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Effect of Micropollutants (Organic Xenobiotics and Heavy Metals) on the Activated Sludge Process Davide Dionisi,*,† Caterina Levantesi,‡ Mauro Majone,† Lorena Bornoroni,† and Marco De Sanctis† Department of Chemistry, SapienzasUniVersity of Rome, P.le A. Moro 5, 00185 Rome, Italy, and Water Research Institute, National Research Council, Via Reno 1, 00198 Rome, Italy
Four experiments were performed in sequencing batch reactors, two by two in parallel, to study the effect of micropollutants (organic xenobiotics and heavy metals) on the performance of activated sludge processes. The reactors were operated for long times (at least 11 months each) and in a wide range of sludge ages (5-30 days). It was observed that production of biological solids, COD removal, and settling properties were not affected by the presence of micropollutants significantly. On the other hand, ammonia removal was much lower in the reactors fed with micropollutants (29-37% removal) than in the reactor without micropollutants (82% removal). Batch tests allowed the measurement of maximal activities of heterotrophic and autotrophic biomass. The activity of nitrifying microorganisms grown without micropollutants was greatly reduced (about 50%) by the addition of the micropollutants, but the residual micropollutants after treatment did not exert any inhibiting effect. Nitrate balances on the four runs confirmed that the fraction of nitrifying microorganisms was much higher in the reactor without micropollutants (4.5% of overall VSS) than in the reactors fed with micropollutants (maximum 0.5% of VSS). In spite of the fact that in the reactors fed with micropollutants the fraction of nitrifying microorganisms increased at increasing sludge age, a satisfactory nitrogen removal was not achieved in the whole tested range of experimental conditions, even after the 11-month acclimation. With regard to nitrogen removal in processes operated with micropollutants, the results obtained in this study seem to indicate the effectiveness both of processes with separate nitrification after a first treatment stage and of single-sludge processes carried out at very high sludge ages, as acheivable, e.g., by the use of membranes or of attached growth systems. 1. Introduction The presence in natural waters of micropollutants, both organic xenobiotics (polycyclic aromatic hydrocarbons, polychlorinated biphenyls, surfactants, pharmaceuticals, etc.) and heavy metals, is causing increasing concern (e.g., European Directive 2000/60/EC and the following decision 2455/2001/ EC). These substances may be potentially hazardous for humans and/or ecosystems due to their carcinogenic or endocrinedisrupting effect. Micropollutants are present in many industrial discharges but can also occur in municipal wastewaters, either in the case of mixed industrial-municipal sewer systems or due to their presence in household discharges (surfactants or pharmaceutical products). When these micropollutant-contaminated wastewaters reach biological treatment systems, they may affect the performance of the treatment plant negatively, due to the possible inhibition of biological activity. Although much research is being directed toward the study of the removal mechanisms of micropollutants,1-3 little attention is being given to the effect of micropollutants on the performance of the plants. Most of the studies on the effect of micropollutants on microorganism activity are batch tests measuring immediate effect on nitrification activity.4-8 However, the actual nitrogen removal in a continuous process fed with micropollutants cannot be directly predicted by short-term batch tests because of two main reasons. First, for a given extent of inhibition, the actual nitrogen removal in the continuous process depends on the steady-state amount of autotrophic microorganisms, which in turn depends on process parameters such as ammonia load and * To whom correspondence should be addressed. Tel.: +39 06 49693224. Fax: +39 06 490631. E-mail:
[email protected]. † SapienzasUniversity of Rome. ‡ Water Research Institute.
