2001_lourenço_effect of some operational parameters on textile dye biodegradation in a sequential...

Journal of Biotechnology 89 (2001) 163–174

Effect of some operational parameters on textile dyebiodegradation in a sequential batch reactor

N.D. Lourenco, J.M. Novais, H.M. Pinheiro *Centro de Engenharia Biologica e Quımica, Instituto Superior Tecnico, A�. Ro�isco Pais, 1049-001 Lisbon, Portugal

Received 26 June 2000; received in revised form 8 January 2001; accepted 15 January 2001

Abstract

The combination of anaerobic and aerobic periods in the operation cycle of a Sequencing Batch Reactor (SBR) waschosen to study biological color removal from simulated textile effluents containing reactive, sulfonated, monoazoand diazo dyes, respectively, Remazol Brilliant Violet 5R and Remazol Black B. 90% color removal was obtained forthe violet dye in a 24-h cycle with a Sludge Retention Time (SRT) of 15 days and an aerated reaction phase of 10h. For the black dye only 75% color removal was achieved with the same operational conditions and no improvementwas observed with the increase of the SRT to 20 days. For the violet dye a reduction of the color removal values from90 to 75% was observed with the increase of the aerated reaction phase from 10 to 12 h. However, this increase didnot promote the aerobic biodegradation of the produced aromatic amines. Abiotic tests were performed withsterilized SBR samples and no color removal was observed in cell-free supernatants. However color removal valuesof 30 and 12% were observed in the presence of sterilized cells and supernatants with violet and black dye, respectivelyand could be attributed to the presence of active reducing principles in the sterilized samples. © 2001 Elsevier ScienceB.V. All rights reserved.

Keywords: Textile effluents; Anaerobic-aerobic biotreatment; Sequencing batch reactor; Sulfonated azo dyes; Reactive dyes; Colorremoval

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1. Introduction

Large amounts of dyes are annually producedand applied in many different industries, includ-ing the textile, cosmetic, paper, leather, pharma-ceutical and food industries (Zollinger, 1987;Meyer et al., 1992). The textile industry accountsfor two-thirds of the total dyestuff market (Riu et

al., 1998), consuming a large proportion of reac-tive azo dyes due to the actual high demand forcotton fabrics with brilliant colors (Phillips, 1996).The reactive dyes bind to the cotton fibers byaddition or substitution mechanisms under alka-line conditions and high temperature. Under theseconditions a significant fraction of the dye ishydrolyzed and released into the environmentwith the rejected dyebaths or wash waters(Ganesh et al., 1994).

The decolorization of textile wastewater is stilla major environmental concern because the syn-

* Corresponding author. Tel.: +351-21-841-9125; fax: +351-21-841-9062.

E-mail address: [email protected] (H.M. Pinheiro).

0168-1656/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.

