quick assessment of genotypic changes in the communityover time, or to compare communities subject to differentenvironmental conditions.
Materials and methods
Description of the WWTP configurations
The activated sludgesamples were collected from the oxic and the anoxic reactors of three WWTPs located in Girona, Spain. The WWTP of Vidreres-Sils (D1) has an anoxic tank (D1-an) of 350 m
, and an oxic tank (D1-ox) of 1500 m
, arranged in a modified Ludzack-Ettinger(MLE) configuration. It treats about 1500 m
/day of domesticwastewater. The WWTP of Ripoll (D2) was built to operate asan Orbal but, due to the low influent load, only the two innerchannels are in use. Of these two, the outer one (4500 m
,designated as D2-an) operates under anoxic conditions, whilethe other (3500 m
, designated as D2-ox) is under oxic conditions.Internal recycling from the inner to the outer channel allows thesystem to work in MLE configuration. The WWTP of Ripolltreats approximately 9000 m
/day of domestic wastewater. Finally,the third WWTP studied is a pilot plant that treats industrialwastewater from a food-processing factory. It has an oxic reactorof 3.5 l (designated as IN-ox), and an anoxic reactor of 2.2 l(designated as IN-an) in an MLE configuration. The influentflow to the pilot plant is 0.83 l/day.
Sampling and analytical determinations
Samples of the inletand the outlet of each reactor were collected in order to determineinfluent characteristics and reactor efficiencies. The conservationof the samples, their processing and all analytical measurementswere carried out according to APHA . Activated sludgesamples of 50 ml for DNA extraction were collected in sterileFalcon tubes and frozen at –20ºC until processing.
16S rDNA amplification
DNA extraction was carried out inNalgene sterile tubes by the phenol-chloroform method describedby Moore . DNA was purified with Bio-Spin chromatographycolumns (Bio-Rad, Hercules, CA, USA) to eliminate proteinsand nucleotides. PCR amplification of 16S rDNA, using theprimer set of fD1 and rP1, was performed as described previously. PCR products were purified using the QIAquick PCRPurification Kit (Quiagen, Valencia, CA, USA).
rDNA restriction fragments separation by electrophoresis
Double restriction endonuclease digestions were performedfor every sample with
I (Promega, Madison, WI, USA)as described previously . Restriction enzymes were chosenon the basis of their high average number of restriction sitesper taxon . Separation of digested products in poly-acrilamide gels were performed as described elsewhere [1, 8].The gel was digitallized using a scanner AGFA Arcus II andthe images were contrasted using the NIH Image Program 1.59(National Institutes of Health, Bethesda, MD, USA).
The patterns of each sample were compared byidentifying, from different samples, fragments of identical sizein the same digestion. Pairwise comparations of the bandpatterns were manually performed, and a presence/absencematrix was constructed. In this way, the Dice similaritycoefficient  was obtained for every pair of samples, enablingus to generate a similarity dendrogram. The data were computedby using the SPSS program version for Macintosh 4.0.
Results and Discussion
Influent characteristics, operational parameters andremoval efficiencies
Table 1 summarizes the principal influentcharacteristics and operational parameters for each of the threeWWTPs. The nitrite and nitrate concentrations in the influentswere always under 1 ppm N. The chemical oxygen demand(COD) removal efficiencies of all WWTPs were over 85%.The nitrification efficiencies were 83% and 94% for reactorsD1 and IN, respectively, while reactor D2 showed completenitrification. Reactors D1 and D2 had denitrification efficienciesof 16% and 64%, respectively. Reactor IN also achievedcomplete denitrification.
Principal characteristics and operational parameters of the treatmentplants studied: D1, urban Vidreres-Sils wastewater; D2, urban Ripoll wastewater;IN, industrial wastewaterOperational parameterReactorD1D2INsCOD (mg/l)1441578539pCOD (mg/l)961491138N-NH
(mg/l)1118655sNorg (mg/l N)23327pNorg (mg/l N)<18–MLSS
(day)>3022>30external recycle (%)75290150internal recycle (%)2002901000T
(°C)101238s: soluble, p: particulate,
: sludge residence timeCOD, chemical oxygen demandMLSS, mixed liquor suspended solidsVSS, volatile suspended solids
Differences between industrial and domestic WWTPcommunities
The restriction patterns obtained by electro-phoresis are shown in Fig. 1. The differences between restrictionpatterns in all the communities subject to domestic wastewaterare smaller than those between domestic wastewater WWTPsand those treating the industrial wastewater, as reflected in thedendrogram and in the Dice similarity coefficient matrix shown
Vol. 3, 2000Gich et al.