sludge age. Second, in a continuous-fed process several factors may modify the extent of inhibition measured by short-term batch tests: the continuous exposure to the micropollutants may increase the inhibiting effect or, on the contrary, acclimation and/or selection may reduce it. For the same reasons, shortterm perturbations with micropollutants of continuous activated sludge processes usually fed with noncontaminated wastewaters9 cannot be directly used to predict continuous process performance in the presence of micropollutants. With regard to the effect on activated sludge processes of continuous micropollutant feeding, heavy metals are the most studied substances: BOD removal was not affected by heavy metal concentrations up to 10 mg/L in the influent,10 whereas 1 mg/L copper, nickel, or zinc was enough to reduce nitrification.11 In another study12 copper concentrations up to 10 mg/L did not affect the extent of ammonia oxidized, whereas concentrations of 20 mg/L decreased the extent of nitrification. In a chemostat study,13 a decreased COD removal due to combined copper and zinc addition was observed at concentrations lower than 10 mg/L. This paper aims to study the effect of the continuous exposure to micropollutants on the performance of activated sludge processes experimentally. The micropollutants used in this study were a synthetic mixture of 10 organic xenobiotics and 2 heavy metals, chosen among the priority substances listed in Decision 2455/2001/EC (Annex X) with the addition of phenol (the simplest phenolic substance) and 4-dodecylbenzenesulfonic acid (representative of anionic surfactants). The considered micropollutants are often found in industrial or municipal wastewaters. In refinery wastewaters,14 polycyclic aromatic hydrocarbons, chlorinated phenols, and polychlorinated biphenyls can be present at concentrations up to dozens of mg/L; their actual concentration in the wastewater which reaches the treatment
10.1021/ie061688c CCC: $37.00 © 2007 American Chemical Society Published on Web 03/23/2007
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plant depends on their dilution with municipal wastewater. In municipal wastewaters anionic and nonionic surfactants can be present at mg/L levels.15 Four long-term experiments (at least 11 months) were performed in sequencing batch reactors (SBRs), two by two in parallel. The first two parallel reactors were run under the same operating conditions, the only difference being feed composition: The first one was fed only with readily biodegradable substrates and nitrogen sources; in the second one the micropollutant mixture was also added. In a comparison of the performance of the two reactors, the effect of micropollutants on the process was evaluated. Biomass production, sludge settling properties, and COD and nitrogen removal were compared. For each of the two biomasses, maximal rates of COD removal and of nitrification with and without micropollutants were also measured through batch tests. With the aim to evaluate the effect of sludge age on process performance in the presence of micropollutants, two other parallel SBRs were operated with the same feed, containing readily biodegradable substrates and micropollutants, but at very different sludge ages (approximately 5 vs 30 d). Process performance was measured by considering the same parameters of the first two reactors. 2. Materials and Methods 2.1. Sequencing Batch Reactors. Four reactors (SBR1, SBR2, SBR3, and SBR4) were operated, two by two in parallel. The first two parallel reactors (SBR1 and SBR2) were run under identical operating conditions, the only difference being feed composition: SBR1 was fed only with readily biodegradable substrates and nitrogen sources; SBR2 also contained a mixture of micropollutants. Operating parameters of SBR1 and SBR2 were the following: organic load rate (referred only to readily biodegradable substrates), (1 g of COD/L)/d; sludge age, 1012 d; length of the cycle, 6 h (unaerated feed, 1.5 min; unaerated phase, 57 min; aerated phase, 246 min; sludge withdrawal, 0.5 min; settling, 45 min; effluent withdrawal, 10 min). The volume of the completely filled reactors was 1.2 L and the daily influent flow rate was 2.4 L/d (0.6 L/cycle); thus, the volumetric exchange ratio (volume fed per cycle/maximum reactor volume) was 0.5. Temperature was set at 25 °C through a thermostatic bath, and pH was maintained