《水污染控制原理》课程教学资源（文献资料）Changes of microbial substrate metabolic patterns through a wastewater reuse process, including WWTP and SAT concerning depth

WATERRESEARCH60(20I4)I05-II7Availableonlineatwww.sciencedirect.comWESTERCHScienceDirectjournalhomepage:www.elsevier.com/locate/watresELSEVIERChanges of microbial substrate metabolicpatternsCrossMarkthrough a wastewater reuse process, includingWWTP andSATconcerningdepthYugo Takabea,b,1, Ippei Kameda a,s1, Ryosuke SuzukiaiFumitake Nishimura a,1, Sadahiko Itoh a,1armniromentaginering,yiesityoigaku-auraNhkyuyoapbRecycling Research Team,Materials and Resources ResearchGroup,Public Works ResearchInstitute,6Minamihara,Tsukuba,Ibaraki305-8516,JapanTokyoEngineering Consultants Co.,Ltd.,3-7-1,Kasumigaseki,Chiyoda-ku,Tokyo100-0013,JapanARTICLEINFOＡBSTRACTArticle history:In this study, changes of microbial substrate metabolic patterns by BIOLOG assay were dis-Received 22 January2014cussed through a sequential wastewater reuse process, which includes activated sludge andReceived in revised formtreated effluent in wastewater treatment plant and soil aquifer treatment (SAT), especially4 April 2014focussing on the surface sand layer in conjunction with the vadose zone, concerning sandAccepted19April2014depth.ASATpilot-scalereactor,in whichtheheightofpackedsandwas237cm (vadosezoneAvailableonline4May201417cmandsaturatedzone220cm),was operatedandfedcontinuouslybydischarged anaerobic-anoxic-oxic(A2O)treatedwater.Continuouswaterqualitymeasurementsoveraperiodof10Keywords:months indicated that the treatment performance of the reactor, such as 83.2% dissolvedWastewater reuseorganiccarbonremoval,appearedtobestable.CoresamplingwasconductedforthesurfaceSoil aquifer treatmentsand toa 30 cm depth,and thesample was divided into six5 cm sections.Microbialactivities, asSurface sand layerevaluatedbyfluoresceindiacetate,sharplydecreased withincreasingdistancefromthesurface of the 30 cm core sample, which included significant decreases only 5 cm from the topActivated sludgeBIOLOGassaysurface.Asimilarmicrobial metabolicpattemcontainingahighdegreeofcarbohydrateswasobtained among the activated sludge,A2Otreated water (influentto the SATreactor)and the0Microbial substratemetabolic-5cmlayerofsand.Meanwhile,the10-30cmsandcorelayersshoweddramaticallydifferentpatternmetabolicpatterns containinga highdegreeofcarboxylicacid andesters,and itis possible thatthe metabolic pattern exhibited by the 5-10 cm layer is at a midpoint of the changing pattern.Thissuggeststhattheremovalof different organiccompoundsbybiodegradationwouldbeexpected to occur in the activated sludge and in the SAT sand layers immediately below5cmfromthetopsurface.Itispossiblethatchangesinthecompositionoftheorganicmatterand/ortransit of the limiting factor for microbial activities from carbon to phosphorus might havecontributedtotheobserveddramaticchangesinSATmetabolicpatterns@2014 Elsevier Ltd. All rights reserved* Corresponding author. Recycling Research Team, Materials and Resources Research Group, Public Works Research Institute, 1-6Minamihara,Tsukuba,Ibaraki305-8516,Japan.Tel:+81298796765;fax:+81298796797E-mail addresses:yu-takabe@pwri.go.jp,takabe.yugo@to2.mbox.media.kyoto-u.ac.jp (Y.Takabe).1TeL/fax:+81753833256.http://dx.doi.org/10.1016/j.watres.2014.04.0360043-1354/@2014Elsevier Ltd.All rights reserved

Changes of microbial substrate metabolic patterns through a wastewater reuse process, including WWTP and SAT concerning depth Yugo Takabe a,b, * ,1 , Ippei Kameda a,c,1 , Ryosuke Suzuki a,1 , Fumitake Nishimura a,1 , Sadahiko Itoh a,1 a Department of Environmental Engineering, Kyoto University, Kyoto-Daigaku-Katsura, Nishikyo-ku, Kyoto 6158540, Japan b Recycling Research Team, Materials and Resources Research Group, Public Works Research Institute, 1-6 Minamihara, Tsukuba, Ibaraki 305-8516, Japan c Tokyo Engineering Consultants Co., Ltd., 3-7-1, Kasumigaseki, Chiyoda-ku, Tokyo 100-0013, Japan article info Article history: Received 22 January 2014 Received in revised form 4 April 2014 Accepted 19 April 2014 Available online 4 May 2014 Keywords: Wastewater reuse Soil aquifer treatment Surface sand layer Activated sludge BIOLOG assay Microbial substrate metabolic pattern abstract In this study, changes of microbial substrate metabolic patterns by BIOLOG assay were dis￾cussed through a sequential wastewater reuse process, which includes activated sludge and treated effluent in wastewater treatment plant and soil aquifer treatment (SAT), especially focussing on the surface sand layer in conjunction with the vadose zone, concerning sand depth. A SAT pilot-scale reactor, in which the height of packed sand was 237 cm (vadose zone: 17 cm and saturated zone 220 cm), was operated and fed continuously by discharged anaerobic eanoxiceoxic (A2O) treated water. Continuous water qualitymeasurements over a period of 10 months indicated that the treatment performance of the reactor, such as 83.2% dissolved organic carbon removal, appeared to be stable. Core sampling was conducted for the surface sand to a 30 cm depth, and the sample was divided into six 5 cm sections.Microbial activities, as evaluated by fluorescein diacetate, sharply decreased with increasing distance from the sur￾face of the 30 cm core sample, which included significant decreases only 5 cm from the top surface. A similar microbial metabolic pattern containing a high degree of carbohydrates was obtained among the activated sludge, A2O treated water (influent to the SAT reactor) and the 0 e5 cm layer of sand. Meanwhile, the 10e30 cm sand core layers showed dramatically different metabolic patterns containing a high degree of carboxylic acid and esters, and it is possible that the metabolic pattern exhibited by the 5e10 cm layer is at a midpoint of the changing pattern. This suggests that the removal of different organic compounds by biodegradation would be expected to occur in the activated sludge and in the SAT sand layers immediately below 5 cm from the top surface. It is possible that changes in the composition of the organicmatter and/or transit of the limiting factor for microbial activities from carbon to phosphorus might have contributed to the observed dramatic changes in SAT metabolic patterns. ª 2014 Elsevier Ltd. All rights reserved. * Corresponding author. Recycling Research Team, Materials and Resources Research Group, Public Works Research Institute, 1-6 Minamihara, Tsukuba, Ibaraki 305-8516, Japan. Tel.: þ81 29 879 6765; fax: þ81 29 879 6797. E-mail addresses: yu-takabe@pwri.go.jp, takabe.yugo@t02.mbox.media.kyoto-u.ac.jp (Y. Takabe). 1 Tel./fax: þ81 75 383 3256. Available online at www.sciencedirect.com ScienceDirect journal homepage: www.elsevier.com/locate/watres water research 60 (2014) 105 e117 http://dx.doi.org/10.1016/j.watres.2014.04.036 0043-1354/ª 2014 Elsevier Ltd. All rights reserved

