poly(3-hydroxybutyrate-co-3-hydroxyvalerate)(PHBV)granules as bioﬁlm carrier,carbon source and electron donor [51].Flavobacterium sp.S6was reported as amylolytic bacterial [52].Most species of Desulfovibrio can oxidize organic compounds such as volatile fatty acids (VFA)and lactate incompletely to acetate [53],and Cellulomonas as cellulose-hydrolysing bacterial was found in sludge from a methanogenic reactor treating paper mill wastewater [54].Rarefaction analysis was used to estimate the richness of total bacterial communities of SPCL bioﬁlm.The steepness curves were received (Fig.7),suggesting that the sampling completeness is low and a large fraction of the species diversity has not yet beensampled.Fig.7.Rarefaction curves of SPCL bioﬁlm sample.The number of OTUs with different cutoff values was plotted as a function of the number of sequences sampled.The 0.03,0.05and 0.1curves contain OTUs with differences that do not exceed 3%,5%and 10%,respectively.3.6.Mass balance of carbon and nitrogen for denitriﬁcationAfter operation for 280d,nitrogen and carbon mass balances were calculated according to the carbon consumption and the removed nitrogen,the results are given in Table 2.For carbon bal-ance,the input and residual mass of SPCL in column,the carbon content of SPCL (42.65%)and the loss of C from efﬂuent DOC were used to calculate the total carbon utilized by microorganism.For nitrogen balance,inﬂuent nitrate was total input N,while efﬂuent nitrate,nitrite and ammonium concentration were integrated to calculate total output N,thus the total removed N was obtained by total input N subtracting total output N.The total output N mass was 31.14%of total input,and it mainly due to the over-loading nitrate in phase 2(Fig.2).A large amount loss of C was observed (from efﬂuent DOC),probably mainly due to the high release and accumulation of dissolved organic carbon at start-up period.According to the carbon consumed by microorgan-isms,the removal of 1g nitrogen required 1.01g carbon (utilized by microorganism),which is equivalent of 2.36g SPCL.However,due to the release of organic compounds in efﬂuent,the required mass of SPCL increased to 4.72g/g N.The consumption PCL for removing 1kg nitrate-N was calculated to be 1.33–1.77kg [1].Table 2Mass balance of nitrogen and carbon for denitriﬁcation.ItemMass (g)Nitrogen In N from inﬂuent nitrate 104.66OutN from efﬂuent nitrate 26.42N from efﬂuent nitrite4.01N from efﬂuent ammonium 2.16TotalTotal N input 104.66Total N output 32.59Total N removed72.08Carbon In C from SPCL202.96OutC measured from DOC 72.46Residual Residual C in column 57.80TotalTotal C utilized72.69。

高级氧化

电化学氧化法
电化学氧化法主要是通过电极作用产生·O2-、 H2O2、·OH等活性基团来氧化降解有机物。
优点是处理效率高、操作简便易于控制、条件温和，不需要另加催化剂；有析氧、析氢副反应、能耗大缺点。
The total organic carbon measurements indicate an efficie 94% respectively for photo-Fenton and electro-Fenton processes after 480 min of treatment. Calculating the cost has shown that the electro-Fenton process is much more interesting than the photo-Fenton process. （Aida Kesraoui Abdessalem，2010）
Fenton法存在问题 Fenton法存在问题
所用试剂量大，处理废水时间较长；反应要求在较低pH范围进行； Fe2+加入可能会增大废水中COD含量而造成二次污染； H202价格昂贵，单独使用成本太高；同时也是自由基淬灭剂，大量存在也可成为已产生的OH自由基的抑制剂，降低Fenton氧化的动力学速率。
Ayouba,2011)
The optimum dose of Fenton reagent (Fe2+/H2O2) was 10 mg/L Fe2+ and 60 mg/L H2O2 for an initial MeS concentration range of 0–80 mg/L. The Fenton process was effective under pH 3. The degradation of MeS at optimum dose of Fenton reagent was more than 95% for MeS of 0–40 mg/L and the degradation time was less than 30 min. The degradation kinetics of MeS was modelled by