in the range 7.4-7.6 through addition of acid or base solutions. The other two parallel reactors (SBR3 and SBR4) were run with the same feed as SBR2 (i.e., containing readily biodegradable substrates and micropollutants). All the other operating parameters were the same as for SBR1 and SBR2, with the exception of sludge age, which was 30.5 days for SBR3 and 4.9 days for SBR4. Sludge age was varied by adjusting the flow rate of the sludge-withdrawal pump. Table 1 reports feed composition of the four runs, and Table 2 summarizes the research scheme. Besides substances indicated in Table 1, mineral elements were also added to the reactors. During the runs the reactors were regularly sampled for determination of suspended solids, readily biodegradable substrates, sludge volume index (SVI), ammonia, nitrite, and nitrate. 2.2. Batch Tests. The maximal activity of the microorganisms in the four reactors was measured through batch tests. Aliquots of mixed liquor were withdrawn from each reactor at the end of the cycle, centrifuged, and washed twice with the mineralelement solution and then resuspended in a final volume of 50 mL. Then, 50 mL of the feed was added and the tests were started. The 50 mL feed was composed in the following way: (1) tests with SBR1 feed (performed with biomass from all the runs), 50 mL of SBR1 feed; (2) tests with SBR2 feed (with biomass from all the runs), 50 mL of SBR2 feed;
Table 1. Composition of the Feed of the Reactors all the reactors
SBR2, SBR3, and SBR4 only
readily biodegradable substrates
mg/L
micropollutants
acetate ethanol glucose glutamic acid tot. readily tot. nitrogen
215 43 84 92 500 51 (as N)
organic benzene 1,3,5-trichlorobenzene phenol pentachlorophenol naphthalene pyrene 4-nonylphenol 4-dodecylbenzenesulfonic acid 2,4-dichlorobiphenyl decachlorobiphenyl heavy metals CdCl2‚H2O PbCl2
mg/L 1.8 1 5 4 4.2 0.1 4 10 0.18 0.0002 10 (as Cd) 10 (as Pb)
Table 2. Research Schemea
a Runs highlighted with the same gray tone were operated in parallel. An asterisk indicates aerobic sludge age.
(3) tests with SBR1 feed and SBR2 effluent (only with biomass from SBR1 and SBR2), 25 mL of SBR1 feed + 25 mL of SBR2 effluent. The aliquots of mixed liquor withdrawn from each reactor were chosen to have in all the tests a similar biomass concentration (referred to the volume after the feed addition), approximately 1500 mg of VSS/L. The volume withdrawn in each test was based on VSS measurements in the days before the test (as orientative values, from SBR1, SBR2, and SBR4 withdrawn volumes were in the range 60-100 mL, whereas those for SBR3 were in the range 25-35 mL). The length of the tests was 4 h, and they were carried out at same temperature and pH of the SBRs. During the tests the mixed liquor was regularly sampled for analytical determination of the four substrates and of ammonia, nitrate, and nitrite. 2.3. Analytical Methods. Suspended solids were determined according to standard methods,16 and SVI was according to the procedure described by Wanner.17 Readily biodegradable substrates were analyzed as following: acetate and ethanol through GC analysis (PE 8410 with FID and nitrogen as carrier gas, stationary phase was 4% Carbowax 20M on 80/120 Carbopack B-DA, column temperature 175 °C for acetate and 110 °C for ethanol, injector and detector temperature 200 °C); glucose and glutamic acid through specific enzimatic kits (hexokinase for glucose, glutamate dehydrogenase for glutamic acid). Ammonia was measured through the Nessler method, and nitrite and nitrate were measured through ionic chromatography (Dionex, AG14 e AS14 columns, eluent 4.8 mM Na2CO3/0.6 mM NaHCO3, flow 0.8 mL/min, regenerant H2SO4 (50 mM), flow rate 1.2 mL/min). 2.4. Calculation of the Sludge Age. The sludge age was calculated according to the following relationship:
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ϑC(d) )
VmaxXend react
tcycle ×
[(QWtw + Vsampl)Xend react + QeffteffXeff] tcycle - tsett - teff (1) tcycle
(
)
This relationship takes into account the manually sampled volumes/cycle (Vsampl, L/cycle) and the losses of volatile suspended solids with the effluent (Xeff). In eq 1, “Xend react” indicates volatile suspended solids at the end of the reaction phase. The unactive phases of the cycle (settling and effluent withdrawal) were subtracted from the length of the cycle.18 Relationship (1) was calculated every day for the two reactors, by assuming, in the days where no sampling were made, that Xend react and Xeff values were average values of the previous and subsequent measured value. The sludge age calculated according to relationship (1) includes also the unaerated phases. Aerobic sludge age, referred only to aerated phases, was calculated according to