PII: S0168-1656(01)00313-3

N.D. Lourenco et al. / Journal of Biotechnology 89 (2001) 163–174164

thetic dyes used are difficult to remove by theconventional wastewater treatment systems basedon adsorption and aerobic biodegradation (Bum-pus, 1995; Vandevivere et al., 1998; O’Neill et al.,1999). In spite of the low toxic effect on receivingbodies, the dyes constitute an aesthetic problemwith great impact in the public opinion(Goncalaves et al., 1996) and color restricts thedownstream use of the wastewater (O’Neill et al.,1999). Furthermore, a great number of the re-ported examples of azo dye decolorization bymicroorganisms start by reductive cleavage of theazo bond, under anaerobic conditions, with theformation of the corresponding aromatic amines(Wuhrmann et al., 1980; Haug et al., 1991; Car-liell et al., 1995; Nigam et al., 1996; Zissi andLyberatos, 1996). This step is responsible forcolor removal but it does not remove the dye-re-lated hazard from the wastewater since the anaer-obic mineralization of aromatic amines has notbeen reported, except for a few hydroxyl- andcarboxyl-substituted amines (Razo-Flores et al.,1996), and some of these aromatic amines can betoxic and carcinogenic (Pasti-Grigsby et al., 1992;Carliell et al., 1995; Field et al., 1995). However,some aromatic amines can be aerobically de-graded (Nortemann et al., 1986; Bumpus, 1995;Zissi and Lyberatos, 1996). Recent studies haveused the combination of anaerobic and aerobicsteps in an attempt to achieve not only decoloriza-tion but also mineralization of reactive azo dyes(Seshadri and Bishop, 1994; Field et al., 1995;FitzGerald and Bishop, 1995; Tan et al., 1999a,c).The Sequencing Batch Reactor (SBR) technologyhas been recently implemented for nitrogen andphosphorus removal (Banas et al., 1999; Helmre-ich et al., 2000), particularly for piggery wastewa-ter treatment (Bernet et al., 2000; Edgerton et al.,2000) due to its good operational flexibility, sim-ple running and compact layout. Lab-scale SBRsystems were used in the present study with se-quenced anaerobic and aerobic phases for col-orant biodegradation in a simulated cotton textileeffluent containing sulfonated azo and diazo reac-tive dyes. Different operational parameters wereevaluated including the Sludge Retention Time(SRT) and the relative duration of the anaerobicand aerobic phases. Abiotic tests were included to

check for the possibility of chemical dye reductionor simple adsorption in oxygen-depletedconditions.

2. Materials and methods

2.1. Chemicals

2.1.1. Preparation of the carbon source stocksolution

The starch-based sizing agent Emsize E1 wasobtained from Emsland-Starke GmbH, Germany,and was hydrolyzed by a procedure based on theoxidative desizing conditions described by themanufacturer. A 100 g of Emsize E1 and 40 g ofsodium hydroxide were dissolved in distilled waterand stirred for 15 h at room temperature. Thesolution was then neutralized with concentratedHCl and diluted to 1 l with distilled water.

2.1.2. Feed solutionThe feed solution had a COD composition of

750 mg l−1 and was prepared in aerated tap waterusing 862 mg l−1 hydrolyzed Emsize E1 (from itsstock solution), 143 mg l−1 NH4Cl, 760 mg l−1

KH2PO4 and 915 mg l−1 Na2HPO4. The saltswere analytical grade and purchased from Merck,Germany.

2.1.3. Hydrolyzed dye stock solutionsThe reactive dyes chosen for the present study

were Remazol Brilliant Violet 5R and RemazolBlack B (Fig. 1), azo and diazo reactive dyes,respectively. The dyes were of commercial quality,supplied by Dystar Anilinas Texteis SA, Portugal,and were used without further purification. Thehydrolyzed stock solutions (5 g l−1) were pre-pared according to the dyebath conditions de-scribed by the manufacturer. Five grams of theselected dye were dissolved in distilled water, thepH was adjusted to 12 with 1N NaOH solutionand the solution was stirred for 1 h at 80 °C.After reaching room temperature the solution wasneutralized with concentrated HCl and diluted to1 l with distilled water.

N.D. Lourenco et al. / Journal of Biotechnology 89 (2001) 163–174 165

2.2. SBR inocula

The four SBR were inoculated with sludge col-lected in a full-scale, continuous activated sludgeplant (Beirolas, Loures, Portugal) treating mixeddomestic-industrial wastewater and acclimatizedto the described feed solution during one monthbefore the decolorization experiments started.

2.3. Experimental system

2.3.1. SBR operationThe experimental system used was composed of

four 1 l reactors operating in a sequencing batchmode with 24-h cycles. SBR 1 was used for stud-ies with Remazol Brilliant Violet 5R and SBR 2was operated in the same conditions but in theabsence of dye and was used as a control. SBR 3and SBR 4 (control) were used in the same waybut for the Remazol Black B dye. The experimen-tal operational conditions used in the color re-moval studies are summarized in Table 1.