106WATERRESEARCH60(20I4)I05-II7fromdifferentlocations,includingnatural soil (Campbell etal.1.Introduction1997;GarlandandMills,1991),soilfromconstructedwetlandsandsAT(Salomoetal.,2009;WeberandLegge,2011;ZhangInadequate water supply and deterioration of water qualityetal,2012),freshwater (Garland and Mills,1991)and acti-areseriousproblemsinmanypartsoftheworld.Thesevated sludge(Al-Mutairi,2009;Guckertetal.,1996)tocompareproblems areattributedtopopulationgrowthin urban areas,and discuss thesubstratemetabolic patterns.contamination of surface water and groundwater, unevenBased on the abovementioned background, treateddistributionofwatersourcesandfrequentdroughtsbecausewastewaterwas continuouslydischarged to a SAT pilot-scaleofextremeglobal weatherpatterns (AsanoandCotruvo,2004)reactor in an actual wWTp,and thefollowing objectives wereWastewater reuse is one of the realistic solutions to theconsidered using the BIOLOG assay: (1) to comprehend theabovementionedproblems.changes in microbial transformation processes through aSoil aquifertreatment (SAT)offers advantages suchaslowsequential wastewater reuse process, including activatedcost,underground storage of reclaimed water and the poten-sludge and treatedeffluent in WWTP and SAT,looking at thetial forwater qualityimprovementthrough infiltration (Asanosoil depth, surface layer and the vadose zone, and (2) toandCotruvo,2004;Asano etal.,2007).Actual wastewaterdiscuss relations between changes in the processes and waterreuse systems using SAT include the'Groundwater Replen-qualityparameters.Inaddition,Hazen etal. (1991)determinedishment System'in California and the'Sweetwater Rechargethat the numberof bacteria in sediment (1.00×105-5.01×x108Facilities' in Arizona (USA) and the Dan Region Sewagebacteria/g dry)was much higher than that in the adjacentReclamationProject'in Israel(ChalmersandPatel,2013;Orengroundwater (1.00×103-6.31x10*bacteria/mL).Therefore,etal.,2007;Quanrud etal.,2003).Reclaimed waterhas beenmicrobes in SAT soil were evaluated in this study.used for such applications as potable and irrigation water.It is widelyknown that water replenishmentusing thewater reuse systemsstronglydepends on microbial decom-position, including not only wastewater treatment plant2.Materials and methods(WWTP)butalsoSAT(DrewesandFox,1999,Xueetal.,2009;Zhang et al.,2012).Alot of past studies focused on microbial2.1.Pilot-scalereactortransformation processes in WWTP(e.g.Al-Mutairi,2009;Guckert et al., 1996; Xue et al., 2010) and SAT (Alfreider et al.,A stainless steel pilot-scalereactor was setinan actual WWTP1997;Kolehmainen et al.,2009;Langmark et al.,2004; Zhangin Kyoto Prefecture, and a diagram of the reactor is shown inet al.,2012),respectively.Moreover,thetransformation pro-Fig-1:The reactor was cuboidal and its width, depth andcesseswouldchangeinWWTPandSAT,whichconsistsofheightwere 150,150and 300cm,respectively (volume:sequential water reuse that replenishes the reused water6.75×105cm).qualities.However,there werefew studies thatfocused on theSand, which was collected in Shiga Prefecture, was pur-changes in the microbial processes in WWTP and SAT fromchased and packed in the reactor, and its characteristics arethe viewpoint of WWTP and SAT being a sequential waterlisted in TableA1.Thereactoroperations began on20Octoberreuseprocess.2011.Theheightof thepacked sand was initially250cm,butMicrobialtransformation processesin SATwerecharac-the surface sank immediately after the initiation of opera-terized by the conversion rate of certain chemicals by mi-tions. The height of the sand surface appeared to be stablecrobes (Alfreider et al.,1997; Langmark et al.,2004)after approximately 1 month at237 cm.A final effluent portextracellularenzymeactivities (Kolehmainenetal.,2009)andwassetatanelevationof220cmfromthebottomoftheplantthe range of used substances using the BIOLOG assay(ZhangTherefore,the thicknesses of thevadose zone and saturatedet al, 2012). These studies analysed the changes in thezone in the reactor were 17 and 220 cm, respectively. Thetransformation processes throughout theentire SAT system,reactoralsohad twosideports atdepths of87(Port1)andand samples, including water and soil, were collected at187 cm (Port2),respectively,from the sand surface to collectdiscrete distances. In addition, the removal of organic matterwater and sand samples at thegiven depths.in SAT occurred in the soil surface layer includingthe vadoseThe influent water to the reactor was effluent of an anae-zone(DrewesandFox,1999;Quanrudetal.,1996,2003;Zhangrobic-anoxic-oxic (A20)process,which is mainly used toet al., 2012).Therefore, dynamic changes in the microbialtreat domestic wastewater.The influent to the SAT reactortransformationprocessesareexpectedintheselayers.How-was collected by a hose attached to a 0.05 cm mesh from aever,thisisalso not well understood (Schitz etal.,2010)100cmdepthinthefinal sedimentationtankbeforechlori-TheBIOLOGassayissimpleandyieldsagreatdeal of in-nation.Thehydraulicretentiontime(HRT)oftheA2Oprocessformationforthecharacterizationofmicrobialtransformationwas 2.8 h in the anaerobic tank, 2.8h in the anoxic tank andprocesses (Campbell et al.,1997;Salomo etal,2009).A single5.6 h in the aerobic tank, and the solid retention time (SRT)substrate, redoxdyetetrazolium violet and nutrients are supwas 9.5 days.plied in well plates.The colour production from thereductionFirst, the influent to the SAT reactor was piped intoaoftetrazoliumvioletis usedasan indicatorofthemetabolismstorage tank made of polyethylene and exposed to the atmo-of the substrates.Moreover, the microbial transformationsphere inthe tank.And then,itwas continuouslypumpedprocesses are characterized on the basis of the substrateinto thereactorthrough a PTMG tube (Aoi,Japan).The influentmetabolicpattem(GarlandandMills,1991;Salomoetal.,2009)to the SAT reactorwas dripped using a final port,which wasTheBIOLOGassay has been used in different media,collectedlocated at a height of 60 cm from the centre of thesand

1. Introduction Inadequate water supply and deterioration of water quality are serious problems in many parts of the world. These problems are attributed to population growth in urban areas, contamination of surface water and groundwater, uneven distribution of water sources and frequent droughts because of extreme global weather patterns (Asano and Cotruvo, 2004). Wastewater reuse is one of the realistic solutions to the abovementioned problems. Soil aquifer treatment (SAT) offers advantages such as low cost, underground storage of reclaimed water and the poten￾tial for water quality improvement through infiltration (Asano and Cotruvo, 2004; Asano et al., 2007). Actual wastewater reuse systems using SAT include the ‘Groundwater Replen￾ishment System’ in California and the ‘Sweetwater Recharge Facilities’ in Arizona (USA) and the ‘Dan Region Sewage Reclamation Project’ in Israel (Chalmers and Patel, 2013; Oren et al., 2007; Quanrud et al., 2003). Reclaimed water has been used for such applications as potable and irrigation water. It is widely known that water replenishment using the water reuse systems strongly depends on microbial decom￾position, including not only wastewater treatment plant (WWTP) but also SAT (Drewes and Fox, 1999; Xue et al., 2009; Zhang et al., 2012). A lot of past studies focused on microbial transformation processes in WWTP (e.g. Al-Mutairi, 2009; Guckert et al., 1996; Xue et al., 2010) and SAT (Alfreider et al., 1997; Kolehmainen et al., 2009; La˚ngmark et al., 2004; Zhang et al., 2012), respectively. Moreover, the transformation pro￾cesses would change in WWTP and SAT, which consists of sequential water reuse that replenishes the reused water qualities. However, there were few studies that focused on the changes in the microbial processes in WWTP and SAT from the viewpoint of WWTP and SAT being a sequential water reuse process. Microbial transformation processes in SAT were charac￾terized by the conversion rate of certain chemicals by mi￾crobes (Alfreider et al., 1997; La˚ngmark et al., 2004), extracellular enzyme activities (Kolehmainen et al., 2009) and the range of used substances using the BIOLOG assay (Zhang et al., 2012). These studies analysed the changes in the transformation processes throughout the entire SAT system, and samples, including water and soil, were collected at discrete distances. In addition, the removal of organic matter in SAT occurred in the soil surface layer including the vadose zone (Drewes and Fox, 1999; Quanrud et al., 1996, 2003; Zhang et al., 2012). Therefore, dynamic changes in the microbial transformation processes are expected in these layers. How￾ever, this is also not well understood (Schu¨tz et al., 2010). The BIOLOG assay is simple and yields a great deal of in￾formation for the characterization of microbial transformation processes (Campbell et al., 1997; Salomo et al., 2009). A single substrate, redox dye tetrazolium violet and nutrients are sup￾plied in well plates. The colour production from the reduction of tetrazolium violet is used as an indicator of the metabolism of the substrates. Moreover, the microbial transformation processes are characterized on the basis of the substrate metabolic pattern (Garland and Mills, 1991; Salomo et al., 2009). The BIOLOG assay has been used in different media, collected from different locations, including natural soil (Campbell et al., 1997; Garland and Mills, 1991), soil from constructed wetlands and SAT (Salomo et al., 2009; Weber and Legge, 2011; Zhang et al., 2012), freshwater (Garland and Mills, 1991) and acti￾vated sludge (Al-Mutairi, 2009; Guckert et al., 1996) to compare and discuss the substrate metabolic patterns. Based on the abovementioned background, treated wastewater was continuously discharged to a SAT pilot-scale reactor in an actual WWTP, and the following objectives were considered using the BIOLOG assay: (1) to comprehend the changes in microbial transformation processes through a sequential wastewater reuse process, including activated sludge and treated effluent in WWTP and SAT, looking at the soil depth, surface layer and the vadose zone, and (2) to discuss relations between changes in the processes and water quality parameters. In addition, Hazen et al. (1991) determined that the number of bacteria in sediment (1.00 106 e5.01 108 bacteria/g dry) was much higher than that in the adjacent groundwater (1.00 103 e6.31 104 bacteria/mL). Therefore, microbes in SAT soil were evaluated in this study. 2. Materials and methods 2.1. Pilot-scale reactor A stainless steel pilot-scale reactor was set in an actual WWTP in Kyoto Prefecture, and a diagram of the reactor is shown in Fig. 1. The reactor was cuboidal and its width, depth and height were 150, 150 and 300 cm, respectively (volume: 6.75 106 cm3 ). Sand, which was collected in Shiga Prefecture, was pur￾chased and packed in the reactor, and its characteristics are listed in Table A1. The reactor operations began on 20 October 2011. The height of the packed sand was initially 250 cm, but the surface sank immediately after the initiation of opera￾tions. The height of the sand surface appeared to be stable after approximately 1 month at 237 cm. A final effluent port was set at an elevation of 220 cm from the bottom of the plant. Therefore, the thicknesses of the vadose zone and saturated zone in the reactor were 17 and 220 cm, respectively. The reactor also had two side ports at depths of 87 (Port 1) and 187 cm (Port 2), respectively, from the sand surface to collect water and sand samples at the given depths. The influent water to the reactor was effluent of an anae￾robiceanoxiceoxic (A2O) process, which is mainly used to treat domestic wastewater. The influent to the SAT reactor was collected by a hose attached to a 0.05 cm mesh from a 100 cm depth in the final sedimentation tank before chlori￾nation. The hydraulic retention time (HRT) of the A2O process was 2.8 h in the anaerobic tank, 2.8 h in the anoxic tank and 5.6 h in the aerobic tank, and the solid retention time (SRT) was 9.5 days. First, the influent to the SAT reactor was piped into a storage tank made of polyethylene and exposed to the atmo￾sphere in the tank. And then, it was continuously pumped into the reactor through a PTMG tube (Aoi, Japan). The influent to the SAT reactor was dripped using a final port, which was located at a height of 60 cm from the centre of the sand 106 water research 60 (2014) 105 e117