微生物英文文献及翻译—原文

Dynamic and distribution of ammonia-oxidizing bacteria communities during sludge granulation in an anaerobic e aerobic sequencing batch reactorZhang Bin a ,b ,Chen Zhe a ,b ,Qiu Zhigang a ,b ,Jin Min a ,b ,Chen Zhiqiang a ,b ,Chen Zhaoli a ,b ,Li Junwen a ,b ,Wang Xuan c ,*,Wang Jingfeng a ,b ,**aInstitute of Hygiene and Environmental Medicine,Academy of Military Medical Sciences,Tianjin 300050,PR China bTianjin Key Laboratory of Risk Assessment and Control for Environment and Food Safety,Tianjin 300050,PR China cTianjin Key Laboratory of Hollow Fiber Membrane Material and Membrane Process,Institute of Biological and Chemical Engineering,Tianjin Polytechnical University,Tianjin 300160,PR Chinaa r t i c l e i n f oArticle history:Received 30June 2011Received in revised form 10September 2011Accepted 10September 2011Available online xxx Keywords:Ammonia-oxidizing bacteria Granular sludgeCommunity development Granule sizeNitrifying bacteria distribution Phylogenetic diversitya b s t r a c tThe structure dynamic of ammonia-oxidizing bacteria (AOB)community and the distribution of AOB and nitrite-oxidizing bacteria (NOB)in granular sludge from an anaerobic e aerobic sequencing batch reactor (SBR)were investigated.A combination of process studies,molecular biotechniques and microscale techniques were employed to identify and characterize these organisms.The AOB community structure in granules was substantially different from that of the initial pattern of the inoculants sludge.Along with granules formation,the AOB diversity declined due to the selection pressure imposed by process conditions.Denaturing gradient gel electrophoresis (DGGE)and sequencing results demonstrated that most of Nitrosomonas in the inoculating sludge were remained because of their ability to rapidly adapt to the settling e washing out action.Furthermore,DGGE analysis revealed that larger granules beneﬁt more AOB species surviving in the reactor.In the SBR were various size granules coexisted,granule diameter affected the distribution range of AOB and NOB.Small and medium granules (d <0.6mm)cannot restrict oxygen mass transfer in all spaces of the rger granules (d >0.9mm)can result in smaller aerobic volume fraction and inhibition of NOB growth.All these observations provide support to future studies on the mechanisms responsible for the AOB in granules systems.ª2011Elsevier Ltd.All rights reserved.1.IntroductionAt sufﬁciently high levels,ammonia in aquatic environments can be toxic to aquatic life and can contribute to eutrophica-tion.Accordingly,biodegradation and elimination of ammonia in wastewater are the primary functions of thewastewater treatment process.Nitriﬁcation,the conversion of ammonia to nitrate via nitrite,is an important way to remove ammonia nitrogen.It is a two-step process catalyzed by ammonia-oxidizing and nitrite-oxidizing bacteria (AOB and NOB).Aerobic ammonia-oxidation is often the ﬁrst,rate-limiting step of nitriﬁcation;however,it is essential for the*Corresponding author .**Corresponding author.Institute of Hygiene and Environmental Medicine,Academy of Military Medical Sciences,Tianjin 300050,PR China.Tel.:+862284655498;fax:+862223328809.E-mail addresses:wangxuan0116@ (W.Xuan),jingfengwang@ (W.Jingfeng).Available online atjournal homepage:/locate/watresw a t e r r e s e a r c h x x x (2011)1e 100043-1354/$e see front matter ª2011Elsevier Ltd.All rights reserved.doi:10.1016/j.watres.2011.09.026removal of ammonia from the wastewater(Prosser and Nicol, 2008).Comparative analyses of16S rRNA sequences have revealed that most AOB in activated sludge are phylogeneti-cally closely related to the clade of b-Proteobacteria (Kowalchuk and Stephen,2001).However,a number of studies have suggested that there are physiological and ecological differences between different AOB genera and lineages,and that environmental factors such as process parameter,dis-solved oxygen,salinity,pH,and concentrations of free ammonia can impact certain species of AOB(Erguder et al., 2008;Kim et al.,2006;Koops and Pommerening-Ro¨ser,2001; Kowalchuk and Stephen,2001;Shi et al.,2010).Therefore, the physiological activity and abundance of AOB in waste-water processing is critical in the design and operation of waste treatment systems.For this reason,a better under-standing of the ecology and microbiology of AOB in waste-water treatment systems is necessary to enhance treatment performance.Recently,several developed techniques have served as valuable tools for the characterization of microbial diversity in biological wastewater treatment systems(Li et al., 2008;Yin and Xu,2009).Currently,the application of molec-ular biotechniques can provide clariﬁcation of the ammonia-oxidizing community in detail(Haseborg et al.,2010;Tawan et al.,2005;Vlaeminck et al.,2010).In recent years,the aerobic granular sludge process has become an attractive alternative to conventional processes for wastewater treatment mainly due to its cell immobilization strategy(de Bruin et al.,2004;Liu et al.,2009;Schwarzenbeck et al.,2005;Schwarzenbeck et al.,2004a,b;Xavier et al.,2007). Granules have a more tightly compact structure(Li et al.,2008; Liu and Tay,2008;Wang et al.,2004)and rapid settling velocity (Kong et al.,2009;Lemaire et al.,2008).Therefore,granular sludge systems have a higher mixed liquid suspended sludge (MLSS)concentration and longer solid retention times(SRT) than conventional activated sludge systems.Longer SRT can provide enough time for the growth of organisms that require a long generation time(e.g.,AOB).Some studies have indicated that nitrifying granules can be cultivated with ammonia-rich inorganic wastewater and the diameter of granules was small (Shi et al.,2010;Tsuneda et al.,2003).Other researchers reported that larger granules have been developed with the synthetic organic wastewater in sequencing batch reactors(SBRs)(Li et al., 2008;Liu and Tay,2008).The diverse populations of microor-ganisms that coexist in granules remove the chemical oxygen demand(COD),nitrogen and phosphate(de Kreuk et al.,2005). However,for larger granules with a particle diameter greater than0.6mm,an outer aerobic shell and an inner anaerobic zone coexist because of restricted oxygen diffusion to the granule core.These properties of granular sludge suggest that the inner environment of granules is unfavorable to AOB growth.Some research has shown that particle size and density induced the different distribution and dominance of AOB,NOB and anam-mox(Winkler et al.,2011b).Although a number of studies have been conducted to assess the ecology and microbiology of AOB in wastewater treatment systems,the information on the dynamics,distribution,and quantiﬁcation of AOB communities during sludge granulation is still limited up to now.To address these concerns,the main objective of the present work was to investigate the population dynamics of AOB communities during the development of seedingﬂocs into granules,and the distribution of AOB and NOB in different size granules from an anaerobic e aerobic SBR.A combination of process studies,molecular biotechniques and microscale techniques were employed to identify and char-acterize these organisms.Based on these approaches,we demonstrate the differences in both AOB community evolu-tion and composition of theﬂocs and granules co-existing in the SBR and further elucidate the relationship between distribution of nitrifying bacteria and granule size.It is ex-pected that the work would be useful to better understand the mechanisms responsible for the AOB in granules and apply them for optimal control and management strategies of granulation systems.2.Material and methods2.1.Reactor set-up and operationThe granules were cultivated in a lab-scale SBR with an effective volume of4L.The effective diameter and height of the reactor was10cm and51cm,respectively.The hydraulic retention time was set at8h.Activated sludge from a full-scale sewage treat-ment plant(Jizhuangzi Sewage Treatment Works,Tianjin, China)was used as the seed sludge for the reactor at an initial sludge concentration of3876mg LÀ1in MLSS.The reactor was operated on6-h cycles,consisting of2-min inﬂuent feeding,90-min anaerobic phase(mixing),240-min aeration phase and5-min efﬂuent discharge periods.The sludge settling time was reduced gradually from10to5min after80SBR cycles in20days, and only particles with a settling velocity higher than4.5m hÀ1 were retained in the reactor.The composition of the inﬂuent media were NaAc(450mg LÀ1),NH4Cl(100mg LÀ1),(NH4)2SO4 (10mg LÀ1),KH2PO4(20mg LÀ1),MgSO4$7H2O(50mg LÀ1),KCl (20mg LÀ1),CaCl2(20mg LÀ1),FeSO4$7H2O(1mg LÀ1),pH7.0e7.5, and0.1mL LÀ1trace element solution(Li et al.,2007).Analytical methods-The total organic carbon(TOC),NHþ4e N, NOÀ2e N,NOÀ3e N,total nitrogen(TN),total phosphate(TP) concentration,mixed liquid suspended solids(MLSS) concentration,and sludge volume index at10min(SVI10)were measured regularly according to the standard methods (APHA-AWWA-WEF,2005).Sludge size distribution was determined by the sieving method(Laguna et al.,1999).Screening was performed with four stainless steel sieves of5cm diameter having respective mesh openings of0.9,0.6,0.45,and0.2mm.A100mL volume of sludge from the reactor was sampled with a calibrated cylinder and then deposited on the0.9mm mesh sieve.The sample was subsequently washed with distilled water and particles less than0.9mm in diameter passed through this sieve to the sieves with smaller openings.The washing procedure was repeated several times to separate the gran-ules.The granules collected on the different screens were recovered by backwashing with distilled water.Each fraction was collected in a different beaker andﬁltered on quantitative ﬁlter paper to determine the total suspended solid(TSS).Once the amount of total suspended solid(TSS)retained on each sieve was acquired,it was reasonable to determine for each class of size(<0.2,[0.2e0.45],[0.45e0.6],[0.6e0.9],>0.9mm) the percentage of the total weight that they represent.w a t e r r e s e a r c h x x x(2011)1e10 22.2.DNA extraction and nested PCR e DGGEThe sludge from approximately8mg of MLSS was transferred into a1.5-mL Eppendorf tube and then centrifuged at14,000g for10min.The supernatant was removed,and the pellet was added to1mL of sodium phosphate buffer solution and aseptically mixed with a sterilized pestle in order to detach granules.Genomic DNA was extracted from the pellets using E.Z.N.A.äSoil DNA kit(D5625-01,Omega Bio-tek Inc.,USA).To amplify ammonia-oxidizer speciﬁc16S rRNA for dena-turing gradient gel electrophoresis(DGGE),a nested PCR approach was performed as described previously(Zhang et al., 2010).30m l of nested PCR amplicons(with5m l6Âloading buffer)were loaded and separated by DGGE on polyacrylamide gels(8%,37.5:1acrylamide e bisacrylamide)with a linear gradient of35%e55%denaturant(100%denaturant¼7M urea plus40%formamide).The gel was run for6.5h at140V in 1ÂTAE buffer(40mM Tris-acetate,20mM sodium acetate, 1mM Na2EDTA,pH7.4)maintained at60 C(DCodeäUniversal Mutation Detection System,Bio-Rad,Hercules,CA, USA).After electrophoresis,silver-staining and development of the gels were performed as described by Sanguinetti et al. (1994).These were followed by air-drying and scanning with a gel imaging analysis system(Image Quant350,GE Inc.,USA). The gel images were analyzed with the software Quantity One,version4.31(Bio-rad).Dice index(Cs)of pair wise community similarity was calculated to evaluate the similarity of the AOB community among DGGE lanes(LaPara et al.,2002).This index ranges from0%(no common band)to100%(identical band patterns) with the assistance of Quantity One.The Shannon diversity index(H)was used to measure the microbial diversity that takes into account the richness and proportion of each species in a population.H was calculatedusing the following equation:H¼ÀPn iNlogn iN,where n i/Nis the proportion of community made up by species i(bright-ness of the band i/total brightness of all bands in the lane).Dendrograms relating band pattern similarities were automatically calculated without band weighting(consider-ation of band density)by the unweighted pair group method with arithmetic mean(UPGMA)algorithms in the Quantity One software.Prominent DGGE bands were excised and dissolved in30m L Milli-Q water overnight,at4 C.DNA was recovered from the gel by freeze e thawing thrice.Cloning and sequencing of the target DNA fragments were conducted following the estab-lished method(Zhang et al.,2010).2.3.Distribution of nitrifying bacteriaThree classes of size([0.2e0.45],[0.45e0.6],>0.9mm)were chosen on day180for FISH analysis in order to investigate the spatial distribution characteristics of AOB and NOB in granules.2mg sludge samples wereﬁxed in4%para-formaldehyde solution for16e24h at4 C and then washed twice with sodium phosphate buffer;the samples were dehydrated in50%,80%and100%ethanol for10min each. Ethanol in the granules was then completely replaced by xylene by serial immersion in ethanol-xylene solutions of3:1, 1:1,and1:3by volume andﬁnally in100%xylene,for10min periods at room temperature.Subsequently,the granules were embedded in parafﬁn(m.p.56e58 C)by serial immer-sion in1:1xylene-parafﬁn for30min at60 C,followed by 100%parafﬁn.After solidiﬁcation in parafﬁn,8-m m-thick sections were prepared and placed on gelatin-coated micro-scopic slides.Parafﬁn was removed by immersing the slide in xylene and ethanol for30min each,followed by air-drying of the slides.The three oligonucleotide probes were used for hybridiza-tion(Downing and Nerenberg,2008):FITC-labeled Nso190, which targets the majority of AOB;TRITC-labeled NIT3,which targets Nitrobacter sp.;TRITC-labeled NSR1156,which targets Nitrospira sp.All probe sequences,their hybridization condi-tions,and washing conditions are given in Table1.Oligonu-cleotides were synthesized andﬂuorescently labeled with ﬂuorochomes by Takara,Inc.