The feed solution was supplied to the fourreactors with a flow of 800 ml d−1 each. Mixingwas provided by magnetic stirrers and air wassupplied by an air compressor through ceramic

diffusers. The dissolved oxygen concentration wasmaintained above 5 mg l−1. A PC computerprogram automatically controlled the pumping,discharge, aeration and agitation functions. Afterthe acclimatization period, hydrolyzed RemazolBrilliant Violet 5R and Remazol Black B stocksolutions were daily injected into SBR 1 and 3,respectively, immediately after the end of the cor-responding fill phase.

2.3.2. Abiotic testsAbiotic color removal tests were conducted us-

ing 50 ml samples taken from SBR 1 and 3 at theend of the fill phase, during operational periods 4and 6, respectively. All samples were sterilized byautoclaving at 120 °C during 20 min. Three dif-ferent tests were performed. The first one con-sisted in autoclaving the entire sample. Thesecond and the third tests consisted in separatingthe cells from the supernatant by centrifugationand resuspending the cells in fresh feed solutionbefore autoclaving. In the last test, however, thecentrifugal separation of the cells from the super-natant was performed in the presence of a liquidparaffin layer in order to minimize contact of thesample with oxygen. Hydrolyzed violet or black

Fig. 1. Chemical structure of the reactive dyes used in the present study.


Table 1Summary of the four SBR operational conditions used for color removal studies with Remazol Brilliant Violet 5R (SBR 1 and 2)and Remazol Black B (SBR 3 and 4)

SBR 1 and 2 SBR 3 and 4

Period 2 Period 3 Period 4Period 1 Period 5Cycle times Period 6

Fill (min) 50 50 50 50 53 53Mixed react (h) 1313 11 9 11 11

8 10 128 10Aerated react (h) 1060Settle (min) 60 60 60 50 5055Draw (min) 55 55 55 67 67

15 15 1515 10Idle (min) 10

Other parameters100 100 10060 30Fed dye conc. (g l−1)a 30

10SRT (d) 15 15 15 15 2090 130 150 220Period duration (d) 160100

a Only on SBR 1 and 3.

dye were injected in the corresponding samples(cell suspensions or cell-free supernatants) in or-der to achieve initial dye concentrations of 100and 30 mg l−1, respectively. The dye-containingsamples were incubated under mild magnetic stir-ring and no aeration, for periods up to 75 h.

2.4. Analyses

Color measurements in clarified (centrifuged)samples of the mixed liquor from SBR 1 and 3and from the abiotic tests were performed in aHitachi U-2000 spectrophotometer in the UV-visi-ble range against a baseline defined by clarifiedsamples from the corresponding dye-free reactor.Absorbance of the samples was measured at themaximum absorption wavelength (�max) in thevisible region for each dye (�max=560 nm forRemazol Brilliant Violet 5R and �max=590 nmfor Remazol Black B). Volatile Suspended Solids(VSS), Chemical Oxygen Demand (COD) and pHwere determined according to standard proce-dures (American Public Health Association,1995). Oxidation-Reduction Potential (ORP) wasmeasured with an ORP electrode (platinum withsilver/silver chloride as reference electrode, 3MKCl) connected to a digital pH meter (WTW, pH538), (pH 7.0 and 25 °C). The relationship be-tween the silver/silver chloride electrode and the

standard hydrogen electrode is Eh (mV)=EAg/AgCl

(mV) +207 at 25 °C. Dye degradation andmetabolite formation were followed by reversed-phase HPLC (Merck-Hitachi LC-organizer, L-4250 UV-VIS detector and L-6200A intelligentpump; Hewlett Packard HP3395 integrator) usinga LichroCART Purospher RP-18e column(250 mm×4 mm), (Merck, Germany). The eluentconsisted of water and acetonitrile with 0.2% oftetrabutylammonium hydrogen sulfate (gradientelution with 20–40% acetonitrile in 35 min), theflow was 1.0 ml min−1 and the spectrophotomet-ric detection was performed at 224 nm.