107WATERRESEARCH60(20I4)I05-II7:WWTP-5cmlayer(A20process)AerobicVadose-10cmlayertankzoneActivated sludg10-15cmlayer.a)15-20cmlayer-aSaturated20-25emlayeraFinal sedimentzonetank2530cmlayera)Influent to the SAT reactor aj.b)Effluent (25cm)fromthe SAT reactor b)Final Eflluent (237 cm)187cm:fromtheSATreactorb)Finalieffluent Port87cm87cmlayer+Effluent (87cm)fromPortWaterSandthe SAT reactor b)220cm237cm187cm layer Port2Effluent (187cm)from50cmthe SAT teactor b)t++Fig.1-Diagram of the SAT reactor and designations of various treated water and layer samples: (a) samples used for thedeterminationofsubstratemetabolicpatternsand(b)samplesusedforthedeterminationofwaterqualitiessurface. The influent to the SAT reactor was continuouslyJapan),respectively.Thespecificultravioletabsorbancedischarged withoutinterruption(SUVA)wascalculatedbydividingtheUVz54valuebytheDOCThe HRT was set at 30 days, and the inflow rate was 70 L/DTN and DTP were measured by an AACS-II auto-analyzerday.(Bran+Luebbe,Germany).The nitrogen component NH wasmeasured by an AA-II auto-analyzer (Bran + Luebbe, Ger-2.2.Continuous water quality measurementmany),and NO2and NOg weremeasured byanAA-III autoanalyzer(Bran+Luebbe,Germany).WatertemperaturewasWater samples of the influent to the SAT reactor and efflu-measured byan HC-763 detector (TOA-DKK,Japan).entsfromPort1,Port2andthefinaleffluentportwereThe effluents from Port 1, Port 2 and the final effluent portcollected once in every two weeks, in principal, from 4are referred to as effluent (87 cm) from the SAT reactor,November 2012 to 6 September 2013. The measured watereffluent (187 cm) from the SAT reactor and final effluentquality parameters included pH (n =26),dissolved oxygen(237 cm)from the SAT reactor,respectively,in this study(DO) (n = 26), dissolved organic carbon (DOC) (n = 29), UV254(n = 29), nitrogen species (dissolved total nitrogen [DTN],2.3.NHt, NO2, NO: and organic N [as the difference betweenEvaluationof microbial substratemetabolicDTN and total inorganic N) (n =20), dissolved total phos-patternsphorus (DTP) (n = 20), as well as water temperature (n = 126)of the influent to the SAT reactor and the final effluent from2.3.1.Sampling schemethe SAT reactor.With respect to theinfluent to the SATSamples,includingthewaterand sandsamplesdescribedreactor,a samplefor each parameter, except pH and DO, wasbelow, were collected four times in the summer forcollected just after thefinal port,whichwas located at areproducibility.heightof6ocmfromthecentreofthesandsurface,whereasActivated sludgefromthe aerobictank of theA20process,thesampleforpHandDOwascollected inthestoragetanktoinfluenttotheSAT reactorand sand samplesfrom theSATprevent DO from increasing during sampling. Prior to thereactor were collected on 29 July and 5,12 and 19 August 2013measurement of the water qualities,except for pH and DO,The watertemperatures at eachsampling eventwere similar,the water samples had been filtered by GF/B with a pore sizerangingfrom 27.3to 30.9°C.of1μm (Whatman,USA)The activated sludge was collected with a plasticladleandThe pH and DO were measured by D-52 and D-55 (Horiba,preserved in a sterilized polypropylene (PP) vial (Vioramo, ASJapan)multi-parametermetres, respectively.TheDOC andONE, Japan).The collected samples were used for BIOLOGUV254 were measured by a TOc-L analyzer (Shimadzu,Japan)assay, fluorescein diacetate (FDA) assay and for the wateranda Multi-Spec-1500s spectrophotometer (Shimadzu,qualitymeasurements,describedbelow

surface. The influent to the SAT reactor was continuously discharged without interruption. The HRT was set at 30 days, and the inflow rate was 70 L/ day. 2.2. Continuous water quality measurement Water samples of the influent to the SAT reactor and efflu￾ents from Port 1, Port 2 and the final effluent port were collected once in every two weeks, in principal, from 4 November 2012 to 6 September 2013. The measured water quality parameters included pH (n ¼ 26), dissolved oxygen (DO) (n ¼ 26), dissolved organic carbon (DOC) (n ¼ 29), UV254 (n ¼ 29), nitrogen species (dissolved total nitrogen [DTN], NH4 þ, NO2 , NO3 and organic N [as the difference between DTN and total inorganic N]) (n ¼ 20), dissolved total phos￾phorus (DTP) (n ¼ 20), as well as water temperature (n ¼ 126) of the influent to the SAT reactor and the final effluent from the SAT reactor. With respect to the influent to the SAT reactor, a sample for each parameter, except pH and DO, was collected just after the final port, which was located at a height of 60 cm from the centre of the sand surface, whereas the sample for pH and DO was collected in the storage tank to prevent DO from increasing during sampling. Prior to the measurement of the water qualities, except for pH and DO, the water samples had been filtered by GF/B with a pore size of 1 mm (Whatman, USA). The pH and DO were measured by D-52 and D-55 (Horiba, Japan) multi-parameter metres, respectively. The DOC and UV254 were measured by a TOC-L analyzer (Shimadzu, Japan) and a Multi-Spec-1500S spectrophotometer (Shimadzu, Japan), respectively. The specific ultraviolet absorbance (SUVA) was calculated by dividing the UV254 value by the DOC. DTN and DTP were measured by an AACS-II auto-analyzer (Bran þ Luebbe, Germany). The nitrogen component NH4 þ was measured by an AA-II auto-analyzer (Bran þ Luebbe, Ger￾many), and NO2 and NO3 were measured by an AA-III auto￾analyzer (Bran þ Luebbe, Germany). Water temperature was measured by an HC-763 detector (TOA-DKK, Japan). The effluents from Port 1, Port 2 and the final effluent port are referred to as effluent (87 cm) from the SAT reactor, effluent (187 cm) from the SAT reactor and final effluent (237 cm) from the SAT reactor, respectively, in this study. 2.3. Evaluation of microbial substrate metabolic patterns 2.3.1. Sampling scheme Samples, including the water and sand samples described below, were collected four times in the summer for reproducibility. Activated sludge from the aerobic tank of the A2O process, influent to the SAT reactor and sand samples from the SAT reactor were collected on 29 July and 5, 12 and 19 August 2013. The water temperatures at each sampling event were similar, ranging from 27.3 to 30.9 C. The activated sludge was collected with a plastic ladle and preserved in a sterilized polypropylene (PP) vial (Vioramo, AS ONE, Japan). The collected samples were used for BIOLOG assay, fluorescein diacetate (FDA) assay and for the water quality measurements, described below. Fig. 1 e Diagram of the SAT reactor and designations of various treated water and layer samples: (a) samples used for the determination of substrate metabolic patterns and (b) samples used for the determination of water qualities. water research 60 (2014) 105 e117 107