(Dalian,China).Hybridizations were performed at46 C for2h with a hybridization buffer(0.9M NaCl,formamide at the percentage shown in Table1,20mM Tris/HCl,pH8.0,0.01% SDS)containing each labeled probe(5ng m LÀ1).After hybrid-ization,unbound oligonucleotides were removed by a strin-gent washing step at48 C for15min in washing buffer containing the same components as the hybridization buffer except for the probes.For detection of all DNA,4,6-diamidino-2-phenylindole (DAPI)was diluted with methanol to aﬁnal concentration of1ng m LÀ1.Cover the slides with DAPI e methanol and incubate for15min at37 C.The slides were subsequently washed once with methanol,rinsed brieﬂy with ddH2O and immediately air-dried.Vectashield(Vector Laboratories)was used to prevent photo bleaching.The hybridization images were captured using a confocal laser scanning microscope (CLSM,Zeiss710).A total of10images were captured for each probe at each class of size.The representative images were selected andﬁnal image evaluation was done in Adobe PhotoShop.w a t e r r e s e a r c h x x x(2011)1e1033.Results3.1.SBR performance and granule characteristicsDuring the startup period,the reactor removed TOC and NH 4þ-N efﬁciently.98%of NH 4þ-N and 100%of TOC were removed from the inﬂuent by day 3and day 5respectively (Figs.S2,S3,Supporting information ).Removal of TN and TP were lower during this period (Figs.S3,S4,Supporting information ),though the removal of TP gradually improved to 100%removal by day 33(Fig.S4,Supporting information ).To determine the sludge volume index of granular sludge,a settling time of 10min was chosen instead of 30min,because granular sludge has a similar SVI after 60min and after 5min of settling (Schwarzenbeck et al.,2004b ).The SVI 10of the inoculating sludge was 108.2mL g À1.The changing patterns of MLSS and SVI 10in the continuous operation of the SBR are illustrated in Fig.1.The sludge settleability increased markedly during the set-up period.Fig.2reﬂects the slow andgradual process of sludge granulation,i.e.,from ﬂocculentsludge to granules.3.2.DGGE analysis:AOB communities structure changes during sludge granulationThe results of nested PCR were shown in Fig.S1.The well-resolved DGGE bands were obtained at the representative points throughout the GSBR operation and the patterns revealed that the structure of the AOB communities was dynamic during sludge granulation and stabilization (Fig.3).The community structure at the end of experiment was different from that of the initial pattern of the seed sludge.The AOB communities on day 1showed 40%similarity only to that at the end of the GSBR operation (Table S1,Supporting information ),indicating the considerable difference of AOB communities structures between inoculated sludge and granular sludge.Biodiversity based on the DGGE patterns was analyzed by calculating the Shannon diversity index H as204060801001201401254159738494104115125135147160172188Time (d)S V I 10 (m L .g -1)10002000300040005000600070008000900010000M L S S (m g .L -1)Fig.1e Change in biomass content and SVI 10during whole operation.SVI,sludge volume index;MLSS,mixed liquid suspendedsolids.Fig.2e Variation in granule size distribution in the sludge during operation.d,particle diameter;TSS,total suspended solids.w a t e r r e s e a r c h x x x (2011)1e 104shown in Fig.S5.In the phase of sludge inoculation (before day 38),H decreased remarkably (from 0.94to 0.75)due to the absence of some species in the reactor.Though several dominant species (bands2,7,10,11)in the inoculating sludge were preserved,many bands disappeared or weakened (bands 3,4,6,8,13,14,15).After day 45,the diversity index tended to be stable and showed small ﬂuctuation (from 0.72to 0.82).Banding pattern similarity was analyzed by applying UPGMA (Fig.4)algorithms.The UPGMA analysis showed three groups with intragroup similarity at approximately 67%e 78%and intergroup similarity at 44e 62%.Generally,the clustering followed the time course;and the algorithms showed a closer clustering of groups II and III.In the analysis,group I was associated with sludge inoculation and washout,group IIwithFig.3e DGGE proﬁle of the AOB communities in the SBR during the sludge granulation process (lane labels along the top show the sampling time (days)from startup of the bioreactor).The major bands were labeled with the numbers (bands 1e15).Fig.4e UPGMA analysis dendrograms of AOB community DGGE banding patterns,showing schematics of banding patterns.Roman numerals indicate major clusters.w a t e r r e s e a r c h x x x (2011)1e 105startup sludge granulation and decreasing SVI 10,and group III with a stable system and excellent biomass settleability.In Fig.3,the locations of the predominant bands were excised from the gel.DNA in these bands were reampliﬁed,cloned and sequenced.The comparative analysis of these partial 16S rRNA sequences (Table 2and Fig.S6)revealed the phylogenetic afﬁliation of 13sequences retrieved.The majority of the bacteria in seed sludge grouped with members of Nitrosomonas and Nitrosospira .Along with sludge granula-tion,most of Nitrosomonas (Bands 2,5,7,9,10,11)were remained or eventually became dominant in GSBR;however,all of Nitrosospira (Bands 6,13,15)were gradually eliminated from the reactor.3.3.Distribution of AOB and NOB in different sized granulesFISH was performed on the granule sections mainly to deter-mine the location of AOB and NOB within the different size classes of granules,and the images were not further analyzed for quantiﬁcation of cell counts.As shown in Fig.6,in small granules (0.2mm <d <0.45mm),AOB located mainly in the outer part of granular space,whereas NOB were detected only in the core of granules.In medium granules (0.45mm <d <0.6mm),AOB distributed evenly throughout the whole granular space,whereas NOB still existed in the inner part.In the larger granules (d >0.9mm),AOB and NOB were mostly located in the surface area of the granules,and moreover,NOB became rare.4.Discussion4.1.Relationship between granule formation and reactor performanceAfter day 32,the SVI 10stabilized at 20e 35mL g À1,which is very low compared to the values measured for activated sludge (100e 150mL g À1).However,the size distribution of the granules measured on day 32(Fig.2)indicated that only 22%of the biomass was made of granular sludge with diameter largerthan 0.2mm.These results suggest that sludge settleability increased prior to granule formation and was not affected by different particle sizes in the sludge during the GSBR operation.It was observed,however,that the diameter of the granules ﬂuctuated over longer durations.The large granules tended to destabilize due to endogenous respiration,and broke into smaller granules that could seed the formation of large granules again.Pochana and Keller reported that physically broken sludge ﬂocs contribute to lower denitriﬁcation rates,due to their reduced anoxic zone (Pochana and Keller,1999).Therefore,TN removal efﬁciency raises ﬂuctuantly throughout the experiment.Some previous research had demonstrated that bigger,more dense granules favored the enrichment of PAO (Winkler et al.,2011a ).Hence,after day 77,removal efﬁciency of TP was higher and relatively stable because the granules mass fraction was over 90%and more larger granules formed.4.2.Relationship between AOB communities dynamic and sludge granulationFor granule formation,a short settling time was set,and only particles with a settling velocity higher than 4.5m h À1were retained in the reactor.Moreover,as shown in Fig.1,the variation in SVI 10was greater before day 41(from 108.2mL g À1e 34.1mL g À1).During this phase,large amounts of biomass could not survive in the reactor.A clear shift in pop-ulations was evident,with 58%similarity between days 8and 18(Table S1).In the SBR system fed with acetate-based synthetic wastewater,heterotrophic bacteria can produce much larger amounts of extracellular polysaccharides than autotrophic bacteria (Tsuneda et al.,2003).Some researchers found that microorganisms in high shear environments adhered by extracellular polymeric substances (EPS)to resist the damage of suspended cells by environmental forces (Trinet et al.,1991).Additionally,it had been proved that the dominant heterotrophic species in the inoculating sludge were preserved throughout the process in our previous research (Zhang et al.,2011).It is well known that AOB are chemoau-totrophic and slow-growing;accordingly,numerous AOBw a t e r r e s e a r c h x x x (2011)1e 106populations that cannot become big and dense enough to settle fast were washed out from the system.As a result,the variation in AOB was remarkable in the period of sludge inoculation,and the diversity index of population decreased rapidly.After day 45,AOB communities’structure became stable due to the improvement of sludge settleability and the retention of more biomass.These results suggest that the short settling time (selection pressure)apparently stressed the biomass,leading to a violent dynamic of AOB communities.Further,these results suggest that certain populations may have been responsible for the operational success of the GSBR and were able to persist despite the large ﬂuctuations in pop-ulation similarity.This bacterial population instability,coupled with a generally acceptable bioreactor performance,is congruent with the results obtained from a membrane biore-actor (MBR)for graywater treatment (Stamper et al.,2003).Nitrosomonas e like and Nitrosospira e like populations are the dominant AOB populations in wastewater treatment systems (Kowalchuk and Stephen,2001).A few previous studies revealed that the predominant populations in AOB communities are different in various wastewater treatment processes (Tawan et al.,2005;Thomas et al.,2010).Some researchers found that the community was dominated by AOB from the genus Nitrosospira in MBRs (Zhang et al.,2010),whereas Nitrosomonas sp.is the predominant population in bioﬁlter sludge (Yin and Xu,2009).In the currentstudy,Fig.5e DGGE proﬁle of the AOB communities in different size of granules (lane labels along the top show the range of particle diameter (d,mm)).Values along the bottom indicate the Shannon diversity index (H ).Bands labeled with the numbers were consistent with the bands in Fig.3.w a t e r r e s e a r c h x x x (2011)1e 107sequence analysis revealed that selection pressure evidently effect on the survival of Nitrosospira in granular sludge.Almost all of Nitrosospira were washed out initially and had no chance to evolve with the environmental changes.However,some members of Nitrosomonas sp.have been shown to produce more amounts of EPS than Nitrosospira ,especially under limited ammonia conditions (Stehr et al.,1995);and this feature has also been observed for other members of the same lineage.Accordingly,these EPS are helpful to communicate cells with each other and granulate sludge (Adav et al.,2008).Therefore,most of Nitrosomonas could adapt to this challenge (to become big and dense enough to settle fast)and were retained in the reactor.At the end of reactor operation (day 180),granules with different particle size were sieved.The effects of variation in granules size on the composition of the AOBcommunitiesFig.6e Micrographs of FISH performed on three size classes of granule sections.DAPI stain micrographs (A,D,G);AOB appear as green ﬂuorescence (B,E,H),and NOB appear as red ﬂuorescence (C,F,I).Bar [100m m in (A)e (C)and (G)e (I).d,particle diameter.(For interpretation of the references to colour in this ﬁgure legend,the reader is referred to the web version of this article.)w a t e r r e s e a r c h x x x (2011)1e 108were investigated.As shown in Fig.5,AOB communities structures in different size of granules were varied.Although several predominant bands(bands2,5,11)were present in all samples,only bands3and6appeared in the granules with diameters larger than0.6mm.Additionally,bands7and10 were intense in the granules larger than0.45mm.According to Table2,it can be clearly indicated that Nitrosospira could be retained merely in the granules larger than0.6mm.Therefore, Nitrosospira was not present at a high level in Fig.3due to the lower proportion of larger granules(d>0.6mm)in TSS along with reactor operation.DGGE analysis also revealed that larger granules had a greater microbial diversity than smaller ones. This result also demonstrates that more organisms can survive in larger granules as a result of more space,which can provide the suitable environment for the growth of microbes(Fig.6).4.3.Effect of variance in particle size on the distribution of AOB and NOB in granulesAlthough an inﬂuence of granule size has been observed in experiments and simulations for simultaneous N-and P-removal(de Kreuk et al.,2007),the effect of granule size on the distribution of different biomass species need be revealed further with the assistance of visible experimental results, especially in the same granular sludge reactors.Related studies on the diversity of bacterial communities in granular sludge often focus on the distribution of important functional bacteria populations in single-size granules(Matsumoto et al., 2010).In the present study,different size granules were sieved,and the distribution patterns of AOB and NOB were explored.In the nitriﬁcation processes considered,AOB and NOB compete for space and oxygen in the granules(Volcke et al.,2010).Since ammonium oxidizers have a higheroxygen afﬁnity(K AOBO2<K NOBO2)and accumulate more rapidly inthe reactor than nitrite oxidizers(Volcke et al.,2010),NOB are located just below the layer of AOB,where still some oxygen is present and allows ready access to the nitrite produced.In smaller granules,the location boundaries of the both biomass species were distinct due to the limited existence space provided by granules for both microorganism’s growth.AOB exist outside of the granules where oxygen and ammonia are present.Medium granules can provide broader space for microbe multiplying;accordingly,AOB spread out in the whole granules.This result also conﬁrms that oxygen could penetrate deep into the granule’s core without restriction when particle diameter is less than0.6mm.Some mathematic model also supposed that NOBs are favored to grow in smaller granules because of the higher fractional aerobic volume (Volcke et al.,2010).As shown in the results of the batch experiments(Zhang et al.,2011),nitrite accumulation temporarily occurred,accompanied by the more large gran-ules(d>0.9mm)forming.This phenomenon can be attrib-uted to the increased ammonium surface load associated with larger granules and smaller aerobic volume fraction,resulting in outcompetes of NOB.It also suggests that the core areas of large granules(d>0.9mm)could provide anoxic environment for the growth of anaerobic denitriﬁcans(such as Tb.deni-triﬁcans or Tb.thioparus in Fig.S7,Supporting information).As shown in Fig.2and Fig.S3,the removal efﬁciency of total nitrogen increased with formation of larger granules.5.ConclusionsThe variation in AOB communities’structure was remarkable during sludge inoculation,and the diversity index of pop-ulation decreased rapidly.Most of Nitrosomonas in the inocu-lating sludge were retained because of their capability to rapidly adapt to the settling e washing out action.DGGE anal-ysis also revealed that larger granules had greater AOB diversity than that of smaller ones.Oxygen penetration was not restricted in the granules of less than0.6mm particle diameter.However,the larger granules(d>0.9mm)can result in the smaller aerobic volume fraction and inhibition of NOB growth.Henceforth,further studies on controlling and opti-mizing distribution of granule size could be beneﬁcial to the nitrogen removal and expansive application of granular sludge technology.AcknowledgmentsThis work was supported by grants from the National Natural Science Foundation of China(No.51108456,50908227)and the National High Technology Research and Development Program of China(No.2009AA06Z312).Appendix.Supplementary dataSupplementary data associated with this article can be found in online version at doi:10.1016/j.watres.2011.09.026.r e f e r e n c e sAdav,S.S.,Lee, D.J.,Show,K.Y.,2008.Aerobic granular sludge:recent advances.Biotechnology Advances26,411e423.APHA-AWWA-WEF,2005.Standard Methods for the Examination of Water and Wastewater,ﬁrst ed.American Public Health Association/American Water Works Association/WaterEnvironment Federation,Washington,DC.de Bruin,L.M.,de Kreuk,M.,van der Roest,H.F.,Uijterlinde,C., van Loosdrecht,M.C.M.,2004.Aerobic granular sludgetechnology:an alternative to activated sludge?Water Science and Technology49,1e7.de Kreuk,M.,Heijnen,J.J.,van Loosdrecht,M.C.M.,2005.Simultaneous COD,nitrogen,and phosphate removal byaerobic granular sludge.Biotechnology and Bioengineering90, 761e769.de Kreuk,M.,Picioreanu,C.,Hosseini,M.,Xavier,J.B.,van Loosdrecht,M.C.M.,2007.Kinetic model of a granular sludge SBR:inﬂuences on nutrient removal.Biotechnology andBioengineering97,801e815.Downing,L.S.,Nerenberg,R.,2008.Total nitrogen removal ina hybrid,membrane-aerated activated sludge process.WaterResearch42,3697e3708.Erguder,T.H.,Boon,N.,Vlaeminck,S.E.,Verstraete,W.,2008.Partial nitriﬁcation achieved by pulse sulﬁde doses ina sequential batch reactor.Environmental Science andTechnology42,8715e8720.w a t e r r e s e a r c h x x x(2011)1e109。