3. Results

3.1. Remazol Brilliant Violet 5R

The first biodegradation studies with the violetdye were performed in an SBR with two imposedvalues of SRT (operational periods 1 and 2 ofTable 1) and an aerated reaction phase of 8 h.Average results obtained after an initial 100-dayperiod of sludge acclimatization are presented inTable 2.

During operational period 2 (Tables 1 and 2) itwas observed that in spite of the high color re-moval performance of the SBR system, the HPLC


Table 2Average SBR 1 performance parameter values for RemazolBrilliant Violet 5R biodegradation with SRT values of 10 and15 days, corresponding to operational periods 1 and 2 (Table1), respectively

SRT 10 d SRT 15 d

60Feed dye concentration 100(mg l−1)

80Residual color in drawn 10effluent (%)

COD removal in the anaerobic 10 30phase (%)

COD removal in the total cycle 70 80(%)

−350Lowest ORP value in −430anaerobic phase (mV)

Mixed liquor VSS (g l−1) 2.01.2

tively. The effluent residual color values obtainedduring these operational periods are presented inFig. 2. The mixed liquor VSS (g l−1) values in theSBR were around 2.0, 1.8 and 2.0 for periods 2, 3and 4, respectively.

Typical examples of SBR performance in termsof color and COD removal during 24-h cycleswith increasing duration of the aerated reactionphase are presented in Fig. 3. The correspondingORP profiles are shown in Fig. 4.

The HPLC analysis performed to clarifiedmixed liquor samples taken during cycles with thethree different duration of the aerated reactionphase indicated that there was no improvement inthe aerobic removal of intermediates formed dur-ing the anaerobic phase of operation. Fig. 5shows examples of the evolution of chromato-graphic peak areas corresponding to the dye andits metabolites, during SBR 1 cycles in periods 2and 3 of Table 1.

Abiotic tests were performed in conditionsmimicking the anaerobic phase of SBR 1, ex-tended up to 75 h, with the objective of detectingpossible chemical or adsorptive color removalprocesses. The first tests, carried out with auto-

analyses seem to indicate that the intermediatesproduced during the non-aerated phase of reac-tion were not mineralized in the subsequent aero-bic phase. In an attempt to promote the aerobicbiodegradation of these intermediates, the dura-tion of the aerated phase of reaction was in-creased from 8 to 10 and 12 h, corresponding tooperational periods 2, 3 and 4 of Table 1, respec-

Fig. 2. Effluent residual color during 150 days of operation of the SBR 1, fed with dye Remazol Brilliant Violet 5R with 15 d SRTand aerated reaction phases of 8 (�), 10 (�) and 12 h (�), corresponding to periods 2, 3 and 4 of Table 1, respectively.


Fig. 3. Typical color (A) and COD (B) removal profiles during24-h SBR 1 cycles operated with 15 d SRT and aeratedreaction phases of 8 (�), 10 (�) and 12 h (�), correspondingto periods 2, 3 and 4 of Table 1, respectively. The vertical lines1, 2 and 3 indicate the onset of aeration in operational periods2, 3 and 4, respectively.

absence of liquid paraffin was exactly the sameand only the latter is presented.

3.2. Remazol Black B

Since the SBR 1 revealed a very good decol-orization performance with dye Remazol BrilliantViolet 5R when operating with a SRT of 15 daysand an aerated reaction phase of 10 h, the sameexperimental conditions were tested in SBR 3 withthe diazo dye Remazol Black B. A comparisonbetween the residual color in the drawn effluentsfrom these two SBR is given in Fig. 7. TypicalSBR cycle profiles for color and COD removal foreach dye are represented in Fig. 8.

On the basis of the influence of SRT on decol-orization observed for the violet dye (Table 2),two different values of SRT were studied forRemazol Black B color removal, namely 15 and20 days, corresponding to periods 5 and 6 ofTable 1, respectively. The average results obtainedfor the two operational periods are summarized inTable 3.