ϑC aer(d) )
VmaxXend react
× t [(QWtw + Vsampl)Xend react + QeffteffXeff] cycle tcycle - tfeed - tunaer - tsett - teff (2) tcycle
(
)
2.5. Calculation of the Concentration of Nitrifying Microorganisms in the Reactors. Steady-state data for nitrate concentration at the end of the cycle were used to determine the concentration of nitrifying (autotrophic) microorganisms in the reactors. Nitrate balance was made on the basis of daily average values of the influent and effluent flow rates. The balance of nitrate nitrogen can be written as follows:
Q[NO3]) rXAVmax‚5.92
58.5 246 - rdenVmax 360 360
Here [NO3] is the nitrate concentration in the effluent, the factor 5.92 (mg of N-NO3 produced/mg of autotrophic biomass produced) derives from the stoichiometry of autotrophic microorganisms growth, according to typical growth yields of nitrifying microorganisms,19 and rden ((mg of N/L)/d) represents the nitrate removal rate due to denitrification in the unaerated phase (the factors 246/360 and 58.5/360 take into account that nitrification and denitrification only occurred during a fraction of the cycle). rden can be calculated by considering that in both reactors nitrate concentration at the end of the unaerated phase was negligible (complete denitrification occurred), thus
58.5 ) rdenVmax 360
rXAVmax‚5.92
246 360
2
(factor 2 in the right-hand side of the equation takes into account the volumetric exchange ratio of the SBR, i.e., that only half of the nitrate produced during the aerated phase was actually removed during the unaerated phase). Thus, the nitrate balance becomes
Q[NO3] )
rXAVmax‚5.92 2
246 360
(3)
To calculate rXA, it can be observed that the amount of nitrifiers produced/day, should be, at steady state, equal to the amount of nitrifiers removed from the reactors; thus
rXA
(QWtw + Vsampl)XAend react + QeffteffXAeff 246 Vmax ) 360 tcycle
which, assuming the same settling properties for autotrophic and heterotrophic microorganisms, can be rewritten as
rXA
(
)
XAend react tcycle - tsett - teff 246 Vmax ) 360 ϑC tcycle
(4)
With insertion of relationship (4) into (3), it follows that
XAend react )
(
2Q[NO3] tcycle ϑC Vmax‚5.92 tcycle - tsett - teff
)
(5)
which allows the calculation of the amount of nitrifying microorganisms with average data of parameters measured during the runs of the reactors. In this calculation, denitrification in the aerated phase and in the settling phase was not considered. Indeed, this phenomenon was prevented by the high oxygen concentration in the aerated phase and by the lack of readily biodegradable substrates during the settling phase. 2.6. Morphological Observation and FISH Analysis. The biomass of the four reactors was monitored by microscopic phase contrast observation at 100 and 1000 magnifications. Size and shape of the aggregates produced, morphological composition of these biomasses, and presence of filamentous bacteria were recorded. Samples from the four reactors were collected at the end of the aerobic phase and fixed for later FISH analyses in both formaldehyde and ethanol (3% and 48% final concentration, respectively) or with ethanol only (50% final concentration) and stored at -20 °C. FISH was performed according to a protocol derived from Manz et al.20 and described in Levantesi et al.21 The probe NSO1225, specific for ammonia oxidizing betaproteobacteria,22 was used. The probes for Eubacteria EUB I 23 and EUB II and EUB III24 used in equimolar mix (EUB mix) were also applied. 3. Results and Discussion The effect of micropollutants on the activated sludge process was evaluated by comparing the performance of SBR1 and SBR2. Afterward, the effect of sludge age on process performance with micropollutants was evaluated by comparing SBR3 and SBR4. Most of the micropollutants were removed from the liquid phase (with removal percentages in the range 80-100%), with the exception of 4-dodecylbenzenesulfonic acid, whose removal was only 20%. The removal mechanisms of the micropollutants included, with different relevance for the different substances, adsorption on sludge flocs, biodegradation, volatilization, and stripping. A detailed quantification of these different removal mechanisms will be the subject of a separate paper (manuscript in preparation). 