ASM1

Process, j
• Aerobic growth of autotrophs:
ˆA

S NH K NH S NH
SO K S X B, A O O, A
• Death and lysis of heterotrtivated Sludge Model No.1
ASM1 IAWPRC Task Group
M. Henze, Denmark C.P.L. Grady, Jr. , USA, W. Gujer, Switzerland G.v. R. Marais, South Africa T. Matsuo, Japan
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11/18/2009
Process, j
• Death and lysis of autotrophs :
bLA X B, A
• Ammonification of soluble organic nitrogen:
ka S NS X B, H
Process, j
• Hydrolysis of particulate organics:
Process, j
• Aerobic growth of heterotrophs:
ˆH Ss SO X B,H K s S s K O , H SO
• Anoxic growth of heterotrophs:
ˆH KO,H Ss S NO g X B , H K s S s K O , H SO K NO S NO
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Stoichiometric and Kinetic Parameters

酵母细胞壁YCW在花鲈饲料中的有效性和耐受性

Ef ﬁcacy and tolerance of yeast cell wall as an immunostimulant in the diet of Japanese seabass (Lateolabrax japonicus )H.H.Yu a ,F.Han a ,M.Xue a ,b ,⁎,J.Wang a ,P.Tacon c ,Y.H.Zheng a ,X.F.Wu a ,Y.J.Zhang daNational Aquafeed Safety Assessment Station,Feed Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081,ChinabKey Laboratory of Feed Biotechnology of Ministry of Agriculture,Feed Research Institute,Chinese Academy of Agricultural Sciences,Beijing,China cSociétéIndustrielle Lesaffre,Lesaffre Additive Division,Marcq en Baroeul,France dThe Key Laboratory of Mariculture,Ministry of Education,Ocean University of China,5Yushan Road,Qingdao,Shandong 266003,Chinaa b s t r a c ta r t i c l e i n f o Article history:Received 26December 2013Received in revised form 28April 2014Accepted 30April 2014Available online 19May 2014Keywords:Yeast cell wallLateolabrax japonicas Ef ﬁcacy ToleranceImmune response Bacterial challengeThe effects of dietary yeast cell wall (YCW)on the growth,immune response and cumulative survival rate after Aeromonas veronii challenge were investigated in Japanese seabass,Lateolabrax japonicus .Six high soybean meal (SBM)inclusion diets were prepared with YCW levels at 0(Y0),250(Y1),500(Y2),1000(Y3),2000(Y4),and 20,000(Y5)mg/kg,respectively.The highest YCW level (20,000mg/kg)was designed as the 10fold of the highest recommended level (2000mg/kg)for tolerance evaluation.In addition,a positive control (FM)with higher ﬁshmeal level and a negative control (Fla)with 4mg/kg ﬂavomycin based on Y0diet were designed.Each diet was fed to six replicates of 30Japanese seabass (18.3±0.01g)for 72days.The growth performance of Y0group was lower than that of FM group.Optimal levels (1000–2000mg/kg)of baker's YCW improved the growth performance and intestinal mucus development of Japanese seabass,while 500mg/kg YCW enhanced immune response and cumulative survival after challenged with A.veronii .Flavomycin inclusion negatively affected the growth and immune response of ﬁsh.The present study proved that the highest recommended dose of YCW was 2000mg/kg,and 10fold of safety margin was obtained.©2014Elsevier B.V.All rights reserved.1.IntroductionOmnivorous Cyprinids (common carp,grass carp,gibel carp.black carp etc.)are the main ﬁsh species cultured in China.However,the culture of marine and fresh water carnivorous species is expanding rapidly.This expansion requires the development of high quality feed formulae.Japanese seabass (Lateolabrax japonicus ),a euryhaline species,is one of the most important species for marine aquaculture in China and East Asia.With the rapid development of intensive culture with high density,diseases occur frequently and become increasingly serious.Except in Europe,antibiotics have been used in ﬁsh diets to prevent bacterial infection and promote growth performance for years in most countries.However,the abuse of antibiotics has caused prob-lems with antibiotic residuals in ﬂesh and the risk of developing drug resistance in animals and human pathogenic bacteria.WHO (the World Health Organization)has announced that it is urgently regarding the replacement of antibiotics by safer substances for the control of infectious diseases in farm animals.In addition,with the increasing demand for ﬁshmeal in aquaculture,ﬁshmeal shortage has become a worldwide problem.Soybean meal (SBM)has been recognized as one of the most cost-effective alternative protein sources for ﬁshmeal in aqua-feed because of its favorable protein content,comparatively balanced amino acid pro ﬁle,availability and reasonable price (Gatlin et al.,2007;Hardy,2010).However,anti-nutritional factors in soybean meal can cause decreased disease resis-tance and soybean-induced enteritis,which is one of most important reasons for less usage of soybean meal in carnivorous ﬁsh diets.Some studies about nutritional requirements and cost-effective feed formulation have been conducted on Japanese seabass.In our lab,we had performed several studies in ﬁshmeal and ﬁsh oil replacement on this species (Hu et al.,2010,2013;Wang et al.,2012).Our ﬁndings suggest that at least 24%–25%of high quality ﬁshmeal is needed for the feed palatability,digestion and growth of Japanese seabass.Yeast cell wall (YCW),a β-glucan and mannan rich substance derived from bakery or brewer's yeast,have been used as prebiotics in ﬁsh feed and produce a positive effect on the ﬁsh immune system and resistance to diseases (Abdel-Tawwab et al.,2008;El-Boshy et al.,2010;Rodríguez et al.,2009).β-Glucan enhances the immunity by increasing tissue lysozyme activity and the respiratory burst activity (Ai et al.,2007),or enhances the disease resistance by directly binding and activating macrophages,neutrophils and natural killer (NK)cellsAquaculture 432(2014)217–224⁎Corresponding author at:National Aquafeed Safety Assessment Station,Feed Research Institute,Chinese Academy of Agricultural Sciences,Beijing,China,100081.Tel./fax:+861082109753.E-mail address:xuemin@ (M.Xue)./10.1016/j.aquaculture.2014.04.0430044-8486/©2014Elsevier B.V.All rightsreserved.Contents lists available at ScienceDirectAquaculturej o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m/l o c a t e /a q u a -o n l i n e(Gantner et al.,2003;Herre et al.,2004;Robertsen,1999).The most described effect of mannan is the elimination of pathogenic bacteria from the intestinal tract by binding of mannose particles to the lectin-type receptors of enteropathogenic bacteria.Besides,these positive effects of mannan,anti-oxidant,anti-mutagenic and anti-cancer activi-ties,were also reported(Križkováet al.,2001;Miadokováet al.,2006). However,an overdose ofβ-glucan and mannan may have negative effects on growth performance and immune system in aquatic animals (Ai et al.,2007;Castro et al.,1999;Gu et al.,2010).The objectives of the present study were to evaluate the efﬁcacy and tolerance of dietary YCW by its effects on growth performance,intestinal histology,hemato-logical proﬁles,immune parameters and resistance to the challenge of Aeromonas veronii in Japanese seabass based on a high soy protein diet.2.Materials and methods2.1.Experimental dietsThe tested YCW was supplied by Lesaffre Feed Additives,France,and extracted from baker's yeast(Saccharomyces cerevisiae),containing28% glucan and24%mannan.Six experimental diets were prepared with YCW levels at0(Y0,control),250(Y1),500(Y2),1000(Y3),2000 (Y4),and20,000(Y5)mg/kg,respectively.The highest inclusion level (20,000mg/kg)was designed as10fold of the highest recommended level(2000mg/kg)for the tolerance test to obtain the safety margin of YCW utilized inﬁsh feed.In addition,a positive control(FM)using a higher level of low temperature steam driedﬁshmeal(Triple Nine Fish protein Co.Ltd.Esbjerg,Denmark)and a negative control(Fla)with 4mg/kgﬂavomycin based on Y0diet were designed.Each diet was ex-truded into2mm diameter pellets under the following extrusion condi-tion as:feeding section(90°C/5s),compression section(150°C/5s) and metering section(60°C/4s)using a Twin-screwed extruder (EXT50A,YANGGONG MACHINE,Beijing,China).The diet formulation and analyzed chemical compositions were shown in Table1.2.2.Experimentalﬁsh,feeding and samplingJuvenile Japanese seabass were obtained from Yulong Aquafarm (Weihai,Shandong,China),and gradually acclimated from seawater (salinity:19ppt)to freshwater over a period of2weeks.Allﬁsh were acclimated in laboratory conditions and fed the control experimental diet without YCW(Y0)for two more weeks before the commencement of trial.Fish(initial body weight=18.3±0.01g)were selected and distributed into280L tanks after24h starvation with30ﬁsh per tank,and six tanks per treatment.The water temperature was main-tained at26±1°C,pH=7–8,dissolved oxygen(DO)N6mg/L and NH4-N b0.5mg/L.Aeration was supplied to each tank24h per day and photoperiod was12D:12L.Fish were fed to apparent satiation twice daily at08:00and15:00for72days.One hour later,uneaten feed was removed,dried to constant weight at70°C and reweighed. Leaching loss of the uneaten diet was estimated by leavingﬁve samples of each diet in tanks withoutﬁsh for1h,recovering,drying and reweighing.Theﬁsh from each tank were batch weighed at the end of the growth trial.Fourﬁsh of each tank were randomly selected and anesthetized with chlorobutanol(300mg/mL)and the body weight,body length, liver,viscera and spleen weight were recorded individually to calculate condition factor(CF),hepatosomatic index(HSI),viscerosomatic index (VSI)and spleensomatic index(SSI),respectively.Then the blood samples(for the hematological parameters and immune parameters before the bacterial challenge determinations)were withdrawn from the caudal part of the sedatedﬁsh using anticoagulant syringes with 2%NaF and4%potassium oxalate.1mL of the whole blood was stored at4°C for respiratory burst activity and analyzed within24h.The rest were centrifuged(1500g for10min)at4°C,and then the plasma was collected and stored at−80°C until analysis.Hematological parameters were only determined in the plasma of Y0,Y4and Y5groups for the tolerance test.2.3.Chemical analysisAll chemical analyses of diets were carried out in duplicate according to AOAC(2006).Dry matter was analyzed by drying the samples to constant weight at105°C.Crude protein(CP)was determined by a KjeltecTM2300Unit(Foss,Hillerød,Denmark)by the method of Kjeldahl,and CP content was estimated by multiplying nitrogen by 6.25.Crude lipid was analyzed by acid hydrolysis with a Soxtex System HT1047Hydrolyzing Unit(Foss,Hillerød,Denmark),followed by Soxhlet extraction using a Soxtex System1043(Foss,Hillerød,Denmark).Ash was analyzed by combustion in a mufﬂe furnace(CWF1100,Carbolite, Derbyshire,UK)at550°C for16h.Gross energy was determined by IKA C2000Calorimeter(C2000,IKA,Staufen,Germany).Table1Formulation and compositions of experimental diets(%).FM Fla Y0Y1Y2Y3Y4Y5IngredientsFishmeal38.525252525252525 Soybean protein concentrate2020202020202020 Soybean meal021212121212121 Wheatﬂour2121212121212120 Fish oil6 6.4 6.4 6.4 6.4 6.4 6.4 6.4 Monocalcium phosphate(Ca(H2PO4)2)1 2.1 2.1 2.1 2.1 2.1 2.1 2.1 Microcrystalline cellulose10.1110.9750.950.90.80 Phospholipid(93%)22222222 Choline chloride(50%)0.40.40.40.40.40.40.40.4 Vitamin and mineral Premix a11111111 Methionine hydroxy analog-Ca(98%)00.10.10.10.10.10.10.1 Flavomycin b(mg/kg)04000000 Yeast cell wall b(mg/kg)0002505001000200020,000Analyzed chemical compositions(dry matter basis%)Crude protein47.648.448.248.747.948.448.549.4 Crude lipid12.111.612.011.711.611.811.611.9 Crude ash7.868.518.518.468.528.598.418.54 Gross energy(MJ/kg)21.521.221.321.421.521.421.621.6a Vitamin premix(mg·kg−1diet):vitamin A20;vitamin B112;vitamin B210;vitamin B615;vitamin B128;niacinamide100;ascorbic acid1000;calcium pantothenate40;biotin2;folic acid10;vitamin E400;vitamin K320;vitamin D310;inositol200;corn protein powder150.Mineral premix(mg·kg−1diet):CuSO4·5H2O10;FeSO4·H2O300;ZnSO4·H2O200; MnSO4·H2O100;KI(10%)80;Na2SeO3(10%Se)67;CoCl2·6H2O(10%Co)5;NaCl100;zeolite638.Vitamin premix:mineral premix=2:1.5.b Flavomycin and yeast cell wall were premixed with vitamin and mineral premix.218H.H.Yu et al./Aquaculture432(2014)217–2242.4.Hematological and immune parametersThe respiratory burst activity was measured by a nitroblue tetrazolium(NBT)assay following the protocol of Anderson and Siwicki(1995).Brieﬂy,0.1mL whole blood was taken to mix with 0.1mL0.2%NBT(AMRESCO,Solon,USA)solution,and then the mixture was incubated for30min at25°C.After incubation,15μL of the mixture was taken,added to0.3mL N,N-dimethyl formamide(Xilong Chemical Co.,Ltd,Guangzhou,China)and centrifuged at2000rpm for5min. Supernatant(0.2mL)was measured at an optimal density(OD)of 540nm in a microplate reader(PowerWave XS2,BioTek,Vermont, USA)with96-well quartz plates,and N,N-dimethyl formamide was used as blank.Myeloperoxidase(MPO)was measured according to Quade and Roth(1997)with slight modiﬁcation.15μL plasma was diluted with 135μL Hank's balanced salt solution(Sigma,Saint Louis,USA)without Ca2+or Mg2+.Then52.5μL3,3′,5,5′-tetramethylbenzidine hydrochlo-ride system(TMB,Sigma,Saint Louis,USA)was added.The color change reaction was stopped after2min by adding52.5μL of0.5mmol/L sulphuric acid(H2SO4).The OD was read at450nm in a microplate reader with96-well plates.Hematological and immune parameters,including plasma ALT (alanine aminotransferase),AST(aspartate aminotransferase),BUN (blood urea nitrogen),ALB(albumin),TPRO(total protein),TBILI (total bilirubin),TC(total cholesterol),TG(triglyceride),ALP(alkaline phosphatase),CRE(creatinine),NO(nitric oxide)and anti-superoxideanion(superoxide anion scavenging activity)were determined by assay kits(Nanjing Jiancheng Co.,Nanjing,China)following the instruc-tions given by the supplier.The C3(complement3)and IgM(immuno-globulin M)were used as the kits of enzyme linked immunosorbent assay(ELISA)forﬁsh with product Nos.of A7251and CSB-E12045F by Shanghai Boyao Co.Shanghai,China.2.5.HistologyIntestine samples from the FM,Y0,Y4and Y5groups were removed from2ﬁsh each replicate tank at the end of trial(12ﬁsh per treatment). All samples were rinsed with physiological saline water andﬁxed in a 10%phosphate-buffered formalin solution with a pH of7.2.After dehydrated by the standard procedures,the samples were embedded in parafﬁn and cut to approximately7μm sections.Intestine sections were stained with hematoxylin and eosin(H&E)and observed under light microscopy(Leica DM2500,Leica,Solms,Germany).In addition, morphological parameters associated with SBM-induced enteritis of anterior and distal intestines,including the height of mucosal folds (HMF),width of mucosal folds(WMF),lamina propria(LP),and connective tissue(CT),were quantiﬁed as the methods shown in Fig.1.2.6.Bacterial challengeAfter all samples were taken,40ﬁsh of each treatment(6–7ﬁsh per tank)were divided into2groups and transferred into a still water system with temperature at26±1°C.Theﬁsh were fed as before and recovered from weighing and sampling stress by a2-week acclima-tion.Then they were challenged by intramuscular injection with A.veronii(CGMCC No.4274)at8×104cells/100g body weight.Ten ﬁsh from each tank was sampled for plasma immune parameters two days after challenge and the others(20ﬁsh per treatment)were recorded for7-day cumulative survival rate without any food.There was no ﬁsh died24h after injection,which means that noﬁsh died caused by injection stress.2.7.Statistical analysisThe data of immune parameters before and after the challenge were analyzed by two-way ANOVA.All other data were subjected to one-way analysis of variance(one-way ANOVA).Differences between the means were analyzed by Duncan's multiple-range test,and the level of signiﬁ-cance chosen was P b0.05and the results were presented as means±S.E.