108WATERRESEARCH60(2014)I05-117The influent to the SAT reactor was collected using the PPfor microbes in the5g-wet sample of the 0-5 cm sand layer,vial for BIOLOG assay and FDAassay and glass bottles for thewhose activitywas thehighest.Immediatelyafter shakingfor1 h, the samplewas filtered using a No.1 filter (Advantec,waterqualitymeasurements.Core sampling was conducted to collect the surface sandJapan)followed bya polytetrafluoroethylene(PTFE)mem-down to a 30 cm depth using a liner sampler (DIK-110C, Daikibrane flter with a pore size of o.2 μm (Advantec,Japan).TheRikaKogyo,Japan)witha5cmdiameterand30cm height.Theamountoffluoresceinwasmeasured astheabsorbanceatliner sampler and its sampling tube were sterilized at250°C490 nmbythe Multi-Spec-1500S.The same process usingfor2h and using10mg-NaHOCl/Lovernight,respectively.Thesamples sterilized by autoclave at 120 C for 20 min was150cmx150cmareaofthesandsurfacewasdividedintorepeated for use as blank samples, and the absorbance of the10cm×10cmsections,andthe30cmdepthsandcoresamplesterile sample was subtracted. A strong relationship waswas collected ata grid point.The four sample collections werefound betweenthe microbial amount and the absorbanceconducted at different grid points within 15 cmfrom thepo(y= 0.0192x, R2=0.995:x=g-wet of sand,y=absorbance)sition of the final port.using 1,2,3,4and 5g-wetof the sand samples takenfromaThe sand core sample was divided into six segments of1 cm depth fromthe topsurface.5 cm lengths (denoted as the 0-5 cm layer, 5-10 cm layer,2.3.4.BIOLOG assay with EcoPlate10-15cmlayer15-20cmlayer,2025cmlayerand25-30cmlayer) in the laboratory,and each layer was collected in aEcoPlate (Biolog Inc.U.s.)was used to evaluate and comparesterilized glass beaker.The sand was well mixed with steril-microbial substratemetabolicpatterns among the samples.izedstainlessspoonsinthebeakerandusedassandsamples.Theactivated sludgeand influenttotheSATreactorwereThe beakers and spoonswere sterilized at 250c for2h indiluted100and10 timeswiththe60mM sterile sodiumadvance.phosphatebuffer,respectively,and150μL of thedilutedA30-cm-deep holewas also createdby the liner sampler atsamples were inoculated to the plates.a point that waslocated 70 cm from theposition of thefinalThe suspension of microbes in the sand was conductedport,and a water sampletakenfromanapproximately25cmwithaWaringblender(AcehomogenizerAM-3,Nihonseikidepth from the top surface (denoted as effluent (25 cm) fromJapan).A mixture of 10 g-wet of each sand sample and 40 mLof thephosphatebufferwereblended in theblenderfor3min.theSATreactor)wascollectedwithaPPsyringeandtubesandstored in the PP vial.This sampling was conducted five timesThehomogenatewascentrifuged for 1min at 1500rpmfrom12Augustto26August2013.(KUBOTA5200,Kubota,Japan)toremovethesand particles,Sand and water samples were collected from Port 1 andand 150 μL of the supernatant was inoculated to the platePort2withPP vials.After thesand mixed with thetreatedEcoPlateswereincubatedat25cinanincubator(IS-41water,whichflowedawayfromPort1andPort2,waslefttoYamato, Japan) for 7 days, and wet papers were set at therestfor 30 s, the supernatant was decanted to another PP vial,bottom of the incubator to prevent dryness.Absorbance was measured using a Powerscan-Pc (Dsand theresulting sand and supernatantwere used as sandandwater samples,respectively.The sandand water samplesPharmaBiomedical, Japan)at590nm after the inoculation offrom Port 1 were denotedas87 cm layerand effluent (87cm)the plates every3 h during the first period of 5days and 6hfrom the SAT reactor,respectively, and the sand and waterduring the last 2 days.The absorbance was also measuredsamplesfromPort2weredenotedas187cmlayerandeffluentevery1.5h during the first period for samples, in which the(187cm)from the SATreactor,respectivelyabsorbancerapidlyincreased.The absorbance data were corrected by subtraction of the2.3.2.TOC measurement in sandblank well data at each sampling time. Average well colourThetotal organic carbon (ToC) of each collected sand sampledevelopment (AWCD) was calculated for each sample atwas measured using an SSM-5000A solid sample combustioneach time, and the absorbance data in each well wasunit (Shimadzu,Japan).normalized by dividingbythe AWCD to compensatefor theinfluences of inoculum density (Garland,1996; Rutgers etal.,2.3.3.FDAassay2008).Several enzymes produced by microbes, such as proteaseSalomo et al. (2009) determined microbial substratelipase and esterase, can catalyse the transformation of fluo-metabolic patterns from a constructed wetland using therescein diacetate (3',6'-diacetylfluorescein (FDA)to fluores-data at AWCD =0.2when the substrate utilization ratescein, and the FDA assay was used to assess the totalwere situated in thetransition from lagphase toexponentialmicrobiological activity (Ichikawa et al.,2002; Schntirerandphase. In this study, the normalized absorbance data forRosswall,1982;Weber andLegge,2011).FDAassaywaseachsampleateachsamplingdatewithAWCD=0.2wasappliedto theactivated sludge,influenttotheSATreactorandanalysed byprincipal component analysis to evaluateandeach sand layer sample, in reference to Ichikawa et al. (2002)comparemicrobialsubstratemetabolicpatternsamongtheandSchnurerandRosswall (1982)samples.A20mLvolumeof60mM sterile sodiumphosphatebufferIt isknown thatbacteria are one component ofbiomass in(pH 7.6) was added to each 5 g-wet sand sample, 1 mL acti-activated sludge and soil (Thawornchaisit and Pakulanon,vated sludge and 5 mL influent to the SAT reactor. After2007;Wardle,1992). Therefore,BIOLOG assay was alsoadditionof 0.3mLofFDA solution(2mgFDAand2mLapplied to the activated sludge with different dilution levelsacetone),the sampleswere shakenfor1h.TheamountofFDAto ensurethat normalization bytheAWCDcompensatedforto be added was determined based on the amount sufficienttheinfluences ofinoculumdensities.Nippon Steel &Sumikin

The influent to the SAT reactor was collected using the PP vial for BIOLOG assay and FDA assay and glass bottles for the water quality measurements. Core sampling was conducted to collect the surface sand down to a 30 cm depth using a liner sampler (DIK-110C, Daiki Rika Kogyo, Japan) with a 5 cm diameter and 30 cm height. The liner sampler and its sampling tube were sterilized at 250 C for 2 h and using 10 mg-NaHOCl/L overnight, respectively. The 150 cm 150 cm area of the sand surface was divided into 10 cm 10 cm sections, and the 30 cm depth sand core sample was collected at a grid point. The four sample collections were conducted at different grid points within 15 cm from the po￾sition of the final port. The sand core sample was divided into six segments of 5 cm lengths (denoted as the 0e5 cm layer, 5e10 cm layer, 10e15 cm layer, 15e20 cm layer, 20e25 cm layer and 25e30 cm layer) in the laboratory, and each layer was collected in a sterilized glass beaker. The sand was well mixed with steril￾ized stainless spoons in the beaker and used as sand samples. The beakers and spoons were sterilized at 250 C for 2 h in advance. A 30-cm-deep hole was also created by the liner sampler at a point that was located 70 cm from the position of the final port, and a water sample taken from an approximately 25 cm depth from the top surface (denoted as effluent (25 cm) from the SAT reactor) was collected with a PP syringe and tubes and stored in the PP vial. This sampling was conducted five times from 12 August to 26 August 2013. Sand and water samples were collected from Port 1 and Port 2 with PP vials. After the sand mixed with the treated water, which flowed away from Port 1 and Port 2, was left to rest for 30 s, the supernatant was decanted to another PP vial, and the resulting sand and supernatant were used as sand and water samples, respectively. The sand and water samples from Port 1 were denoted as 87 cm layer and effluent (87 cm) from the SAT reactor, respectively, and the sand and water samples from Port 2 were denoted as 187 cm layer and effluent (187 cm) from the SAT reactor, respectively. 2.3.2. TOC measurement in sand The total organic carbon (TOC) of each collected sand sample was measured using an SSM-5000A solid sample combustion unit (Shimadzu, Japan). 2.3.3. FDA assay Several enzymes produced by microbes, such as protease, lipase and esterase, can catalyse the transformation of fluo￾rescein diacetate (30 ,60 -diacetylfluorescein (FDA)) to fluores￾cein, and the FDA assay was used to assess the total microbiological activity (Ichikawa et al., 2002; Schnu¨ rer and Rosswall, 1982; Weber and Legge, 2011). FDA assay was applied to the activated sludge, influent to the SAT reactor and each sand layer sample, in reference to Ichikawa et al. (2002) and Schnu¨ rer and Rosswall (1982). A 20 mL volume of 60 mM sterile sodium phosphate buffer (pH 7.6) was added to each 5 g-wet sand sample, 1 mL acti￾vated sludge and 5 mL influent to the SAT reactor. After addition of 0.3 mL of FDA solution (2 mg FDA and 2 mL acetone), the samples were shaken for 1 h. The amount of FDA to be added was determined based on the amount sufficient for microbes in the 5 g-wet sample of the 0e5 cm sand layer, whose activity was the highest. Immediately after shaking for 1 h, the sample was filtered using a No.1 filter (Advantec, Japan) followed by a polytetrafluoroethylene (PTFE) mem￾brane filter with a pore size of 0.2 mm (Advantec, Japan). The amount of fluorescein was measured as the absorbance at 490 nm by the Multi-Spec-1500S. The same process using samples sterilized by autoclave at 120 C for 20 min was repeated for use as blank samples, and the absorbance of the sterile sample was subtracted. A strong relationship was found between the microbial amount and the absorbance (y ¼ 0.0192x, R2 ¼ 0.995: x ¼ g-wet of sand, y ¼ absorbance) using 1, 2, 3, 4 and 5 g-wet of the sand samples taken from a 1 cm depth from the top surface. 2.3.4. BIOLOG assay with EcoPlate EcoPlate (Biolog Inc. U.S.) was used to evaluate and compare microbial substrate metabolic patterns among the samples. The activated sludge and influent to the SAT reactor were diluted 100 and 10 times with the 60 mM sterile sodium phosphate buffer, respectively, and 150 mL of the diluted samples were inoculated to the plates. The suspension of microbes in the sand was conducted with a Waring blender (Ace homogenizer AM-3, Nihonseiki, Japan). A mixture of 10 g-wet of each sand sample and 40 mL of the phosphate buffer were blended in the blender for 3 min. The homogenate was centrifuged for 1 min at 1500 rpm (KUBOTA 5200, Kubota, Japan) to remove the sand particles, and 150 mL of the supernatant was inoculated to the plate. EcoPlates were incubated at 25 C in an incubator (IS-41, Yamato, Japan) for 7 days, and wet papers were set at the bottom of the incubator to prevent dryness. Absorbance was measured using a Powerscan-PC (DS Pharma Biomedical, Japan) at 590 nm after the inoculation of the plates every 3 h during the first period of 5 days and 6 h during the last 2 days. The absorbance was also measured every 1.5 h during the first period for samples, in which the absorbance rapidly increased. The absorbance data were corrected by subtraction of the blank well data at each sampling time. Average well colour development (AWCD) was calculated for each sample at each time, and the absorbance data in each well was normalized by dividing by the AWCD to compensate for the influences of inoculum density (Garland, 1996; Rutgers et al., 2008). Salomo et al. (2009) determined microbial substrate metabolic patterns from a constructed wetland using the data at AWCD ¼ 0.2 when the substrate utilization rates were situated in the transition from lag phase to exponential phase. In this study, the normalized absorbance data for each sample at each sampling date with AWCD ¼ 0.2 was analysed by principal component analysis to evaluate and compare microbial substrate metabolic patterns among the samples. It is known that bacteria are one component of biomass in activated sludge and soil (Thawornchaisit and Pakulanon, 2007; Wardle, 1992). Therefore, BIOLOG assay was also applied to the activated sludge with different dilution levels to ensure that normalization by the AWCD compensated for the influences of inoculum densities. Nippon Steel & Sumikin 108 water research 60 (2014) 105 e117