3.1. Effect of Micropollutants (Comparison of SBR1 and SBR2). 3.1.1. Overall Performance of the Two Reactors. Table 3 compares average values of the measured parameters during steady-state operation of the two reactors. No important difference was observed between the two reactors with regard to production of biological solids and settling properties. VSS were slightly higher in SBR1 than in SBR2 (2267 vs 1728 mg of VSS/L, respectively), but SBR2 was characterized by slightly higher solid losses in the effluent than SBR1 (58 mg of VSS/L for SBR2 and 40 mg of VSS/L for SBR1). As a result, the overall observed yield (0.16 for SBR1 and 0.18 for SBR2, COD/ COD) and the sludge age (12.0 vs 10.5 days) were very similar. Moreover, the presence of micropollutants had no important effect on the settling properties of the sludge, which were
Ind. Eng. Chem. Res., Vol. 46, No. 21, 2007 6765 Table 3. Comparison of the Performance of SBR1 and SBR2a
param VSS (mg/L) SVI (mL/g of VSS) VSSeff (mg/L) obsd yield (COD/COD) effluent readily biodegradable substrates (mg of COD/L) readily biodegradable substrates removal (%) effluent ammonia (mg of N/L) ammonia removal (%) effluent nitrate (mgN/L) overall nitrogen removal (%) a
SBR1 (without micropollutants)
SBR2 (with micropollutants)
2267 (63) 111 (8) 40 (4) 0.16 (0.02) 0 (0)
1728 (37) 142 (6) 58 (18) 0.18 (0.02) 0 (0)
100 (0)
100 (0)
9.3 (1.3) 82.5 (2.4) 11.0 (0.9) 65.4 (2.3)
32.2 (1.2) 37.3 (2.5) 0.6 (0.07) 36.3 (1.8)
Values in parentheses indicate standard deviation of the mean.
Figure 2. Batch tests with SBR1 and SBR2 biomass (tests with SBR1 feed): typical profile of readily biodegradable COD in a batch test (A); average rate values (B). Error bars indicate standard deviations of the mean of at least four replicates.
Figure 1. Comparison of ammonia (A) and nitrate (nitrite concentration negligible) (B) profiles in the two reactors. The arrows indicate addition of 5 mL of the well nitrifying inoculum sludge (3000 mg of VSS/L) in both reactors.
satisfactory in both reactors (average SVI was 111 mL/g of TSS in SBR1 and 142 mL/g of TSS in SBR2). The removal of readily biodegradable COD was virtually complete in both reactors. On the other hand, ammonia nitrogen in the effluent was 32 mg of N/L in SBR2 (37% ammonium removal) and 9 mg of N/l in SBR1 (82% ammonium removal). As a consequence, nitrate in the effluent (nitrite was usually negligible) was higher in SBR1 (11 mg of N/L) and very low for SBR2 (0.6 mg of N/L). Overall nitrogen removal (considering the sum of ammonia and nitrate nitrogen) was 65% in SBR1 and 36% in SBR2. By material balances and batch tests without biomass (data not shown), it was verified that the removal of ammonia nitrogen which was not due to nitrification was due to both heterotrophic growth and air stripping. To have a more detailed comparison of nitrogen removal in the two reactors, Figure 1 compares ammonia removal and nitrate nitrogen production during the reactors operation. SBR2 biomass maintained appreciable nitrifying activity only for the first 5 days after the start-up, and then nitrate levels dropped down at very low levels. No evidence of acclimation of nitrifying populations to micropollutants during SBR2 run (11 months) was obtained. The
arrows in Figure 1 indicate small additions of the inoculum sludge to both reactors (5 mL of a 3000 mg of VSS/L sludge). These additions were necessary to rapidly restore nitrification activity in SBR1, after it had decreased due to sludge withdrawals for batch tests, whereas in SBR1 nitrifying activity was rapidly restored; the inoculum addition in SBR2 had no detectable effect on nitrification. It is evident therefore that the presence of micropollutants in SBR2 feed did not allow the growth of the new nitrifying microorganisms which had been introduced again. 