(standard error).3.Results3.1.Growth performanceThe results of growth performance were presented in Table2.Allﬁsh showed high survival(N97.8%)in the present study,and no signiﬁcant difference was observed between groups(P N0.05).An inclusion of 1000–20,000mg/kg YCW(Y3–Y5)improved the feeding rate of Japanese seabass,which was signiﬁcantly higher than that of the FM and Y0groups(P b0.05).Fish of the FM group had the highest FBW and SGR,which were signiﬁcantly higher than those of Fla and Y0–Y2 groups(P b0.05),but no signiﬁcant differences were observed when YCW level reached1000mg/kg or more(Y3–Y5)(P N0.05).This sug-gested that1000–20,000mg/kg YCW,but notﬂavomycin promoted the growth performance of Japanese seabass.The FM group showed a signiﬁcantly lowest FCR among groups(P b0.05).3.2.Physiological parametersThe physiological parameters of the experimental groups were shown in Table3.There were no signiﬁcant differences on CF,HSI and SSI among groups.The VSI of the Y1group was the highest among groups,and signiﬁcantly higher than that of FM and Y0groups (P b0.05).3.3.Bacterial challenge and immune responsesAfter the bacterial challenge,ﬁsh fed diets Y2and Y5had the highest 7-day cumulative survival rate(85%),whileﬁsh of Y0group had the lowest one(40%).Allﬁsh of Fla group died within48h after the chal-lenge,the plasma samples could not be taken(Fig.2;Table4).Besides, we failed to get good quality plasma samples in Y1group,because of the hemolysis.Therefore,the immune parameters after the challenge in these two groups were not determined.The data for the immune parameters before and after bacterial challenge were listed in Table4.HMFWMFLPCTFig.1.Schematic representation of the parameters describing the morphology of anterior and distal intestine,HMF:height of mucosal fold,WMF:width of mucosal fold,LP:lamina propria,CT:connective tissue.219H.H.Yu et al./Aquaculture432(2014)217–224Plasma C3was not affected by both the dietary treatment and the bacterial challenge.Except the C3and anti-superoxide anion,the values of most immune parameters for all groups were signiﬁcantly increased after the bacterial challenge.Before the challenge,theﬁsh of the Fla group had signiﬁcantly higher plasma IgM and NO than other groups, whileﬁsh fed Y0,FM and Y5diet had the highest MPO,NBT and anti-superoxide anion,respectively(P b0.05).After the challenge,ﬁsh fed diet Y2showed the highest IgM among groups,which simultaneously showed the highest cumulative survival(P b0.05).Accordingly,the ﬁsh of Y0group with higher mortality after the challenge showed lower plasma IgM than that ofﬁsh fed Y2diet,and was not different from the value of before challenge.The Y5group had the highest MPO,while the FM group showed the lowest NBT value(P b0.05), and there was no signiﬁcant difference for plasma NO among groups after the challenge(P N0.05).The interaction of the dietary treatment and the challenge affected the plasma MPO and NBT(P b0.05).3.4.Hematological parameters and histology of intestine for tolerance testThe results of hematological parameters for tolerance evaluation were listed in Table5.There were no signiﬁcant differences in plasma ALT,glucose,ALB,TBILI,TC and TG(P N0.05).Plasma AST,BUN and TPRO in Y4and Y5groups were signiﬁcantly higher than thoseﬁsh fed the Y0diet(P b0.05),but generally within the normal values range for Japanese seabass.The content of creatinine in plasma was not detected.The results of histological examination of anterior and distal intes-tine tissues of FM,Y0,Y4and Y5groups were shown in Fig.3.All the sections of distal intestine examined were basically normal with well-developed mucosal folds and lamina propria.The slightly increased number and size of goblet cells(GC)were observed in anterior intestine ofﬁsh fed Y0diet.The results of quantiﬁcation were shown in Table6. YCW inclusion had a signiﬁcant effect on the height of mucosal folds of both the anterior and distal intestines.The height of mucosal folds in Y4group was signiﬁcantly higher than that of the Y0group(P b0.05) both in anterior and distal intestine sections.Although there were no statistical signiﬁcant differences in morphological parameters between FM and Y0,we can observe a clear broaden width of mucosal fold both in anterior and distal intestines(Fig.3).This suggested that21% inclusion of soybean meal had slightly negative effects on intestinal health of Japanese seabass.There were no signiﬁcant differences in other morphological parameters among groups(P N0.05).4.DiscussionIn most cases,replacingﬁshmeal by plant proteins will produce reduced feed intake as one of the important factors for low growth per-formance of carnivorousﬁsh species(Blaufuss and Trushenski,2012; Hill et al.,2013).This effect was observed in Japanese seabass,even when relatively small amounts ofﬁshmeal were replaced(9%ﬁshmeal was replaced)by a blend of plant protein with balanced essential amino acids,fatty acids and minerals(Hu,2010;Zhang et al.,2013). However,in the present study,the feed intake of Japanese seabass was not inﬂuenced by21%inclusion of soybean meal(Y0),to replace 13.5%ﬁshmeal.This could be due to the feeding adaptation during the 4-week acclimation,in which Y0diet was used,a similar phenomenon as we observed in this species in the study ofﬁshmeal substitution (Hu et al.,2010;Zhang et al.,2013).Adaptation to a lowﬁshmeal diet was also reported in rainbow trout(Refstie et al.,1997)and gibel carp (Xue et al.,2004)after a4-week feeding period.However,the growth performance of the Y0group was signiﬁcantly lower than that of the FM group,which may be induced by essential nutrient deﬁciency, such as nucleotides(Abtahi et al.,2013;Tahmasebi-Kohyani et al., 2011),taurine(Gaylord et al.,2006;Lunger et al.,2007;Matsunari et al.,2008;Takagi et al.,2008),glycine,etc.(McGoogan and Gatlin, 1997)or by anti-nutrients from plant protein(Gatlin et al.,2007).In the present study,the YCW inclusion improved the feed intake of Japanese seabass,which accordingly enhanced the growth perfor-mance.The higher height of mucosal folds in the anterior and distal intestines could be another reason for the better growth ofﬁsh of the Y4group by improving the intestinal nutrient absorption area.Although the FCR of the Y4group was still higher than that of the FM group,the cost of2000mg/kg YCW would be much lower than13.5%ofﬁshmeal. Typical morphological changes of the intestine caused by soybean mealTable2Effects of dietary YCW on the growth and feed performance of Japanese seabass(means±S.E.n=6).FM Fla Y0Y1Y2Y3Y4Y5FBW(g)85.3±1.4678.1±0.4476.9±1.5578.7±2.2975.8±1.1879.3±2.8382.0±1.4880.6±1.56 WGR(%)366±7.77324±1.87316±9.13325±12.9310±6.80328±16.9342±9.23333±25.5 SGR(%day−1) 2.13±0.03 2.00±0.01 1.99±0.03 2.02±0.04 1.97±0.02 2.02±0.05 2.08±0.02 2.06±0.03 FCR0.95±0.01 1.04±0.01 1.03±0.01 1.05±0.02 1.07±0.02 1.07±0.02 1.06±0.01 1.04±0.02 FR(%bw day−1) 1.71±0.02 1.78±0.02 1.75±0.02 1.81±0.02 1.80±0.02 1.84±0.02 1.85±0.01 1.82±0.01 SR%100±0.0098.9±1.1098.3±2.7997.8±2.7298.9±1.7098.3±2.8098.4±1.8197.8±2.73Within the same row,values with different superscripts are signiﬁcantly different(P b0.05).FBW:ﬁnal body weight.WGR(weight gain rate,%)=100×(W f+W d−W i)/W i.W f is theﬁnal total weight,W d is the total weight of deadﬁsh,W i is the initial total weight.The same below.GR(speciﬁc growth rate,%)=100×[Ln(FBW/initial body weight)]/days.FCR(feed conversion rate)=feed intake/(W f+W d−W i).FR(feeding rate,%)=100×feed intake/[(W f+W i+W d)/2]/days.SR(survival rate,%)=100×ﬁnalﬁsh number/initialﬁsh number.Table3Effects of dietary YCW on physiological parameters of Japanese seabass(means±S.E.n=24).FM Fla Y0Y1Y2Y3Y4Y5CF 1.24±0.02 1.24±0.03 1.21±0.01 1.28±0.01 1.21±0.02 1.26±0.03 1.23±0.02 1.26±0.04 SSI%0.08±0.000.10±0.010.08±0.000.10±0.010.09±0.010.10±0.010.10±0.010.08±0.01 VSI%9.56±0.4610.5±0.159.46±0.3610.9±0.3510.6±0.3010.3±0.3810.3±0.2710.6±0.35 HSI% 1.82±0.09 1.81±0.10 1.66±0.08 1.86±0.08 1.92±0.09 1.83±0.08 1.86±0.06 1.80±0.14Within the same row,values with different superscripts are signiﬁcantly different(P b0.05).CF(condition factor)=100×(body weight,g)/(body length,cm)3.SSI(spleensomatic index,%)=100×spleen weight/whole body weight.VSI(viscerosomatic index,%)=100×viscera weight/whole body weight.HSI(hepatosomatic index,%)=100×liver weight/whole body weight.220H.H.Yu et al./Aquaculture432(2014)217–224include shortening of mucosal folds,loss of vacuoles in absorptive enterocytes,widening of lamina propria,increasing of connective tissue and in ﬂammatory cell in ﬁltration in lamina propria (Baeverfjord and Krogdahl,1996;Knudsen et al.,2007).We did not ﬁnd intestinal pathol-ogy when 40%dietary soybean protein products (20%soybean protein concentrate and 21%soybean meal)were used in extruded diets of Japanese seabass in previous and present studies (Hu et al.,2010).Bonaldo et al.(2011)and Murray et al.(2010)reported that turbot (Psetta maxima )and Atlantic halibut (Hippoglossus hippoglossus )fed diets with 22.5%–30%of soybean meal did not show gut histological changes.However,we noticed that a diet with 20%of soybean meal induced enteritis in Atlantic salmon (Urán et al.,2008),even if only fed for 7days,which pointed out the species differences in tolerance of dietary plant proteins.The inclusion of YCW in a soybean meal inclusion diet improved the gut morphology in the present study (Table 6).Previous studies also demonstrated that YCW or its sub-components bene ﬁtted the gut development,such as increased micro-villi density and length (Dimitroglou et al.,2010),improved gross morphological absorptive surface area (Dimitroglou et al.,2009)and prevented soybean meal-induced enteritis (Refstie et al.,2010).The mode of action of YCW to improve gut health was not fully understood.However,it was reported that optimal β-glucan inclusion levels in the diet could reduce in ﬂammatory response (Falco et al.,2012)and stimulate the wound healing process of ﬁsh (Przybylska et al.,2013).Mannanoligosaccharides (MOS)improved the growth of bene ﬁcial bacteria and prevent the colonization of pathogenic bacteria in the gut of chicken and Atlantic salmon (Fernandez et al.,2002;Green et al.,2013).The gut is not only responsible for digestion and absorption of nutrients but also involved in the immunity of aquatic animals.The intestinal mucosa is an important part of immune system,rich in differ-ent types of immune cells and elicits local responses (Press and Evensen,1999).Although there are several studies reported that ﬁsh also have IgT (Zhang et al.,2011)and IgD (Hirono et al.,2003),IgM is the most widely studied immunoglobulin in ﬁsh (Bag et al.,2009;Estensoro et al.,2012;Purcell et al.,2012).Results of the present study show that plasma IgM could be a good biomarker for evaluating the immune status of ﬁsh when meeting an exogenous challenge.In contrast,the innate immunity parameters could be reacted in some irregular rules.In fact,a unique characteristic of the immune system resides in its capacity to maintain homeostasis and tightly regulate perturbations induced by endogenous (in ﬂammation)and exogenous (infection)sig-nals.Before the bacterial challenge in the present study,in ﬂammation might induced the higher speci ﬁc and innate immune response of ﬁsh fed Fla and Y0diets,although we did not work on the intestinal histol-ogy of Fla group,we did ﬁnd the slight pathological reaction in anterior intestine of ﬁsh fed Y0diet.The increased number and size of GC,and broaden width of mucosal folds in this group could be related with the requirement of compensatory nutrients absorption under the adventitious stress related with antinutritional factors or unbalanced nutrients of SBM diet.Intestinal goblet cell is a specialized epithelial20 40 60 80 100120 1234567C u m u l a t i v e S u r v i v a l %DaysFM Fla Y0Y1Y2Y3Y4Y5Fig.2.Effects of dietary YCW on 7-day cumulative survival of Japanese seabass challenged with Aeromonas veronii .Table 4Effects of dietary YCW on immune parameters before and after challenged with Aeromonas veronii of Japanese seabass (means n =12for the samples before challenge and n =10for the samples after challenge).FMFlaY0Y1Y2Y3Y4Y5P value CT C*T C3(μg/mL)BC 1.26±0.01 1.29±0.02 1.24±0.04 1.26±0.04 1.29±0.03 1.30±0.03 1.34±0.05 1.30±0.02***AC 1.27±0.05–1.23±0.05–1.29±0.03 1.26±0.04 1.22±0.05 1.24±0.04IgM (μg/mL)BC 12.2±2.05X 25.7±2.6721.2±2.1418.6±0.5617.7±2.07X 17.2±1.35X 13.6±1.15X 16.7±1.90X *****AC 24.9±1.85Y –22.3±1.75–30.8±1.42Y 25.2±1.84Y 20.5±2.52Y 26.6±3.53Y MPO (OD)BC 2.65±0.22X 2.54±0.34 5.33±0.90X 2.84±0.32 2.90±0.23X 2.90±0.21X 2.87±0.49X 2.13±0.30X *****AC 12.6±1.46Y –12.4±2.02Y –9.98±1.78Y 10.5±1.17Y 9.66±1.16Y 15.7±1.83Y NBT (OD)BC 0.35±0.01X 0.31±0.010.27±0.01X 0.28±0.020.22±0.01X 0.30±0.01X 0.26±0.01X 0.21±0.01X ******AC 0.51±0.02Y –0.57±0.02Y –0.54±0.01Y 0.57±0.01Y 0.54±0.01Y 0.54±0.02Y NO (μmol/L)BC 3.24±0.13X 3.82±0.72 2.42±0.25X 2.07±0.42 2.81±0.37X 2.14±0.34X 1.53±0.37X 1.53±0.91X ****AC 38.9±15.9Y –27.7±10.2Y –36.7±9.39Y 33.0±12.1Y 29.6±7.36Y 44.6±14.4Y Anti-superoxide anion (U/L)BC 175±8.39172±5.38172±9.15164±2.92163±7.27189±6.75184±6.40193±7.37****AC178±7.50–199±6.77–169±8.78189±6.65177±5.18180±8.83Within the same row for same parameter,different lowercase superscripts indicate signi ﬁcant differences by dietary treatment and uppercase (X and Y)signi ﬁcant differences by challenge (P b 0.05).BC:before challenge;AC:after challenge;C:challenge;T:treatments.**P b 0.05;*P N 0.05.Table 5Effects of dietary YCW on hematological parameters of Japanese seabass (means ±S.E.n =12).Y0Y4Y5Normal ranges a ALT (U/L) 4.98±1.6013.8±4.248.70±3.362–33AST (U/L)20.9±5.7177.0±40.862.4±16.68–130Glucose (mmol/L) 5.18±0.46 5.48±0.86 4.27±0.4310–18BUN (mmol/L) 3.13±0.17 3.78±0.24 3.65±0.18–TPRO (g/L)26.4±0.4030.6±0.5636.5±0.6436–42ALB (g/L)13.3±0.5113.4±0.5613.4±0.34–TBILI (μmol/L)12.6±0.1512.2±0.2212.6±0.16–TC (mmol/L) 5.60±0.22 5.24±0.19 5.59±0.31 3.8–7.5TG (mmol/L) 4.53±0.36 4.89±0.37 5.63±0.784–6ALP (U/L)19.0±0.6116.3±4.7622.0±2.3130–50CRE (μmol/L)NDNDND–Within the same row,values with different superscripts are signi ﬁcantly different (P b 0.05).ND:Not Detected.aHu (2010),Liu et al.(2010),and Wu et al.(2013).221H.H.Yu et al./Aquaculture 432(2014)217–224。