109WATERRESEARCH60(20I4)I05-II7Eco-Tech(Japan)measured16SrRNAcopiesbyreal-timePCR3.Resultsanddiscussionfor the activated sludge, influent to the SAT reactor and thephosphatebufferaftertheextractionofeachsandlayeron 193.1.Temporal changes in water qualitiesAugust (DNA extraction with Extrap Soil DNAKit Plus ver.2[Nippon Steel & Sumikin Eco-Tech, Japan] and quantificationTemporal changes of thewaterqualitiesofeach watersamplewith PicoGreen dsDNA assay kit [Invitrogen, US]). Thereare shown in Fig.2(1)and (2).The distribution of each qualitywere two digit differences in the copy numbers among theisarrangedinorderoftheinfluenttotheSATreactor,effluentsamples (Table 1). Therefore, activated sludge samples(87cm)from theSAT reactor,effluent (187cm)from theSATdiluted10,100and1000timeswereassayedandanalysedbyreactorandfinaleffluent(237cm)fromthe SATreactorunlessprincipal component analysis with the other samples on 19otherwise noted, and median, minimum and maximumAugust.values for each index are formatted as median (mini-mum-maximum)in this section.2.3.5.WaterqualitymeasurementsThe pH varied 6.58 (6.16-7.67), 6.70 (5.88-7.03), 5.79ThepHand DO (except foreffluent (25cm) from theSAT(5.31-6.52) and 5.64 (5.28-6.03), respectively. There were noreactor) and DOC, UV254, nitrogen species and DTP aftersignificantdifferencesbetween theinfluentto theSATreactorfiltrationwith GF/B weremeasured for eachwater sample.and effluent (87 cm) from the SAT reactor (p > 0.05). MeanThe measurement methods were the same as described inwhile,pH significantlydecreased from effluent (87cm)fromSection2.2.In addition,fluorescencespectrawerecollectedthe SAT reactortoeffluent (187cm)from the SAT reactorandforthefilteredsampleson12August.Thesampleswerefrom effluent (187cm) fromtheSATreactorto final effluentdiluted to 0.7mgC/L and adjusted to pH 7.To obtain excita-(237 cm) from the SAT reactor (p 0.05)layer.A significance level of 0.05 was used for all tests.Table1-RangesofToC andFDA,16SrRNA copynumbers andresults of theWilcoxon rank sumtest, whereranges aregivenasmedian(minimum-maximum).SampleTOC(%)FDA (cmlg-dryor em-mL-)16S rRNA copy numbers(copies/mL)3.3×10Activated sludge0.160 (0.14-0.23)p0.058.2 ×10gInfuen to the SAT reactor0.005P≤0.050.090 (0.051-0.186)2.4 *1070-5 cm layer0.084 (0.0320.094)p0.05p >0.052.1×10710-15 cm layer0.015 (0.012-0.019)0.012 (0.005-0.038)P>0.05p>0.0515-20 cm layer0.005 (40.0050.011)17×1070.013 (0.0082-0.019)p>0.05p0.050.0051.1×10725-30 cm layer0.0072 (0.0063-0.011)px0.055.6×1087cm layer0.0050.0017 (0.00110.0044)p>0.050.0058.2× 105187cm layer0.0020 (0.00480.0057)

Eco-Tech (Japan) measured 16S rRNA copies by real-time PCR for the activated sludge, influent to the SAT reactor and the phosphate buffer after the extraction of each sand layer on 19 August (DNA extraction with Extrap Soil DNA Kit Plus ver. 2 [Nippon Steel & Sumikin Eco-Tech, Japan] and quantification with PicoGreen dsDNA assay kit [Invitrogen, US]). There were two digit differences in the copy numbers among the samples (Table 1). Therefore, activated sludge samples diluted 10, 100 and 1000 times were assayed and analysed by principal component analysis with the other samples on 19 August. 2.3.5. Water quality measurements The pH and DO (except for effluent (25 cm) from the SAT reactor) and DOC, UV254, nitrogen species and DTP after filtration with GF/B were measured for each water sample. The measurement methods were the same as described in Section 2.2. In addition, fluorescence spectra were collected for the filtered samples on 12 August. The samples were diluted to 0.7 mgC/L and adjusted to pH 7. To obtain excita￾tioneemission matrix (EEM) profiles, excitation wavelengths were incremented from 220 to 400 nm in 5 nm steps, and for each excitation wavelength, the emission was detected from 240 to 500 nm in 5 nm steps. Fluorescence of super purified water as a blank sample was subtracted from each spectrum. Suspended solid (SS): 105 C for 2 h and volatile suspended solid (VSS): 600 C for 30 min were measured for the activated sludge and treated wastewater (Japan Sewage Works Association, 1997). 2.4. Statistical analysis The Wilcoxon rank sum test was used to examine the statis￾tical significance of the measured sand and water quality in￾dexes between two samples at subsequent points along the flow path, such as the influent to the SAT reactor vs. effluent (25 cm) from the SAT reactor and 0e5 cm layer vs. 5e10 cm layer. A significance level of 0.05 was used for all tests. 3. Results and discussion 3.1. Temporal changes in water qualities Temporal changes of the water qualities of each water sample are shown in Fig. 2(1) and (2). The distribution of each quality is arranged in order of the influent to the SAT reactor, effluent (87 cm) from the SAT reactor, effluent (187 cm) from the SAT reactor and final effluent (237 cm) from the SAT reactor unless otherwise noted, and median, minimum and maximum values for each index are formatted as median (mini￾mumemaximum) in this section. The pH varied 6.58 (6.16e7.67), 6.70 (5.88e7.03), 5.79 (5.31e6.52) and 5.64 (5.28e6.03), respectively. There were no significant differences between the influent to the SAT reactor and effluent (87 cm) from the SAT reactor (p > 0.05). Mean￾while, pH significantly decreased from effluent (87 cm) from the SAT reactor to effluent (187 cm) from the SAT reactor and from effluent (187 cm) from the SAT reactor to final effluent (237 cm) from the SAT reactor (p 0.05). Table 1 e Ranges of TOC and FDA, 16S rRNA copy numbers and results of the Wilcoxon rank sum test, where ranges are given as median (minimumemaximum). water research 60 (2014) 105 e117 109

110WATERRESEARCH60(20I4)I05-II7lnfluEffluent(187cmnaleffluent(237em)fromthefinal effluen(87cm)from(1)pHDODOC8.012.0355.0to(oam4.0(/08u)9.0AAATA25(1ou)on7.03.020(-) Hd60置菜152.0OC6.0103.0.0?0.00.05.0Oct-12Jan-13Apr-13Jun-13Sep-13Oer-12Sep-13Oct-12Jan-13Apr-13Jun-13Sep-13Jan-13Apr-13Jun-13Date (month-year)Date (month-year)Date (month-year)UV254SUVADTP1.2 0.160.10()0.0840.120.9()中4中 0.06 0.080.60.040.020.30.000.00Sep-130.0Oct-12Jan-13Apr-13Jun-13Oet-12Jan-13Apr-13Jun-13Sep-13Oct-12Jan-13Apr-13Jun-13Sep-13Date (month-year)Date (month-year)Date (month-year)+DTNNH+ANO2X NO,Organic-N(2)(Effluent (87cm)from the SATreactor(INN-oeueON 'NIAInfluent to theSAT reactor9.01.59.00.3(TN)CONe(1/NSu) CON pue,.16.01.06.00.2 .X?0.50.13.03.0+-HN+HN.ONXX(X0.00.0 0.00.0'NLAOct-12Jan-13Apr-13Jun-13Sep-13Oct-12Jan-13Apr-13Jun-13Sep-13DateDateEffluent (187 cm) from the SAT reactorFinal effluent (237cm)from the SAT reactor(TNNOeNNIA9.00.31.5(1/NSu) CON put(/Na)ONpue+HN■+0.26.06.01.0+0. 1 X0.53.03.0XHN4x版邮牌0.00.0H0.0PAOct-12Oct-12Jan-13Apr-13Jun-13Sep-13Jan-13Apr-13Jun-13Sep-13DateDateFig.2-(1)Temporal changes inpH, DO,DOC, UV254,SUVAand DTP for treated water sampled from variouspoints of theSAT reactor, (2) Temporal changes in DTN and its components for treated water sample from various points of the SATreactor.Fig 2-1 also shows that DO in each effluent adverselythrough the percolation path in the SAT reactor (p<0.05).Inincreased and decreased against the water temperature of thecomparisonwiththemedianvaluesof theinfluenttotheSATfinaleffluent (237cm)from theSATreactor.Therefore,changesreactor,DOCremovalpercentageswere73.7%,81.3%andinthe saturated oxygen concentration of waterbecauseof the83.2%. Drewes and Fox (1999) obtained 76% DOC removaltemperaturemightinfluencetheDOofeacheffluent(from11.5mgC/Lto2.8mgC/L)forthesecondaryeffluentafterBased on the results, DO in each effluent mostly exceeds21dayspercolation.Inaddition,Zhangetal.(2o12)obtained2mgOz/L, and it was determined that the entire reactor79%DOCremoval (from4.31mgC/L to0.91mgC/L)forozo-operated under aerobic conditions.nated secondary effluent after 28.8 days retention. The DOCThe distributionof DOCwas 3.91 (2.85-4.55),1.02removal ofthefinal effluent (237cm)from theSATreactor in(0.7811.57), 0.734 (0.5601.32) and 0.658 (0.4820.870) mgC/L,thepresentstudyissimilartothesevaluesoverasimilarHRTrespectively,andDOC significantlydecreasedin eacheffluentDOCwas slightlylow in the influent to the SATreactor in the