The abiotic tests performed with the dye Rema-zol Black B were conducted as described forRemazol Brilliant Violet 5R. Results are shown inFig. 9. Results of tests with the liquid paraffinprocedure were not conclusive due to paraffininterference in the dye solubilization.

Fig. 4. Typical ORP profiles during 24-h SBR 1 cycles oper-ated with 15 d SRT and aerated reaction phases of 8 (······), 10(— ) and 12 h (— ), corresponding to periods 2, 3 and 4 ofTable 1, respectively.

claved portions of the SBR 1 mixed liquor fedwith the violet dye, showed some degree of decol-orization (Fig. 6). Therefore, for the second andthird tests, biomass was centrifugally separatedfrom the SBR 1 medium and anaerobic decol-orization runs were performed both with cell-freesupernatants and with the biomass resuspended infresh feed solution. However, in the third test thecell separation was performed in the presence ofliquid paraffin to minimize contact with oxygen.The decolorization results obtained are summa-rized in Fig. 6. The behavior of the cell-freesupernatant obtained in the presence or in the


Fig. 5. Examples of the evolution of the chromatographic peakareas corresponding to the dye metabolites during SBR 1cycles in periods 2 (A) and 3 (B) of Table 1. Metabolites 1 (�)and 3 (�) correspond to the benzene-based and naphthalene-based amines, respectively. Metabolite 2 (�) is apparently inequilibrium with metabolite 1. The violet dye concentrationscalculated from spectrophotometric analysis (�) are also pre-sented.

tion runs, in which the same value of biomassconcentration (4.5 g l−1 VSS) was used, wereconducted using activated sludge grown in anSBR with different imposed SRT values and indi-cated that a SRT value of at least 15 days in thebiomass production reactor was necessary forhigh color removal efficiencies (results notshown). These results suggest that the adequatemicrobial population could not be installed in theSBR operated with 10 days of SRT but coulddevelop with 15 days of SRT. Rodrigo et al.(1999) also encountered a relationship betweenthe SRT and the development of a minimumnecessary culture of phosphate accumulating mi-croorganisms for enhanced biological phospho-rous removal. In the present case, decolorizationefficiency is apparently related to the capacity tolower the medium ORP in the anaerobic phase,through generation of reducing equivalents andtheir transfer to adequate electron carriers.

4.1.2. SBR operation with different aerated reactphases

During operational period 2 good results wereobtained in terms of color and COD removal inthe non-aerated phase of the SBR operation.However the 8-h aerobic phase was not efficient in

Fig. 6. Color removal profiles in the abiotic tests performedwith 50-ml samples taken from SBR 1 during period 4 (SRT15 d, aeration 12 h, VSS 2.0 g l−1). The samples consisted ofautoclaved mixed liquor (�), harvested cells resuspended infresh feed solution and autoclaved (�), the correspondingcell-free supernatant (�) and cells harvested by centrifugationin the presence of a paraffin layer, resuspended in fresh feedsolution and autoclaved (× ).

4. Discussion

4.1. Remazol Brilliant Violet 5R

4.1.1. SBR operation with different SRT �aluesThe results in Table 2 for the violet dye show

that the increase of the SRT from 10 to 15 daysresulted in an increase of the color and anaerobicCOD removal values, accompanied by a reduc-tion of the ORP values attained during the non-aerated phase. This could be attributed to thecorresponding increase of the biomass concentra-tion level in the SBR system from 1.2 to 2.0 g l−1

of VSS. However, parallel anaerobic decoloriza-


Fig. 7. Comparison between the residual color in the drawn effluents from SBR 1 (�) fed with 100 mg l−1 Remazol Brilliant Violet5R (period 3, VSS 2.0 g l−1) and SBR 3 (�) fed with 30 mg l−1 Remazol Black B (period 5, VSS 1.5 g l−1). Both SBR systemswere operated with 15 d SRT and 10 h of aeration in the 24-h cycle.