给水排水工程专业英语文献翻译原文第三篇

Journal of Membrane Science 376 (2011) 196–206Contents lists available at ScienceDirectJournal of MembraneSciencej o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /m e m s ciFouling and cleaning of RO membranes fouled by mixtures of organic foulants simulating wastewater efﬂuentWui Seng Ang 1,Alberto Tiraferri,Kai Loon Chen 2,Menachem Elimelech ∗Department of Chemical and Environmental Engineering,P.O.Box 208286,Yale University,New Haven,CT 06520-8286,USAa r t i c l e i n f o Article history:Received 6December 2010Received in revised form 7April 2011Accepted 9April 2011Available online 20 April 2011Keywords:Reverse osmosis FoulingWastewater efﬂuent CleaningOrganic foulantsWastewater treatment Efﬂuent organic matter Wastewater reclamation Membranesa b s t r a c tThe fouling and subsequent cleaning of RO membranes fouled by a mixture of organic foulants sim-ulating wastewater efﬂuent has been systematically investigated.The organic foulants investigated included alginate,bovine serum albumin (BSA),Suwannee River natural organic matter,and octanoic acid,representing,respectively,polysaccharides,proteins,humic substances,and fatty acids,which are ubiquitous in efﬂuent organic matter.After establishing the fouling behavior and mechanisms with a mixture of organic foulants in the presence and absence of calcium ions,our study focused on the clean-ing mechanisms of RO membranes fouled by the mixture of organic foulants.The chemical cleaning agents used included an alkaline solution (NaOH),a metal chelating agent (EDTA),an anionic surfactant (SDS),and a concentrated salt solution (NaCl).Speciﬁcally,we examined the impact of cleaning agent type,cleaning solution pH,cleaning time,and fouling layer composition on membrane cleaning efﬁ-ciency.Foulant–foulant adhesion forces measured under conditions simulating chemical cleaning of a membrane fouled by a mixture of the investigated organic foulants provided insights into the chemical cleaning mechanisms.It was shown that while alkaline solution (NaOH)alone is not effective in dis-rupting the complexes formed by the organic foulants with calcium,a higher solution pH can lead to effective cleaning if sufﬁcient hydrodynamic shear (provided by crossﬂow)prevails.Surfactant (SDS),a strong chelating agent (EDTA),and salt solution (NaCl)were effective in cleaning RO membranes fouled by a mixture of foulants,especially if applied at high pH and for longer cleaning times.The observed cleaning efﬁciencies with the various cleaning agents were consistent with the related measurements of foulant–foulant intermolecular forces.Furthermore,we have shown that an optimal cleaning agent con-centration can be derived from a plot presenting the percent reduction in the foulant–foulant adhesion force versus cleaning agent concentration.© 2011 Elsevier B.V. All rights reserved.1.IntroductionAs demand for potable water increases worldwide,the paradigm for selecting water sources to meet this demand is transitioning from conventional sources,such as reservoirs and lakes,to less con-ventional sources,such as treated secondary wastewater efﬂuent.In order to produce water of superior quality,the use of mem-branes in desalination and wastewater reclamation has become more widespread.Membrane fouling is a major impediment to the use of membrane technology for such applications,because fouling is inevitable.Despite research efforts to develop better anti-fouling membranes [1]and improved fouling-control strategies [2,3],membrane fouling still occurs over time.Thus,a long-term∗Corresponding author.Tel.:+12034322789;fax:+12034324387.E-mail address:menachem.elimelech@ (M.Elimelech).1Current address:Public Utility Board of Singapore,Singapore.2Current address:Department of Geography and Environmental Engineering,Johns Hopkins University,Baltimore,MD 21218,United States.solution would be to remove the foulant deposited on the mem-brane via chemical cleaning.To select the appropriate cleaning agents and adopt an effective chemical cleaning protocol for fouled membranes in wastewater reclamation,the implications of wastewater efﬂuent characteris-tics on membrane fouling have to be well-understood.Wastewater efﬂuent contains dissolved organic matter,commonly known as efﬂuent organic matter (EfOM),which comprises polysaccha-rides,proteins,aminosugars,nucleic acids,humic and fulvic acids,organic acids,and cell components [2–4].Organic fouling of the RO membranes by the EfOM can be extensive since EfOM is gener-ally small enough to pass through the pores of pretreatment (MF or UF)membranes [4].In particular,recent ﬁndings suggest that while biofouling can prevail on the tail-element of the membrane module,fouling of the lead-element exposed to reclaimed water is dominated by EfOM adsorption [5].In addition,higher potential of fouling was observed for the higher molecular weight hydropho-bic/aromatic fraction of the EfOM [6,7].The presence of Ca 2+in the feed source for the RO membranes has been reported to form complexes with the constituents of EfOM,such as polysaccharidesW.S.Ang et al./Journal of Membrane Science376 (2011) 196–206197[8]and natural organic matter[9],and to signiﬁcantly enhance membrane fouling.While our previous studies have addressed the fouling of RO membranes by individual organic foulant types,such as polysaccharides[10],proteins[11],and fatty acids[12],only recently have investigations reported on the effects of a combina-tion or mixture of foulants on the fouling of RO membranes[13,14].A variety of chemical cleaning agents are commonly used to clean RO membranes fouled by organic matter[15].Alkaline solutions remove organic foulants on membranes by hydroly-sis and solubilization of the fouling layer.Alkaline solutions also increase the solution pH,and,therefore,increase the negative charges and solubility of the organic foulant.Metal chelating agents remove divalent cations from the complexed organic molecules and weaken the structural integrity of the fouling layer matrix[16]. Surfactants solubilize macromolecules by forming micelles around them[17],thereby facilitating removal of the foulants from the membrane surface.In our earlier study on salt cleaning of organic matter-fouled RO membranes[18],we demonstrated that NaCl and other common inert salts can be used as an effective alternative for the cleaning of RO membranes fouled by gel-forming hydrophilic organic foulants.In the presence of a salt solution,the fouling layer swells and becomes more porous.As a result,this would facil-itate the diffusion of Na+into the fouling layer and breakup of Ca2+–alginate bonds by ion exchange.Understanding the fouling layer characteristics and the interaction of chemical agents with foulants is therefore critical for the effective cleaning of organic matter-fouled RO membranes.Atomic force microscopy(AFM)has been applied in mem-brane fouling/cleaning research to quantify intermolecular forces [10,19–21].Our research has shown that foulant–foulant inter-actions could be determined by performing force measurements using a carboxylate-modiﬁed latex colloid probe in an AFMﬂuid cell[10,20].The technique has been used to quantify the foul-ing behavior of a nanoﬁltration membrane fouled by humic acid and the cleaning efﬁciencies of EDTA and SDS[20],and has been extended to quantify RO membrane fouling by organic foulant in the form of alginate[10],BSA[11],and octanoic acid[12].In this study,the AFM has also been employed as an alternative tool to indicate the optimal concentration of cleaning agent for cleaning fouled membranes.The original protocol[11,12]for using the AFM has been modiﬁed to investigate the intermolecular adhesion force between different foulants.The objective of this study is to explore the mechanisms govern-ing the fouling of RO membranes by mixtures of organic foulants simulating wastewater efﬂuent,and the ensuing chemical cleaning of the fouled membranes by cleaning agents.To make this study rel-evant to wastewater reclamation,we systematically investigate the fouling of RO membranes by each individual organic foulant type (polysaccharides,proteins,humic acids,or fatty acids)and mix-tures containing several types of organic foulants in the absence and presence of calcium ions.Cleaning experiments are performed with the fouled membranes using NaOH,EDTA,SDS,and NaCl as model alkaline solution,metal chelating agent,surfactant,and salt cleaning solution,respectively.The intermolecular adhesion forces between the different foulants and estimated aggregate sizes in foulant mixtures were used to explain the fouling mechanism of RO membranes and the cleaning behavior of a cleaning agent on the fouled membranes.2.Materials and methodsanic foulants Louis,MO),Suwannee River natural organic matter(SRNOM) (International Humic Substances Society,St.Paul,MN),bovine serum albumin(BSA)(Sigma–Aldrich,St.Louis,MO),and octanoic acid(OA)(Sigma–Aldrich,St.Louis,MO),respectively.According to the manufacturer,the molecular weight of the sodium alginate ranges from12to80kDa.Other characteristics of SRNOM,includ-ing molecular weight and mass fraction of hydrophobic NOM,can be found elsewhere[22,23].According to the manufacturer,the molecular weight of the BSA is about66kDa.BSA is reported to have an isoelectric point at pH4.7[24].Octanoic acid(Sigma–Aldrich,St. Louis,MO)was selected to model fatty acids in EfOM because of its presence in food and solubility in water(saturation concentration of4.7mM at20◦C)[12].Sodium alginate,BSA,and SRNOM were received in powder form,and stock solutions(2g/L)were prepared by dissolving each of the foulants in deionized(DI)water.DI water was supplied from a Milli-Q ultrapure water puriﬁcation system(Millipore,Billerica, MA).Mixing of the stock solutions was performed for over24h to ensure complete dissolution of the foulants,followed byﬁl-tration with a0.45-␮mﬁlter(Durapore,Millipore,Billerica,MA). Theﬁltered stock solutions were stored in sterilized glass bottles at4◦C.Octanoic acid was received in solution(≥98%concentra-tion)and was stored at room temperature.To achieve the intended octanoic acid concentration during fouling,octanoic acid was dis-solved separately for at least8h prior to fouling so that,at the initiation of fouling,octanoic acid could be introduced as a solu-tion.A few hours before the initiation of fouling,the ionic strength of the stock solution was adjusted to the same concentration as that of the feed solution(10mM)and the stock solution pH was elevated,as needed,from ambient pH of3.9–9.0by adding small amounts of1M NaOH.2.2.Chemical cleaning agentsThe chemical cleaning agents used were:NaOH(pH11.0)as an alkaline solution,certiﬁed grade disodium ethylenediaminete-traacetate(Na2–EDTA)as a metal chelating agent,certiﬁed grade sodium dodecyl sulfate(SDS)as an anionic surfactant,and NaCl as a salt cleaning solution.The agents were purchased from Fisher Sci-entiﬁc(Pittsburgh,PA)and used with no further puriﬁcation.The stock chemical solutions were prepared fresh by dissolving each chemical in deionized(DI)water.The pH of the EDTA,SDS,and NaCl cleaning solutions was adjusted with1.0M NaOH as necessary.2.3.RO membraneThe relatively well-characterized thin-ﬁlm composite LFC-1 membrane(Hydranautics,Oceanside,CA)was used as a model RO membrane.The average hydraulic resistance was determined to be 9.16(±0.11)×1013m−1corresponding to a hydraulic permeabil-ity of10.9(±0.13)×10−11m s−1Pa−1.The observed salt rejection was98.7–99.3%,determined with a10mM(584mg/L)NaCl feed solution at an applied pressure of300psi(2068.5kPa)and a cross-ﬂow velocity of8.6cm/s.Membrane samples were received as dry large sheets,and were cut and stored in DI water at4◦C.The membrane has been reported to be negatively charged at solu-tion chemistries typical to wastewater efﬂuents,with an isoelectric point at about pH4.6[25].The membrane has been reported to be coated with a neutral polyalcohol layer rich in–COH functional groups,which renders the surface less charged than the surfaces of other polyamide RO membranes without a coating layer[25,26].2.4.Crossﬂow test unit198W.S.Ang et al./Journal of Membrane Science376 (2011) 196–206unit consists of a membrane cell,pump,feed reservoir,temper-ature control system,and data acquisition system.The membrane cell consisted of a rectangular plate-and-frame unit,which con-tained aﬂat membrane sheet placed in a rectangular channel with dimensions measuring7.7cm long,2.6cm wide,and0.3cm high. Both permeate and retentate were recirculated back to the feed reservoir.Permeateﬂux was registered continuously by a digital ﬂow meter(Optiﬂow1000,Humonics,CA),interfaced with a com-puter.Aﬂoating disc rotameter(King Instrument,Fresno,CA)was used to monitor the retentateﬂow rate.The crossﬂow velocity and operating pressure were adjusted using a bypass valve(Swagelok, Solon,OH)in conjunction with a back-pressure regulator(U.S.Para Plate,Auburn,CA).Temperature was controlled by a recirculating chiller/heater(Model633,Polysciences)with a stainless steel coil submerged in the feed water reservoir.2.5.Fouling and cleaning experimentsThe membrane wasﬁrst compacted with DI water until the permeateﬂux became constant,followed by the initial baseline performance for1h.The membrane was then stabilized and equi-librated with a foulant-free electrolyte solution for2h.Theﬂux at which the baseline run was performed was predetermined so that the initialﬂux would drop to a speciﬁedﬂux of2.3×10−5m s−1(or 83L m−2h−1)after adding the electrolyte solution.The chemistry of the foulant-free electrolyte solution and operating conditions adjusted in this stage were similar to those used for the ensuing fouling runs.As octanoic acid takes time to dissolve completely,the mixture of organic foulant solution has to be prepared8h before the fouling run.The feed foulant solution was prepared separately in another container.The chemistry of the feed foulant solution was adjusted to be identical to that of the foulant-free electrolyte solution so that the overall ionic strength and solution chemistry would not change when the feed foulant solution was added to initiate fouling. Fouling runs were carried out for17h.At the end of the fouling run, the solution in the feed reservoir was disposed off and chemical cleaning solution was added to the feed reservoir to clean the fouled membrane.At the end of the cleaning stage,the chemical cleaning solution in the reservoir was discarded,and both the reservoir and membrane cell were rinsed with DI water toﬂush out the residual chemical cleaning solution.Finally,the cleaned RO membrane was subjected to the second baseline performance with DI water to re-determine the pure waterﬂux.The crossﬂow velocity throughout the experiment,except dur-ing cleaning,was maintained at8.6cm/s.The operating conditions (i.e.,initialﬂux,crossﬂow velocity,and temperature)at this stage were identical to those applied during the initial baseline perfor-mance,so as to determine the cleaning efﬁciency by comparing the pure waterﬂuxes determined before fouling and after clean-ing.Throughout all the fouling/cleaning stages,the feed water in the reservoir,which was located on top of a magnetic stirrer,was mixed rigorously to ensure complete mixing of the feed water and cleaning solution.To conﬁrm the reproducibility of determined cleaning efﬁ-ciency,selected fouling/cleaning runs were duplicated.Results showed that fouling rate and cleaning efﬁciency obtained from the duplicate runs were within less than a5%difference.To investigate the change in the permeate quality during the fouling stage,permeate samples taken before and at the start and end of fouling were analyzed for salt(NaCl)rejection using an ICP-AES(ICP Optima3000,Perkin Elmer,Waltham,MA).Permeate and feed samplings obtained before the fouling run