Fig. 2-1 also shows that DO in each effluent adversely increased and decreased against the water temperature of the final effluent (237 cm) from the SAT reactor. Therefore, changes in the saturated oxygen concentration of water because of the temperature might influence the DO of each effluent. Based on the results, DO in each effluent mostly exceeds 2 mgO2/L, and it was determined that the entire reactor operated under aerobic conditions. The distribution of DOC was 3.91 (2.85e4.55), 1.02 (0.781e1.57), 0.734 (0.560e1.32) and 0.658 (0.482e0.870) mgC/L, respectively, and DOC significantly decreased in each effluent through the percolation path in the SAT reactor (p < 0.05). In comparison with the median values of the influent to the SAT reactor, DOC removal percentages were 73.7%, 81.3% and 83.2%. Drewes and Fox (1999) obtained 76% DOC removal (from 11.5 mgC/L to 2.8 mgC/L) for the secondary effluent after 21 days percolation. In addition, Zhang et al. (2012) obtained 79% DOC removal (from 4.31 mgC/L to 0.91 mgC/L) for ozo￾nated secondary effluent after 28.8 days retention. The DOC removal of the final effluent (237 cm) from the SAT reactor in the present study is similar to these values over a similar HRT. DOC was slightly low in the influent to the SAT reactor in the Fig. 2 e (1) Temporal changes in pH, DO, DOC, UV254, SUVA and DTP for treated water sampled from various points of the SAT reactor, (2) Temporal changes in DTN and its components for treated water sample from various points of the SAT reactor. 110 water research 60 (2014) 105 e117

111WATERRESEARCH60(2014)105-1173.2.summer season from June to September.Meanwhile, thereChanges of microbial substrate metabolicpatternswereno noticeable seasonal changes in each effluent throughthesamplingperiod.3.2.1.TOC in the sand along the flow pathUV254 ranged 0.088 (0.0460.128), 0.027 (0.0080.062), 0.024Ranges of TOC for all sand samples are listed in Table 1.Me-(0.008-0.048)and 0.023 (0.005-0.052)cm-1,respectively.dian TOCin the0-25cmlayerswas high in comparisonwithaTherewasasignificantdecreasefromtheinfluenttotheSATTOCof0.0094%inthesandbeforereactorpacking(TableA1)reactor to the effluent (87 cm)from the SAT reactor (p0.05).Therewereoriginally adsorbed werewashed out by percolation of thenonoticeableseasonal changesin eachwatersamplethroughtreated water cleaned by theabove layers.thesamplingperiod.Thedistribution of UVA was 0.022 (0.013-0.035),0.0253.2.2.Microbial activities by FDA assayTheresults of FDAassayare listed inTable 1.Themicrobial(0.0078-0.061),0.030 (0.00990.065)and 0.033 (0.00770.079) L/(mgCcm).There was a significant increase from the influentactivity of the activated sludge and the 0-5 cm layer wastotheSATreactortotheeffluent(87cm)fromtheSATreactorsignificantlyhigherthanthatof theinfluenttothe SATreactor(p0.05).Therereactor,based on a water density of 1 g/mL.Therefore, underwerenonoticeableseasonalchangesineachwatersample.conditionswherethebulkwaterinthe0-5cmlayersampleDTN ranged 4.58(3.36-7.50),4.62(3.56-8.14),4.43wastheinfluenttotheSATreactor,themicrobial activityof(2.90-6.83)and 4.69 (3.79-6.29)mgN/L,respectively,and notheinfluenttotheSATreactorcouldbecalculatedaslessthansignificant differences were observed among the water sam-0.0008 cm-1 in 1g wet of sand.Meanwhile, the minimumples (p > 0.05).In addition, usingmedian values,NOabsorbance of the 0-5 cm layer was 0.044 cm-1g-wet-1accounted for 78.3%, 92.3%, 96.6% and 88.9% of DTN in eachTherefore, the sample should contain some active microbeswater sample.DTN was comparativelyhighin each waterwhich were directly discharged from the influent to the SATsampleduringthewinterseason fromDecembertoMarch.reactor into the0-5 cm layer.However,the assay determinedThedistributionofDTPwas0.206(0.090-1.13)mgP/Linthethat this contribution to the overall activity in the 0-5 cminfluent to the SAT reactor. Meanwhile, it was not detectedlayerwas very small (less than 1/50).(<0.002mgP/L)in each effluent.DTPwashighin the influentTheSATmicrobialactivity sharplydecreased overtheflowto the SAT reactorduring the summer season from June topath,and this correspondsto theToC trend.In comparisontoSeptember.Ak and Gunduz (2013b)showed thatPO-precip-themedianabsorbanceof the0-5cmlayer,the absorbance ofitated in the soil surface.PO-accounted for 56±21%the5-10 cm layerwas47%thereof and significantlylow(average± standard deviation,n =34)in DTP in the influent to(p<0.05).In addition,lowabsorbancewasobtained in thethe SAT reactor (Kyoto City Waterworks Bureau):therefore,10-15cm layer (13%)and 15-20 cm layer (5.6%)the Poz-precipitation might have contributed to the decrease3.2.3.MicrobialsubstratemetabolicpatterninDTPintheSATreactor.The median mole ratio C:N:P of the influent to the SATFig.A1 shows the AWCD of each sampleat each samplingreactorduringtheperiodfromJunetoSeptember,whenDOCdate.AWCD in activated sludge, influent to the SAT reactordecreased andDTP increased, was100:129:4.02,whileforalland o-5 cm layer increased more rapidly than the otherotherperiodsitwas100:115:12.7.Therefore,itwasdeterminedsamples.that carbon was the limiting factorfor microbial activities inFig.3shows theresults of theprincipal component anal-the influent to the SAT reactor, despite the observed decreaseysis.In addition,TableA2showstheprincipalcomponentand increase.Meanwhile,the median C:N:P ratio wasloadingrate of each substrate, and the31 substrateswere100:483:<0.196,100:634:<0.273and100:734:<0.304foreffluentclassified based on their structure, in reference to previous(87cm)fromtheSATreactor,effluent (187cm)fromtheSATreports (Salomo et al.,2009;Zhanget al.,2012).reactorandfinal effluent (237cm)from theSAT reactor,Thecontribution ratioofthefirstand secondprincipalrespectively,through the entire period, and was determinedcomponentranged from61.3%to71.6%andfrom12.3%tothat phosphorus was the limiting element.14.9%, respectively.Thefirst principal component exhibitedBased on these results,it was determined thatthe treatedmuchin commonwith substrates whose loading rate excee-effluents from the SAT reactor exhibited no noticeable sea-ded0.5 (Sala etal.,2005)amongthe samplingdates.Data plotssonal variations,except for slightly high DTN in the winterof microbes with substantial metabolism forthefollowingsubstrates are located in the positive direction on the firstseason,andthetreatmentperformanceappeared tobe stableunder aerobic conditions, including the sampling period forprincipal component axis: carboxylic acids (4-hydroxy ben-the analysis of the metabolic patterns.zonic acid, itaconic acid and -hydroxybutyricacid),amino