the mineralization of metabolites produced duringthe anaerobic phase (Fig. 5). In an attempt toimprove this efficiency, the aeration period wasincreased to 10 h (period 3, Fig. 2). The color andCOD removal capacities of the system were notcompromised (Fig. 3(B)), but HPLC analyses ofmetabolites revealed no improvement in theirmineralization. This is apparent in the chromato-graphic peak area profiles shown in Fig. 5. Itshould be noted that due to the unavailability ofstandards, the chromatographic peaks appearingin samples taken during the anaerobic phasecould not be identified or quantified. Howeverthese peaks did not appear in samples from thedye-free SBR. Through retention time comparisonwith standards of similar structured amines, it canbe proposed that metabolites 1 and 3 correspondto the benzene-based and naphthalene-basedamines, respectively, directly resulting from azobond reduction in the violet dye (Fig. 1). Metabo-lite 2 has a time profile suggesting that it estab-lishes an equilibrium with metabolite 1, dependingon the redox potential (Fig. 5). The differences inthe ORP profiles obtained in operational periods2 and 3 (Fig. 4) could have caused the different

proportions of metabolites 1 and 2 observed inthe anaerobic phases of Fig. 5 parts A and B. Thecolor removal profiles observed during period 3(Fig. 3(A)) suggested that high color removalvalues could still be achieved if the aerated phaseof the SBR cycle started even earlier. However,when the aeration time was increased to 12 h(period 4) there was a significant loss in the colorremoval yield (Fig. 2), accompanied by an in-crease in the anaerobic COD removal (Fig. 3(B))and a less negative profile of ORP values in thenon-aerated phase (Fig. 4). As suggested in theSRT experiments, the change of the relative dura-tion of the anaerobic and aerobic phases couldhave brought about alterations in the microbialpopulation in the SBR, negatively affecting dyereduction rates. Difficulties with the mineraliza-tion of azo dye intermediates after the anaerobicreduction are frequently encountered, particularlyfor the substituted aminobenzenesulfonic acids(Takeo et al., 1997; Tan et al., 1999b), and can beattributed to the lack in the SBR mixed biomassof an adequate aerobic microbial population ca-pable of metabolizing these compounds. The sim-ple increase of the aerobic phase of the 24-h cycle


SBR from 8 to 12 h proved to be inefficient forthe development of such a microbial population,suggesting that the addition of specifically-adapted aerobic bacteria may be required for totalmineralization of reactive azo dyes in an anaero-bic/aerobic SBR system. Also, an increase in thedye/carbon source concentration ratio in the SBRfeed could bring about earlier carbon source ex-haustion in the aerobic phase, thus possibly favor-ing the development of an amine-metabolizingmicrobial consortium.

4.2. Remazol Black B

4.2.1. SBR operation with different SRT �aluesTwo different SBR systems were used with the

same operational conditions (SRT of 15 days,10-h-aeration) for comparative color removal ex-periments with an azo (violet) and a diazo (black)

dye. It can be observed in Fig. 7 that along 220days of operation the percentage of unremovedcolor in the effluent of SBR 3, fed with RemazolBlack B, was much higher than in the effluent ofSBR 1, operated with the violet dye. The morecomplex structure of Remazol Black B (Fig. 1)could be responsible for the lower color removallevels obtained for this dye, since no apparentinhibitory effect on the culture could be detectedfrom the COD removal profiles (Fig. 8B). Theimprovement of the color removal yield of SBR 3was attempted through the increase of the SRTfrom 15 to 20 days, on the basis of the favorableresults obtained with the violet dye (Table 2).However, for SBR 3 this improvement was notobserved with the change in SRT (Table 3), inspite of an observed increase in the biomass con-centration level (from 1.5 to 2.0 g l−1 of VSS). Itshould be noted that the ORP values attained in

Fig. 8. Typical color (A) and COD (B) removal profiles obtained during 24-h cycles of SBR 1 fed with 100 mg l−1 Remazol BrilliantViolet 5R (�) and SBR 3, fed with 30 mg l−1 Remazol Black B (�). The SRT was 15 days and the aeration time was 10 h. Thevertical lines represent the onset of aeration. The VSS content was 2.0 and 1.5 g l−1 for SBR 1 and 3, respectively.