were collected at the end were collected during theﬁnal40min of the fouling run.2.6.AFM adhesion force measurementsAtomic force microscopy(AFM)was used to measure the inter-facial force between the foulant in the bulk solution and the foulant in the fouling layer on the membrane.The force measurements were performed with a colloid probe,modiﬁed from a commercial-ized SiN AFM probe(Veeco Metrology Group,Santa Barbara,CA).A carboxylate modiﬁed latex(CML)particle(Interfacial Dynam-ics Corp.,Portland,OR)was used as a surrogate for the organic foulants,because organic foulants(alginate and SRNOM)carry pre-dominantly carboxylic functional groups.To make a colloid probe, a CML particle with a diameter of4.0␮m was attached using Nor-land Optical adhesive(Norland Products,Inc.,Cranbury,NJ)to a tipless SiN cantilever.The colloid probe was cured under UV light for20min.The AFM adhesion force measurements were performed in a ﬂuid cell using a closed inlet/outlet loop.The solution chemistries of the test solutions injected into theﬂuid cell were identical to those used in the bench-scale fouling/cleaning experiments.Once all the air bubbles had beenﬂushed out of theﬂuid cell,the injection would stop and the outlet was closed.The membrane was equilibrated with the test solution for30–45min before force measurements were performed.The force measurements were conducted at three toﬁve different locations,and at least10measurements were taken at each location.Because the focus of this study was on the foulant–foulant interaction(adhesion),only the raw data obtained from the retracting force curves were processed and converted to obtain the force versus surface-to-surface separation curves.The force curves presented were the averages of all the representative force curves obtained at the different locations.The protocol for AFM analysis has been modiﬁed slightly to investigate the interaction between different foulant types. The AFM colloidal probe is soaked in organic foulant solution (2000mg/L alginate,BSA,or SRNOM,or>98%octanoic acid)for at least24h(at4◦C for alginate,BSA,and SRNOM solutions to prevent organic degradation,and at room temperature for octanoic acid). The membrane is fouled with200mg/L organic foulant(alginate, BSA,SRNOM,or octanoic acid)using the crossﬂow unit for about 17h.After transferring the colloidal probe to the AFMﬂuid cell and the membrane to the AFM disc puck,an electrolyte solution con-taining0.5mM CaCl2and8.5mM NaCl(adjusted to pH6.5±0.2) (identical solution chemistry as during fouling)is injected into the ﬂuid cell.The volume of electrolyte solution added is just enough toﬁll up theﬂuid cell so as to minimize the possibility ofﬂush-ing away the foulants on the membrane and probe surfaces.AFM force measurements are taken after20min of equilibration time.To investigate the effect of cleaning agent on the intermolecular adhe-sion force,the cleaning agent was added to the electrolyte solution at the same concentration as that used in the cleaning experiments.2.7.Light scatteringDynamic light scattering experiments were performed on foulant solution to determine the effective hydrodynamic diam-eters of the foulant aggregates in foulant mixtures using a multi-detector light scattering unit(ALV-5000,Langen,Germany). New glass vials(Supelco,Bellefonte,PA)for containing foulant solu-tions under various solution chemistries were cleaned prior to use by soaking overnight in a cleaning solution(Extran MA01,Merck KGaA,Darmstadt,Germany),rinsing with DI water,and drying inW.S.Ang et al./Journal of Membrane Science376 (2011) 196–206199Fig.1.Inﬂuence of individual foulant type on fouling of LFC-1membranes:(a)in the absence of Ca2+and(b)in the presence of0.5mM Ca2+.The total ionic strength of the feed solution wasﬁxed at10mM by adjusting with NaCl and the feed solution pH was adjusted to6.0±0.2,as necessary,by adding NaOH.Fouling conditions: foulant concentration of25mg/L,initial permeateﬂux of23␮m/s(or83L m−2h−1), crossﬂow velocity of8.6cm/s,and temperature of21.0±0.5◦C.of1M NaOH.The vial containing the foulant solution was vortexed (Mini Vortexer,Fisher Scientiﬁc)to homogenize the solution.The vial was then allowed to sit for30min before starting the light scattering experiment.All light scattering measurements were conducted by employ-ing the detector positioned at a scattering angle of90◦from the incident laser beam.The detector signal was fed into the correla-tor,which accumulated each autocorrelation function for15s.The intensity-weighted hydrodynamic radius of the colloidal aggre-gates was determined with second-order cumulant analysis(ALV software)[27].The reported size is the average of theﬁrst20mea-surements.3.Results and discussion3.1.Membrane fouling3.1.1.Fouling with individual foulantsFig.1presents the normalizedﬂux proﬁles for LFC-1mem-branes fouled by each individual foulant(alginate,BSA,SRNOM, or octanoic acid)in the absence(Fig.1a)and presence(Fig.1b)of Ca2+,respectively.In the absence of Ca2+,theﬂux decline proﬁles of membranes fouled by the various foulants are insigniﬁcant.The 2+Fig.2.Inﬂuence of a mixture of(a)2foulants or(b)more than2foulants on fouling of LFC-1membranes in the presence of0.5mM Ca2+.The total ionic strength of the feed solution wasﬁxed at10mM by adjusting with NaCl and the feed solution pH was adjusted to6.0±0.2,as necessary,by adding NaOH.Fouling conditions were identical to those in Fig.1.RO membranes by BSA,SRNOM,or octanoic acid is minimal.How-ever,we have observed that the presence of Ca2+can affect fouling behavior when the foulant concentrations are higher(300mg/L BSA;2mM or288mg/L octanoic acid)[11,12].3.1.2.Fouling with mixture of foulantsTo investigate the implications for wastewater reclamation, the effect of Ca2+on fouling of RO membranes by all possible combinations of two or more foulant types is investigated.The con-centration of each foulant type was maintained at25mg/L.Fig.2a shows the normalizedﬂux proﬁles of membranes fouled by a mix-ture of two foulants in the presence of Ca2+.The effect of Ca2+is most signiﬁcant for feed solutions containing alginate as one of the two foulant types.This mechanism will be further investigated with the aid of DLS and AFM paring theﬂux proﬁles of mem-branes fouled by alginate as a co-foulant,theﬂux-decline proﬁle of membrane fouled by alginate and octanoic acid is the least sig-niﬁcant due to the formation of octanoic acid–calcium complexes, which increase the hydrophilicity of the fouling layer[12].Fig.2b shows the normalizedﬂux proﬁles of membranes fouled by a mixture of three foulant types and all foulant types in the presence of Ca2+.In the presence of Ca2+,for membranes fouled by mixtures containing alginate,the effect of Ca2+onﬂux proﬁles is most signiﬁcant,especially for the membrane fouled by a mixture of alginate,BSA,and SRNOM(without octanoic acid).In compar-ing the latter with theﬂux proﬁle of the membrane fouled by all200W.S.Ang et al./Journal of Membrane Science376 (2011) 196–206Fig.3.Sodium ion(Na+)rejection of RO membranes measured before,and at the start and end of the fouling runs,at an adjusted feed solution pH of6.0.The membranes were fouled by combined foulant types,composed of25mg/L each of alginate,BSA,SRNOM,and octanoic acid.Permeate and feed samples obtained before the fouling run were collected30min before the onset of fouling.Samples taken at the start of the fouling run were initiated afterﬁrst discarding20mL of permeate(duration of8min).Permeate and feed samples taken at the end were collected during theﬁnal40min of the fouling run.Error bars indicate one standard deviation.Fouling conditions were identical to those in Fig.1.contained alginate and octanoic acid in the presence of Ca2+.The inhibitory effect of octanoic acid onﬂux-decline proﬁles can also be observed by comparing theﬂux proﬁle of combined foulant types of alginate,SRNOM,and octanoic acid in Fig.2b with the proﬁle of alginate and SRNOM in Fig.2a.3.1.3.Impact of fouling on salt rejectionFig.3presents Na+rejection of the RO membranes fouled by combined foulant types of alginate,BSA,SRNOM,and octanoic acid in the presence of Ca2+,at the start and end of the fouling runs.The trend of observed Na+rejection is similar both in the absence and presence of Ca2+.At the onset of fouling,the Na+rejection instan-taneously increases.This phenomenon is consistent with previous observations,which attributed the decrease in Na+permeability to the fouling layer acting as an additional selective barrier[12]. Toward the end of the fouling runs,the fouling layer becomes thicker and denser,resulting in even higher Na+rejection.It can be inferred that the presence of Ca2+resulted in a more compact fouling layer,which improves the ability of the fouling layer to fur-ther act as a selective barrier against the transport of Na+across the membrane.3.2.Fouling mechanisms3.2.1.Role of foulant–foulant interaction and foulant sizeRecent studies have demonstrated that the long-term organic fouling of RO membranes and the consequent behavior of water ﬂux are dominated by the feed water chemistry and strong foulant–foulant interactions[4,20,21,28].Quantifying these inter-actions provides a basis for the understanding of the fouling mechanisms and for the rational selection of a suitable cleaning strategy.As discussed in Section3.1.2,fouling behavior becomes signiﬁcant when alginate is one of the co-foulants.When alginate is absent from the feed solution,regardless of the other foulant types present,fouling is relatively insigniﬁcant.This behavior can be explained by evaluating the interaction forces among the differ-ent foulants.To investigate the effect of interactions of alginate with other foulant types,DLS analysis is performed on a solution contain-ing2foulant types(200mg/L alginate plus200mg/L of another foulant type)in the presence of Ca2+.Fig.4a shows that the alginate molecules in the solution have an effective hydrodynamic diame-ter of84nm,which is larger than the effective diameter of51nm of alginate molecules in a solution in which the foulant concentration is halved.The results imply that aggregation of alginate molecules is concentration dependent.The larger effective diameter of aggre-gates formed in400mg/L alginate solution as opposed to those formed in200mg/L alginate solution also implies a more exten-sive gel network at a higher concentration.The effective diameters of the foulant molecules in mixtures of alginate and BSA,alginate and SRNOM,and alginate and octanoic acid are,respectively,48, 63,and73nm.The effective diameter is an indirect indication of the foulant size due to aggregation between the foulants.Because of the varying interactions between alginate and another foulant type in the presence of Ca2+,the aggregate size differs for foulant aggregates of different foulant combinations.Fig.4b shows the intermolecular forces between foulant adsorbed on a colloidal probe and a membrane fouled by algi-nate as determined by AFM.For a membrane fouled by alginate and octanoic acid,the dominant foulant interactions are between alginate and alginate molecules(1.03mN/m)and between octanoic acid and octanoic acid molecules(0.90mN/m).For a membrane fouled by alginate and SRNOM,the dominant foulant interaction is between alginate and alginate molecules(1.03mN/m).For a membrane fouled by alginate and BSA,the dominant foulantinter-Fig.4.(a)Effective diameter of foulant aggregates in solutions of various foulant combinations that contain alginate as co-foulant.The foulant solution consists of200mg/L alginate plus200mg/L of another foulant type in an electrolyte solution of0.5mM CaCl2and8.5mM NaCl(same solution chemistry as that used in fouling experiments).TheW.S.Ang et al./Journal of Membrane Science376 (2011) 196–206201Fig.5.Proposed structure of fouling layer on membrane surface under different combinations of foulants.actions are between alginate and alginate molecules(1.03mN/m) and between alginate and BSA molecules(0.73–0.79mN/m).We observe that when alginate is present in the feed,regardless of the co-foulant,the interaction of alginate molecules among them-selves is most dominant,with the possibility of alginate molecules interacting with other molecules,especially BSA molecules.Comparing the effective diameters of the foulant aggregates in various2-foulant mixtures(Fig.4a)with the intermolecular adhe-sion force between different foulants(Fig.4b)reveals that there is an inverse correlation between the foulant aggregate size and the intermolecular adhesion force(foulant aggregate size generally decreases as intermolecular adhesion force increases).It is hypoth-esized that the interaction among the foulant types within the aggregates would affect the conformation,and hence,the size of the aggregates in the foulant solution.For example,the relatively stronger intermolecular adhesion force between alginate and BSA molecules in the feed solution in the presence of Ca2+results in a more‘compact’or‘tighter’conformation of the foulant aggregates as compared to the foulant aggregates formed from a solution of alginate and SRNOM.The deposition of the smaller and more‘com-pact’alginate–BSA aggregates results in a tighter fouling layer and a lowerﬁnalﬂux(Fig.5)[29].We note that the SA–SA aggregate does not follow the trend of a decrease in aggregate size with increasing adhesion force because alginate molecules tend to form extended gel networks in the presence of calcium ions[29],as opposed to the other combinations of foulants.3.2.2.Proposed structure of fouling layerThe fouling experiments reveal that membrane fouling in the presence of Ca2+is controlled by alginate.From the AFM force mea-surement analysis and the DLS experiments,the proposed structure of the fouling layer when severe fouling occurs under various solu-tion chemistries is schematically shown in Fig.5.The top drawing shows the likely conformation of the cross-linked alginate fouling layer when the feed contains alginate in the presence of Ca2+.In this case,the fouling layer has the typical structure resulting from the formation of an‘egg-box’shaped gel network on the mem-brane surface[29].The middle drawing shows the proposed fouling layer formed by a feed solution containing a mixture of alginate, BSA,SRNOM,and octanoic acid(each foulant has the same concen-tration).The DLS experiments show that the aggregates of foulant mixtures containing alginate as a co-foulant have smaller effective feed solution in which alginate is the sole foulant.When the algi-nate concentration is increased while maintaining the same total foulant concentration,the fouling layer becomes more porous due to the increase in the highly ordered alginate–calcium complexes on the membrane surface.The state of the fouling layer would affect the transfer of a cleaning agent to the fouling layer,and hence,the cleaning efﬁciency of the cleaning agent as delineated in the next sections.3.3.Cleaning of fouled membranes3.3.1.Type of cleaning agentFig.6presents the cleaning efﬁciencies of various cleaning agents on membranes fouled by combined foulant types compris-ing alginate,BSA,SRNOM,and octanoic acid in the presence of 0.5mM Ca2+.Cleaning was performed for15min without an oper-ating pressure(i.e.,no permeate)and at a crossﬂow velocityﬁve times higher than that during fouling.Cleaning the fouled mem-brane with DI water resulted in19%cleaning efﬁciency,which implies that the fouling layer on the membrane surface was largely irreversible.Conventional cleaning agents,such as NaOH(pH11),Fig.6.Cleaning efﬁciencies of various cleaning agents on membranes fouled by combined foulant types comprising alginate,BSA,SRNOM,and octanoic acid,with the concentration of each foulant type at25mg/L,in the presence of0.5mM Ca2+.Cleaning conditions:time,15min;temperature,21±0.5◦C;and no applied。

