《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). There were notion-emission matrix (EEM)profiles,excitation wavelengthsnoticeable seasonal changes in eachwater sample.were incremented from220 to400 nm in5nm steps,and forWhen10g-wetsterilized sandwasshaken with40mL ofeach excitation wavelength, the emission was detected fromsterile influent to the SAT reactor for 12 h, the pH in the240to500nm in5nmsteps.FluorescenceofsuperpurifiedinfluenttotheSATreactorwasfoundtodecreasefrom7.34towaterasablanksamplewassubtractedfromeachspectrum6.16. Basedon this result, the decreaseof pHin the SAT reactorSuspended solid (Ss): 105°C for 2 h and volatile suspendedwaspartiallycausedbytheoriginal sand characteristics,suchsolid (vss):600c for 30min weremeasuredfortheactivatedas ion exchanges.sludge and treated wastewaterWorksJapan SewageThe DO ranged 3.36 (1.12-6.70), 7.87 (3.32-10.6), 4.38Association,1997)(1.28-9.18) and 4.52 (1.30-7.56) mgO2/L, respectively. The DOoftheeffluent (87cm)fromtheSATreactorwas significantlyhigher than that of the influent to the SAT reactor (p < 0.05)2.4.Statistical analysisand theincreasecanbepartiallyattributed tothecontactofthe influent to the SAT reactor with air trapped in the sandThe Wilcoxon rank sum test was used to examine the statis-voids of the vadose zone.DO significantly decreased throughtical significance of the measured sand and water quality in-percolation from the87-187 cmdepth (p<0.05).Meanwhiledexes between two samples at subsequent points along thethere were no significant differences in DO between theflowpath,suchastheinfluenttotheSATreactorvs.effluenteffluent(187cm)fromtheSATreactorandfinal effluent(25cm)fromtheSAT reactor and 0-5cmlayer vs.5-10cm(237cm)fromtheSATreactor(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)p<0.05p<0.051.8×1075-10 cm layer0.027 (0.012-0.038)0.042 (0.010-0.057)β>0.05p >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>0.05p<0.050.0052.0×102025 cm layer0.010 (0.00780.011)p>0.050.0051.1×10725-30 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). There were no noticeable seasonal changes in each water sample. When 10 g-wet sterilized sand was shaken with 40 mL of sterile influent to the SAT reactor for 12 h, the pH in the influent to the SAT reactor was found to decrease from 7.34 to 6.16. Based on this result, the decrease of pH in the SAT reactor was partially caused by the original sand characteristics, such as ion exchanges. The DO ranged 3.36 (1.12e6.70), 7.87 (3.32e10.6), 4.38 (1.28e9.18) and 4.52 (1.30e7.56) mgO2/L, respectively. The DO of the effluent (87 cm) from the SAT reactor was significantly higher than that of the influent to the SAT reactor (p < 0.05), and the increase can be partially attributed to the contact of the influent to the SAT reactor with air trapped in the sand voids of the vadose zone. DO significantly decreased through percolation from the 87e187 cm depth (p < 0.05). Meanwhile, there were no significant differences in DO between the effluent (187 cm) from the SAT reactor and 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