summer season from June to September. Meanwhile, there were no noticeable seasonal changes in each effluent through the sampling period. UV254 ranged 0.088 (0.046e0.128), 0.027 (0.008e0.062), 0.024 (0.008e0.048) and 0.023 (0.005e0.052) cm1 , respectively. There was a significant decrease from the influent to the SAT reactor to the effluent (87 cm) from the SAT reactor (p 0.05). There were no noticeable seasonal changes in each water sample through the sampling period. The distribution of SUVA was 0.022 (0.013e0.035), 0.025 (0.0078e0.061), 0.030 (0.0099e0.065) and 0.033 (0.0077e0.079) L/ (mgC cm). There was a significant increase from the influent to the SAT reactor to the effluent (87 cm) from the SAT reactor (p 0.05). There were no noticeable seasonal changes in each water sample. DTN ranged 4.58 (3.36e7.50), 4.62 (3.56e8.14), 4.43 (2.90e6.83) and 4.69 (3.79e6.29) mgN/L, respectively, and no significant differences were observed among the water sam￾ples (p > 0.05). In addition, using median values, NO3 accounted for 78.3%, 92.3%, 96.6% and 88.9% of DTN in each water sample. DTN was comparatively high in each water sample during the winter season from December to March. The distribution of DTP was 0.206 (0.090e1.13) mgP/L in the influent to the SAT reactor. Meanwhile, it was not detected (<0.002 mgP/L) in each effluent. DTP was high in the influent to the SAT reactor during the summer season from June to September. Ak and Gunduz (2013b) showed that PO4 3 precip￾itated in the soil surface. PO4 3 accounted for 56  21% (average  standard deviation, n ¼ 34) in DTP in the influent to the SAT reactor (Kyoto City Waterworks Bureau); therefore, the PO4 3 precipitation might have contributed to the decrease in DTP in the SAT reactor. The median mole ratio C:N:P of the influent to the SAT reactor during the period from June to September, when DOC decreased and DTP increased, was 100:129:4.02, while for all other periods it was 100:115:12.7. Therefore, it was determined that carbon was the limiting factor for microbial activities in the influent to the SAT reactor, despite the observed decrease and increase. Meanwhile, the median C:N:P ratio was 100:483:<0.196, 100:634:<0.273 and100:734:<0.304 for effluent (87 cm) from the SAT reactor, effluent (187 cm) from the SAT reactor and final effluent (237 cm) from the SAT reactor, respectively, through the entire period, and was determined that phosphorus was the limiting element. Based on these results, it was determined that the treated effluents from the SAT reactor exhibited no noticeable sea￾sonal variations, except for slightly high DTN in the winter season, and the treatment performance appeared to be stable under aerobic conditions, including the sampling period for the analysis of the metabolic patterns. 3.2. Changes of microbial substrate metabolic patterns 3.2.1. TOC in the sand along the flow path Ranges of TOC for all sand samples are listed in Table 1. Me￾dian TOC in the 0e25 cm layers was high in comparison with a TOC of 0.0094% in the sand before reactor packing (Table A1). In addition, TOC in the 0e5 cm layer was significantly higher than that of the 5e10 cm layer (p < 0.05). This indicates that microbial growth and organic adsorption occur in the layers through operation, especially in the 0e5 cm layer. The 87 cm and 187 cm layers exhibited lower TOC values than that before packing, which indicates that organic matter and microbes originally adsorbed were washed out by percolation of the treated water cleaned by the above layers. 3.2.2. Microbial activities by FDA assay The results of FDA assay are listed in Table 1. The microbial activity of the activated sludge and the 0e5 cm layer was significantly higher than that of the influent to the SAT reactor (p < 0.05). The maximum weight of water in the 0e5 cm layer sample was 0.16 g in a 1 g wet sand sample, and the absor￾bance was below 0.005 cm1 g1 in the influent to the SAT reactor, based on a water density of 1 g/mL. Therefore, under conditions where the bulk water in the 0e5 cm layer sample was the influent to the SAT reactor, the microbial activity of the influent to the SAT reactor could be calculated as less than 0.0008 cm1 in 1 g wet of sand. Meanwhile, the minimum absorbance of the 0e5 cm layer was 0.044 cm1 g-wet1 . Therefore, the sample should contain some active microbes which were directly discharged from the influent to the SAT reactor into the 0e5 cm layer. However, the assay determined that this contribution to the overall activity in the 0e5 cm layer was very small (less than 1/50). The SAT microbial activity sharply decreased over the flow path, and this corresponds to the TOC trend. In comparison to the median absorbance of the 0e5 cm layer, the absorbance of the 5e10 cm layer was 47% thereof and significantly low (p < 0.05). In addition, low absorbance was obtained in the 10e15 cm layer (13%) and 15e20 cm layer (5.6%). 3.2.3. Microbial substrate metabolic pattern Fig. A1 shows the AWCD of each sample at each sampling date. AWCD in activated sludge, influent to the SAT reactor and 0e5 cm layer increased more rapidly than the other samples. Fig. 3 shows the results of the principal component anal￾ysis. In addition, Table A2 shows the principal component loading rate of each substrate, and the 31 substrates were classified based on their structure, in reference to previous reports (Salomo et al., 2009; Zhang et al., 2012). The contribution ratio of the first and second principal component ranged from 61.3% to 71.6% and from 12.3% to 14.9%, respectively. The first principal component exhibited much in common with substrates whose loading rate excee￾ded 0.5 (Sala et al., 2005) among the sampling dates. Data plots of microbes with substantial metabolism for the following substrates are located in the positive direction on the first principal component axis: carboxylic acids (4-hydroxy ben￾zonic acid, itaconic acid and g-hydroxybutyricacid), amino water research 60 (2014) 105 e117 111

112WATERRESEARCH60(20I4)I05-II710JSPC10SPC29July5August:(12.3%)(14.3%)6T10-15 cm layer$-10cm layermmea22r-25cn lyeo25_30 cm layerIngient1015 cm lye 2-25 cm lyer.AS5-10cm layer0.87 em layer20.cm-2FPc'0-10lsembeAsT-21(--2187em layerFPC05 cm layer5lafucnt(68.9%)(61.4%)T-487 cm laycr--187em layer--8-10-101010SPCSPC12August19August(12.5%)(14.9%)41510510 cm layer2025 em layer:(-5 em layer10-15 em layer5-10cm aje15-20cm layer.AS (DIL: 100)05 cm layerAS(DIL-10)Influent25=30cm liveeP0 201-25 sm last.10-8-10 .9-8-4-2Yo4-241.6FPCaFPCASInfluent-287 cm layerAS (DIL: 1000)87 cm layer(61.3%)(71.6%)T4.187emligyer1187 cm layer953-10-10Fig.3-Changes of microbial substratemetabolic patterns at each sampling event, whereFPC refers to thefirst principalcomponent, SPC to the second principal component, AS to activated sludge, Influent to influentto the SATreactor and DILtodilution.acids (t-asparagine, L-phenylalanine and L-threonine), poly-and esters to a high degree, and it is possible that the meta-mers (Tween 40 and Tween 80) and esters(pyruvic acidbolicpatternexhibitedbythe5-iocmlayerisatamidpointofmethylester).Meanwhile,thosewithsubstantialmetabolismthechangingpattern from the0-5cmlayer to the10-30cmfor the following substrates are located in the negative direc-layers.This result suggests that the SAT sand layers imme-tion: carbohydrates (p-cellobiose, D-mannitol, N-acetyl-D-glu-diatelybelow5cmfromthetopsurfaceremovedifferentcosamine,α-D-lactoseand β-methyl-p-glucoside),amino acidsorganic compounds by biodegradation than the aerobic tankin WWTP.This also means that several organic compounds,(L-serine),polymers (glycogen)and phoshorylated chemicals(pL-α-glycerol phosphate). Roughly speaking, microbes withwhich are noteasily removed by the microbial activities in thesubstantialmetabolismforcarboxylicacidandestersareactivatedsludge,whichisoperatedwithlimitedHRTandSRT,plotted in the positive direction, while those with substantialareremovedbymicrobialactivitiesintheSATsandlayersmetabolism for carbohydrates are plotted in the negative di-below5cmfrom thetopsurface.In addition,theboundaryrection.Meanwhile,thesecond principal componentexhibi-betweenthevadose zoneand saturated zone was exhibitedbyted little in common with substrates whose loading ratethe 15-20 cm layer. Meanwhile, there were no changesexceeded 0.5 among the sampling dates.observed in the metabolic patterns of adjacent layers. More-Activated sludge having different dilution levels are plottedover, metabolic patterns demonstrated by the 87 cm layer,with the data set taken on 19 August. The fact that the threeespeciallythe187cmare unique.data plots are located at approximately the same position3.2.4.Water qualities along the flow pathindicates that the two digit differences in the copy numbersamongthe sampleshavelittle influence on theresults of theSS and VSS for the activated sludge were (median (mini-mum-maximum)1140(960-1360)and 975(757-1200),principalcomponentanalysis.The distribution of substrate metabolicpatterns for therespectively,while those for the influent to the SAT reactor(2.90-4.75)mg/Land3.60(2.503.65)mg/L,samplesexhibited much in commonamongthe samplingwere3.80date. It was determined that similar substrate metabolic pat-respectively.terns, which metabolized carbohydrates to a high degree,Table2showswater qualities alongtheflow path.Inwere obtained for the activated sludge, influent to the SATaddition, Fig.4 shows theprofiles of the water qualitypa-reactor and 0-5 cm layer.In comparison to these, quitearameters in the SAT reactor.The pH of the water samples,different metabolic pattern was demonstrated by the sandexcept for the effluent (187 cm) from the SAT reactor, waslayersfrom 10 to30 cm,which metabolized carboxylicacidsimilar.Meanwhile,theeffluent (187 cm) from the SAT reactor