Table 3Average SBR 3 performance parameter values for RemazolBlack B biodegradation with SRT values of 15 and 20 days,corresponding to operational periods 5 and 6 (Table 1), re-spectively

SRT 20 dSRT 15 d

Feed dye concentration 30 30(mg l−1)

Residual color in drawn 35 35effluent (%)

45COD removal in the anaerobic 45phase (%)

COD removal in the total cycle 80 85(%)

Lowest ORP value in −340 −340anaerobic phase (mV)

Mixed liquor VSS (g l−1) 2.01.5

4.3. Abiotic tests

The abiotic tests performed with both the violetand the black dye indicated that color removaldid not occur in the absence of biomass (Figs. 6and 9). However, slow decolorization of the violetdye was observed in the presence of autoclavedcells. These results suggest the existence of anactive reducing factor present in the autoclavedbiomass samples and capable of reducing theviolet azo dye in the absence of microbial activity.The results also suggest that this reducing factorcan be oxidized by non-anoxic sample manipula-tion. In fact, there was a higher level of colorremoval when a paraffin layer was used duringcentrifugation, for the minimization of contactwith oxygen (Fig. 6). The presence of electroncarriers capable of azo dye reduction in the ex-tracted membrane fraction of a dye-reducing Sph-ingomonas strain was previously reported(Kudlich et al., 1997).

5. Conclusion

The studied SBR system proved to be poten-tially efficient for color removal from azo dyecontaining feeds when operated in optimized con-ditions in what concerns SRT and relative dura-tion of anaerobic and aerobic reaction phases.The color removal yield with the more complexdiazo dye Remazol Black B was, however, muchlower than that obtained with the monoazo Re-mazol Brilliant Violet 5R, even at higher SRTvalues. The establishment of low ORP values(under −400 mV) in the non-aerated phase seemsto be required for high decolorization rates, ap-parently also interfering with the profile ofmetabolites generated during the reduction pro-cess. For the monoazo violet dye it was observedthat the increase of the duration of the aeratedphase did not promote the aerobic biodegradationof the aromatic amine metabolites produced bydye reduction in the anaerobic phase. This sug-gests that the addition of specific, amine-degrad-ing aerobic bacteria to the SBR mixed culturemay be required for total mineralization of reac-tive azo dyes in this type of anaerobic/aerobicSBR system.

the anaerobic phase were higher than those ob-served for period 2 in Table 2, which gave bestdecolorization yields for the violet dye. It could besuggested that lower ORP values would be neces-sary for complete reduction of the diazo blackdye. Oxspring et al. (1996), for example achievedmore than 95% color removal with this same dyeafter 48 h retention in an upflow anaerobic filterof 1.25 l total volume, with an initial dye concen-tration of 500 mg l−1.

Fig. 9. Color removal profiles in the abiotic tests performedwith 50-ml samples taken from SBR 3 during period 6 (SRT20 d, aeration 10 h, VSS 2.0 g l−1). The samples consisted ofautoclaved mixed liquor (�), harvested cells resuspended infresh feed solution and autoclaved (�) and the correspondingcell-free supernatant (�).


Acknowledgements

This work was partially financed by the Eu-ropean Commission Contract ENV4-CT95-0064and FCT (Portugal, Contract PBIC/C/BIO/2027/95). The help of Dr Horacio Novais (OrganicChemistry Department, IST, Lisboa) with the or-ganic chemistry of the dyes is gratefully acknowl-edged. N.D. Lourenco acknowledges a Ph.D.fellowship from FCT (Portugal).

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