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JournalofEnvironmentalSciences21(2009)1169–1174Denitrifyingphosphorusremovalinastep-feedCASTwithalternatinganoxic-oxicoperationalstrategy

MAJuan1,PENGYongzhen1,∗,WANGShuying1,WANGLi1,LIUYang2,MANingping21.KeyLaboratoryofBeijingforWaterQualityScienceandWaterEnvironmentRecoveryEngineering,CollegeofEnvironmentalandEnergyEngineering,BeijingUniversityofTechnology,Beijing100124,China.E-mail:majuan@emails.bjut.edu.cn2.ChinaWaterHoldingsPrivateLimited,28KakiBukitCrescent,KakiBukitTechparkIS416259,416259,Singpore.

Received26November2008;revised14January2009;accepted24March2009

AbstractAbench-scalecyclicactivatedsludgetechnology(CAST)wasoperatedtostudythebiologicalphosphorusremovalperformanceandaseriesofbatchtestswascarriedouttodemonstratetheaccumulationofdenitrifyingpolyphosphate-accumulatingorganisms(DNPAOs)inCASTsystem.Underalloperatingconditions,step-feedCASTwithenoughcarbonsourcesininﬂuenthadthehighestnitrogenandphosphorusremovaleﬃciencyaswellasgoodsludgesettlingperformance.TheaverageremovalrateofCOD,NH4

+-N,

PO43−-Pandtotalnitrogen(TN)was88.2%,98.7%,97.5%and92.1%,respectively.Theaveragesludgevolumeindex(SVI)was

133mL/g.Theoptimumanaerobic/aerobic/anoxic(AOA)conditionsforthecultivationofDNPAOscouldbeachievedbyalternatinganoxic/oxicoperationalstrategy,thusasigniﬁcantdenitrifyingphosphorusremovaloccurredinstep-feedCAST.ThedenitriﬁcationofNOx−-Ncompletedquicklyduetostep-feedoperationandenoughcarbonsources,whichcouldenhancephosphorusreleaseand

furtherphosphorusuptakecapabilityofthesystem.Batchtestsalsoprovedthatpolyphosphate-accumulatingorganisms(PAOs)inthestep-feedprocesshadstrongdenitrifyingphosphorusremovalcapacity.Bothnitrateandnitritecouldbeusedaselectronacceptorsindenitrifyingphosphorusremoval.LowCODsupplywithstep-feedoperationstrategywouldfavorDNPAOsaccumulation.

Keywords:cyclicactivatedsludgetechnology;biologicalphosphorusremoval;denitrifyingpolyphosphate-accumulatingorganismsDOI:10.1016/S1001-0742(08)62398-0

IntroductionOverthepastseveraldecades,biologicalnutrientre-moval(BNR)processesinconjunctionwithchemicaloxygendemand(COD)havebeenwidelyusedtotreatwastewatercontainingnitrogenandphosphorusduetotheireconomicadvantages.ThedevelopedBNRsystemsincludesequencingbatchreactor(SBR),theUniversityofCapeTown(UCT)system,theBardenphoprocessandtheanaerobic/anoxic/oxic(A2O)system,andsoon

(Tchobanoglousetal.,2003;Pengetal.,2006;Yangetal.,2007).Cyclicactivatedsludgetechnology(CAST),atypeofSBR,hasattractedagreatdealofinterestinrecentyears,becauseithasagoodperformanceinbiologicalnutrientre-movalfromwastewaterandtheabilityofpreventingsludgebulkingwithaselector.Beingdiﬀerentfromcontinuousﬂowactivatedsludgesystems,variousbiologicalreactionsareswitchableinthesamereactor.InCAST,clariﬁersandﬂowequalizationtanksareunnecessary,andthus,thecostsoffacilitiesandoperationmanagementaremuchlowerthanthosewithcontinuousﬂowactivatedsludgesystems.Moreover,operationconditionscanbechangedeasilyin

*Correspondingauthor.E-mail:pyz@bjut.edu.cn

CAST(Irvineetal.,1997).Therefore,CASTisregardedasaneﬀectivetechnology,especiallyforsmallwastewatertreatmentplants.Enhancedbiologicalphosphorusremoval(EBPR)isthemosteconomicalandsustainableprocessforremov-ingphosphorusfromwastewater.EBPRisachievedbyrecyclingpolyphosphate-accumulatingorganisms(PAOs)throughalternatinganaerobicandaerobicconditions.PAOstakeupcarbonsourceswhilereleasingorthophos-phateintheanaerobicphaseandstorethemintheformofpolyhydroxyalkanoates(PHA),usingtheenergyproducedthroughthehydrolysisofintracellularpolyphosphate.Inthesubsequentaerobicphase,PAOsgrowandtakeuporthophosphatebyusingthestoredPHAasthecarbonandenergysources(Zengetal.,2004).Phosphorusuptakealsooccursunderanoxiccondition.PreviousstudieshaveshownthatatleastsomePAOs,calleddenitrifyingPAOs(DNPAOs),areabletooxidizetheirintracellularPHAwithnitrateand/ornitriteastheterminalelectronacceptor,andthusprovideenergyforphosphorusuptake(Kubaetal.,1996,1997;Minoetal.,1998;Ahnetal.,2001,2002;Shojietal.,2003).ThismeansthatthecarbonsourcetakenupbyPAOsintheanaerobicphaseisusedforbothdenitriﬁcationandphosphorusremoval,whichisadvantageouswhen