acids (L-asparagine, L-phenylalanine and L-threonine), poly￾mers (Tween 40 and Tween 80) and esters (pyruvic acid methyl ester). Meanwhile, those with substantial metabolism for the following substrates are located in the negative direc￾tion: carbohydrates (D-cellobiose, D-mannitol, N-acetyl-D-glu￾cosamine,a-D-lactose and b-methyl-D-glucoside), amino acids (L-serine), polymers (glycogen) and phoshorylated chemicals (DL-a-glycerol phosphate). Roughly speaking, microbes with substantial metabolism for carboxylic acid and esters are plotted in the positive direction, while those with substantial metabolism for carbohydrates are plotted in the negative di￾rection. Meanwhile, the second principal component exhibi￾ted little in common with substrates whose loading rate exceeded 0.5 among the sampling dates. Activated sludge having different dilution levels are plotted with the data set taken on 19 August. The fact that the three data plots are located at approximately the same position indicates that the two digit differences in the copy numbers among the samples have little influence on the results of the principal component analysis. The distribution of substrate metabolic patterns for the samples exhibited much in common among the sampling date. It was determined that similar substrate metabolic pat￾terns, which metabolized carbohydrates to a high degree, were obtained for the activated sludge, influent to the SAT reactor and 0e5 cm layer. In comparison to these, quite a different metabolic pattern was demonstrated by the sand layers from 10 to 30 cm, which metabolized carboxylic acid and esters to a high degree, and it is possible that the meta￾bolic pattern exhibited by the 5e10 cm layer is at a midpoint of the changing pattern from the 0e5 cm layer to the 10e30 cm layers. This result suggests that the SAT sand layers imme￾diately below 5 cm from the top surface remove different organic compounds by biodegradation than the aerobic tank in WWTP. This also means that several organic compounds, which are not easily removed by the microbial activities in the activated sludge, which is operated with limited HRT and SRT, are removed by microbial activities in the SAT sand layers below 5 cm from the top surface. In addition, the boundary between the vadose zone and saturated zone was exhibited by the 15e20 cm layer. Meanwhile, there were no changes observed in the metabolic patterns of adjacent layers. More￾over, metabolic patterns demonstrated by the 87 cm layer, especially the 187 cm are unique. 3.2.4. Water qualities along the flow path SS and VSS for the activated sludge were (median (mini￾mumemaximum)) 1140 (960e1360) and 975 (757e1200), respectively, while those for the influent to the SAT reactor were 3.80 (2.90e4.75) mg/L and 3.60 (2.50e3.65) mg/L, respectively. Table 2 shows water qualities along the flow path. In addition, Fig. 4 shows the profiles of the water quality pa￾rameters in the SAT reactor. The pH of the water samples, except for the effluent (187 cm) from the SAT reactor, was similar. Meanwhile, the effluent (187 cm) from the SAT reactor Fig. 3 e Changes of microbial substrate metabolic patterns at each sampling event, where FPC refers to the first principal component, SPC to the second principal component, AS to activated sludge, Influent to influent to the SAT reactor and DIL to dilution. 112 water research 60 (2014) 105 e117

113WATERRESEARCH60(20I4)I05-II7Table:Rangesiterquallnandresult:aregivenasmediallererangesminimumximum)SamplepHi-00.0g0/0)DOC(ngC/L)UVs(en)6.45 (6.32-6.50)2.36(7.20-3,36)3650.41-113)0.104(0.071-1.156)Actiaidsdolg>005-002993643(631-6.30)(0.070-0.128)147 (0.13--25).68(331-4:19)p>0.05p>0.05cnnmuerhe SAT6.52 (6.2%6.90cno32(1180.0550.05afluct55 (6.47-6.704.34 (3.544.7848 (0.88060.P>e0E0U2200.5.51 (534-5.65)Eflaes (187 cm) bom theSAT nmitc3.61:(3.27-1.93),672 (0.6080:740024 (0,01-0.035SUVA(LAmgCcn)omorenL)TNL(egN0.029 (0.0200.038)798 (0.370-1865)0.25 (2.414.2)mada-130.05>0.0C0030.01.023 (0.02(6.0))400 (0.280-0.8)8nons0.0326uenttle SAT tekt0.026(0.024-0.061).34 (:32NO,-NEmN/Lo0>0.05Efhumt (25 an) fromthe SAT racsn0mnthe SATmsdo0.0184(0.9Elfunt (07 cn) t0.89(3.6~1.19)200(0.003 (00.001-0.027)0.30(0.220.41)Efflaeni(t87cm)bomtheSAT.rmc8.64 (3.263.99)was significantlylowerthan that ofthe effluent (87cm)fromreactorwas observed (p0.05).Meanwhile,aSimilar values of UV254 and SUVA were obtained for thesignificant decreasing trend along thepercolation pathin theactivatedsludgeandinfluenttotheSATreactor(p>0.o5)pH(-)DO (mgO,/L)DOC (mgC/L)UV (cm-)02345720.050.10.1556834500000(a) dap pues(a) ydap(s)(o)dappues50505050idap100100100100puespues150150150150200200200200SUVA (L/mgC-cm)DTN (mgN/L)DTP (mgP/L)00.02 0.04 0.06 0.08400.2 0.4 0.6 0.8C--(u) dap(ua)505050udap100100Ipues150150200200200Fig.4-Profiles of the water quality parameters in the SAT reactor.Plot and bars show median, and minimum andmaximumvalues,respectively

was significantly lower than that of the effluent (87 cm) from the SAT reactor, as discussed previously. Median DO of the samples varied from 1.47 to 4.34 mgO2/L indicating that each water sample was under aerobic conditions, as discussed previously. Similar values of DOC were obtained for the activated sludge and influent to the SAT reactor (p > 0.05). Meanwhile, a significant decreasing trend along the percolation path in the reactor was observed (p 0.05). Fig. 4 e Profiles of the water quality parameters in the SAT reactor. Plot and bars show median, and minimum and maximum values, respectively. Table 2 e Ranges of water quality index and results of the Wilcoxon rank sum test, where ranges are given as median (minimumemaximum). water research 60 (2014) 105 e117 113

114WATERRESEARCH60(20I4)I05-II7MedianUV254decreasedfromtheinfluenttotheSATreactorexcitationwavelengths(Ex)/emissionwavelengthtotheeffluent(25cm)fromtheSATreactor;however,itwas(Em)= o.05).Meanwhile,SUVA significantlyproteins.Peaks in the range of Ex/Em=250-280nm/0.05)acid-likeandhumicacid-likecompoundswereobservedinThere were no significant differences for UV254 and SUVAthe activated sludge and influent to the SAT reactor. Mean-betweeneffluent(87cm)fromtheSATreactorandeffluentwhile,effluent(25cm)fromtheSATreactorwasdominatedby(187cm)from theSATreactor (p>0.05)SMP-likecompounds.In addition,peaksrepresentativeofFluorescence EEM profiles of the activated sludge, influentsimplearomaticproteins,fulvicacid-likeandhumicacid-liketotheSATreactor,eacheffluentfromtheSATreactor samplescompoundsin effluent (25cm)fromthe SAT reactorwereare given in Fig.5. In general, peaks in the short range ofstronger than those in the influent to the SAT reactor.In285-300270-285255-270240-255225-240210-225195-210180-195165-180150-165135-150120-135105-12090-10575-9060-7530-4515-300-1545-60Activated sludgeInfluetotheSATcogeo400400aaaea380380#33360340320300280280260260240240220220240270300330360390420450480240270300330360390420 450480Emission wavelength (nm)Emission wavelength (nm)Effluent(25cm)fromtheSATreaetoEffluent(87cm)fromtheSATreact400400aeea380380360360340340320320300300280280260260240240220220240270300330360390420450480240270300330360390420450480Emission wavelength (nm)Emission wavelength (nm)Effluent (187cm)fromtheSAT reactor400(aaee380360340320300280260240220240270 300330360390420450480Emissionwavelength (nm)Fig.5-Fluorescenceemission-excitationmatrices(EEM)profilesoftreatedwatersamplesfromvariouspointsoftheSATreactor

Median UV254 decreased from the influent to the SAT reactor to the effluent (25 cm) from the SAT reactor; however, it was not significant (p > 0.05). Meanwhile, SUVA significantly increased from the influent to the SAT reactor to the effluent (25 cm) from the SAT reactor (p 0.05). There were no significant differences for UV254 and SUVA between effluent (87 cm) from the SAT reactor and effluent (187 cm) from the SAT reactor (p > 0.05). Fluorescence EEM profiles of the activated sludge, influent to the SAT reactor, each effluent from the SAT reactor samples are given in Fig. 5. In general, peaks in the short range of excitation wavelengths (Ex)/emission wavelength (Em) ¼ <250 nm/<350 nm are related to simple aromatic proteins. Peaks in the range of Ex/Em ¼ 250e280 nm/<380 nm are associated with soluble microbial by-product-like (SMP￾like) compounds. Peaks in the range of Ex/Em ¼ 240e260 nm/ 380e480 nm are related to fulvic acid-like compounds. Peaks in the range of Ex/Em ¼ 320e350 nm/400e480 nm are associ￾ated with humic acid-like compounds (Chen et al., 2003; Xue et al., 2009). Peaks representative of simple aromatic proteins, fulvic acid-like and humic acid-like compounds were observed in the activated sludge and influent to the SAT reactor. Mean￾while, effluent (25 cm) from the SAT reactor was dominated by SMP-like compounds. In addition, peaks representative of simple aromatic proteins, fulvic acid-like and humic acid-like compounds in effluent (25 cm) from the SAT reactor were stronger than those in the influent to the SAT reactor. In Fig. 5 e Fluorescence emissioneexcitation matrices (EEM) profiles of treated water samples from various points of the SAT reactor. 114 water research 60 (2014) 105 e117