3.1.2. Batch Tests. The activities of heterotrophic and autotrophic populations in the two reactors were studied in greater detail through batch tests. The maximal activity of SBR1 and SBR2 heterotrophic microorganisms on readily biodegradable substrates without micropollutants is compared in Figure 2 (tests with SBR1 feed). By these tests with SBR1 feed, the activity of the two biomasses could be compared under the same experimental conditions, because residual micropollutants in the liquid phase in SBR2 were removed by biomass washing before the test. The only difference in experimental conditions could be due to the possible presence in SBR2 biomass of strongly sorbed micropollutants, which were not removed by washing. Heterotrophic microorganisms in SBR1 were characterized by higher substrate removal rates than those grown in SBR2. Average values of substrate removal rate were 448 mg of COD/g of COD/h for SBR1-grown biomass and 250 mg of COD/g of COD/h for SBR2-grown biomass. However, both rates were high enough to allow complete removal of readily biodegradable COD during the SBR cycles. The comparison of maximal COD removal rates by the two biomasses shows that in the presence of micropollutants microorganisms with lower maximal substrate uptake rates were selected. However, a possible residual inhibiting effect on SBR2 sludge due to the adsorbed micropollutants cannot be excluded. Figure 3 shows the effect of the addition of micropollutants on SBR1-grown heterotrophic microorganisms. The addition of micropollutants caused a low decrease (about 20%) of maximal activity of heterotrophic
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Figure 3. Substrate uptake rate for SBR1 biomass with and without micropollutants. Error bars indicate standard deviations of the mean of at least four replicates.
Figure 5. Batch tests with SBR1 biomass for measuring the nitrifying activity: typical profiles in batch tests (A); nitrate production rates (B). Error bars indicate standard deviations of the mean of at least four replicates. Table 4. Comparison of the Performance of SBR3 and SBR4a Figure 4. Substrate uptake rate for SBR2 biomass with and without micropollutants. Error bars indicate standard deviations of the mean of at least four replicates.
microorganisms. This decrease was observed both with the micropollutants in SBR2 feed and with those in SBR2 effluent. The same comparison for SBR2-grown microorganisms is reported in Figure 4. In this case no significant effect of micropollutants addition was observed (in the case of SBR2 microorganisms, it is more correct to say that no increase in activity was observed by removing the micropollutants). Thus, SBR1-grown microorganisms were characterized by higher substrate uptake rates but were more sensitive to micropollutants addition. SBR2-grown microorganisms, on the other hand, had lower substrate uptake rates but were completely acclimated to micropollutants. The same comparison of the effect of micropollutants was carried out for SBR1-grown nitrifying microorganisms, whereas a similar comparison was not possible for SBR2-grown nitrifying microorganisms because of their too low activity. Figure 5 compares nitrate production rates by SBR1-grown microorganisms under three conditions: without micropollutants; with the micropollutants in SBR2 feed; with those in SBR2 effluent. It is evident that a significant reduction of activity (approximately 50%) was observed with the SBR2 feed, whereas no inhibition was observed with SBR2 effluent. This evidence can have relevant effects in the design of activated sludge processes treating micropollutant-containing wastewaters, as discussed in section 3.5. 3.2. Effect of Sludge Age (Comparison of SBR3 and SBR4). As sludge age is the most important design parameter of activated sludge processes, its effect on process performance in the presence of micropollutants was investigated. Table 4 summarizes the performance of SBR3 and SBR4, whereas, as expected, a much higher concentration of biological solids and a lower observed yield was obtained for SBR3; the other process parameters were very similar. In particular, nitrogen removal was poor in both reactors, and only a low nitrification activity was detected. Thus, sludge ages up to 30 days (of which 25
param VSS (mg/L) SVI (mL/g of VSS) VSSeff (mg/L) obsd yield (COD/COD) effluent readily biodegradable substrates (mg of COD/L) readily biodegradable substrates removal (%) effluent ammonia (mg of N/L) ammonia removal (%) effluent nitrate (mg of N/L) overall nitrogen removal (%) a
SBR3 (high sludge age)
SBR4 (low sludge age)
4971 (162) 95 (15) 51 (7) 0.14 (0.02) 0 (0)
1536 (46) 140 (11) 50 (3) 0.27 (0.02) 0 (0)
100 (0)
100 (0)
36.2 (2.7) 29.6 (4.0) 1.0 (0.1) 27.7 (2.1)
36.0 (1.2) 30.0 (2.1) 0.2 (0.04) 29.6 (1.7)
Values in parentheses indicate standard deviation of the mean.
Figure 6. Comparison of substrate uptake rates without micropollutants (tests with SBR1 feed) for the four biomasses. Error bars indicate standard deviations of the mean of at least four replicates.
days were aerobic, Table 2) were not enough to significantly improve nitrogen removal. Batch tests with SBR1 feed and SBR2 feed were carried out with both SBR3 and SBR4 biomasses. Only activity of heterotrophic microorganisms could be detected through batch tests, because of the too low activity of nitrifying microorganisms. Regarding maximal COD removal rates in the absence of micropollutants (tests with SBR1 feed), Figure 6 summarizes the values of the four reactors. It is evident that maximal activities of SBR2, SBR3, and SBR4 heterotrophic microorgan-
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Figure 8. Microscopic images of the four biomasses (phase contrast).
Figure 7. Comparison of the nitrifying biomass in the four reactors as a function of aerobic sludge age: mg of VSS/L (A); % of total VSS (B).
isms were very similar, whereas the activity of SBR1 microorganisms was higher. This seems to indicate that continuous exposure to micropollutants prevents the presence of the microorganisms with the highest substrate uptake rates. Similarly to what was observed for SBR2 biomass, the activities of heterotrophic microorganisms of SBR3 and SBR4 were very similar in tests with SBR1 feed and in tests with SBR2 feed (data not shown), indicating complete adaptation to micropollutants. 3.3. Analysis of the Presence of Autotrophic Microorganisms in the Four Reactors. As nitrogen removal was the main parameter affected by the presence of micropollutants, the nitrate balance (eq 5 in Experimental Section) was used to calculate the average amount of autotrophic microorganisms which was present in the four runs (Figure 7). In the well nitrifying sludge (SBR1) the average amount of nitrifiers was 105 mg of VSS/ L, approximately 4.5% of overall biological solids, whereas in the reactors fed with micropollutants the amount of nitrifiers was much lower. However, the concentration of nitrifiers was different among SBR2, SBR3, and SBR4. The highest amount of nitrifying microorganisms, both as absolute number and as percent of the total VSS, was observed for SBR3, i.e., for the reactor operated at the highest sludge age. In this reactor the average concentration of nitrifying microorganisms was 24 mg of VSS/L, which corresponded to approximately 0.5% of the total VSS. In SBR2 and SBR4, on the other hand, nitrifying microorganisms were present at lower concentration and the lowest concentration corresponded to the lowest sludge age. Thus, it can be observed that, in the presence of micropollutants, both the concentration and the fraction in the sludge of nitrifying microorganisms increase at increasing sludge age. The possible effects of this evidence in the treatment of micropollutantcontaining wastewaters are discussed in section 3.5. 3.4. Microscopic Observation and FISH Analysis of the Selected Biomasses. The composition of the microbial biomasses selected in the four SBRs was monitored during reactors runs by phase contrast observation. The three reactors fed with micropollutants were characterized by the production of big compact flocs, while small and irregularly shaped flocs were
Figure 9. Probe NSO1225 positive cells in the biomass of SBR1 (without micropollutants) and SBR3 (with micropollutants, high sludge age): Micrographs show phase contrast (left-hand side) and epifluorescence (righthand side) microscopy images of the same microscopic field.
mostly observed in SBR1 (Figure 8). From the morphological point of view, the biomasses grown with micropollutants were quite homogeneous being mainly composed of Zooglea-like bacteria. A higher biodiversity characterized instead the microbial community of SBR1, comprising various floc-forming morphotypes (bacilli, cocci, and tetrad-forming bacteria) and, although rarely, filamentous bacteria. The presence of ammonia oxidizers belonging to the Betaproteobacteria (BetaAOB) was investigated in the four microbial communities by FISH analysis with the probe NSO1225. According to the previous results, BetaAOB were found abundant in the biomass of SBR1 while they were not detected at all (SBR4) or observed in only minor amount (SBR2 and SBR3) in the biomasses of the reactors fed with micropollutant (Figure 9). Thus, even though qualitatively, FISH analysis confirmed the data of Figure 7 on the relative abundance of nitrifying microorganisms in the four runs. 3.5. Discussion. The results obtained in this study show that when activated sludge processes are operated with continuous inputs of micropollutant mixtures, the most important effect is a reduction of nitrogen removal. This confirms earlier findings.11,12 Moreover, our results indicate that nitrifying microorganisms do not acclimate to micropollutants even after longterm exposure (up to 11 months). Thus, it seems that acclimation alone is not enough to improve nitrogen removal in activated sludge processes fed with micropollutants, at least in case of severe inhibition such as that caused by the substances and at the concentrations used in this study. On the other hand,
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acclimation of nitrifiers to inhibiting substances contained in industrial wastewaters was evident in many Swedish wastewater treatment plants;25 however, also in that case the observed acclimation decreased at increasing inhibition. With regard to heavy metals, acclimation of nitrifiers in soils to zinc and lead26 and to nickel27 was observed. The practical question now is how to efficiently remove nitrogen in systems fed with micropollutants. The results obtained in this study seem to indicate two possible strategies: First of all, we have observed that, with a well nitrifying sludge, micropollutants in the feed significantly inhibit nitrification but residual micropollutants after biological treatment do not exert any inhibiting effect. Thus, as a first strategy, a twostage treatment process could be effective. In the first stage, the activated sludge process fed with micropollutants is operated under a sequence of anoxic and aerobic conditions to remove nitrate, COD, and most of the micropollutants. After the secondary settling tank, the effluent of the first stage is sent to a second stage where nitrification is carried out. Nitrate produced in this stage is recycled to the first stage to perform denitrification. According to a similar approach, a two-stage treatment process, first aerobic carbon and phenol removal and then nitrification, separated by a settling tank, proved to be effective in ammonia removal with a coke wastewater.28 Second, we have shown that even though at sludge ages up to 30 d no relevant improvement of nitrification occurs, nevertheless the fraction of nitrifiers in the sludge increases at increasing sludge ages. When 4-chlorophenol was the only inhibiting substance, an improvement in nitrification was observed when sludge age increased from 5 to 25 days.29 Thus, in the presence of severe inhibition due to mixtures of micropollutants, it can be expected that activated sludge processes operated at very high sludge ages could obtain a significant improvement in nitrogen removal. Very high sludge age in biological wastewater treatment processes can be obtained either with membrane bioreactors or with attached growth systems. In several studies on membrane bioreactors with domestic wastewaters very high sludge ages have been reported: e.g., 50 days,30 more than 100 days,31 or almost infinite values32 always with a good nitrification performance. These processes could be, therefore, more effective than disperse growth systems with secondary settling tanks in nitrogen removal in the presence of micropollutants. Obviously, both strategies need to be verified with laboratoryscale studies. Another important aspect which is evident from our results is that not only nitrifying microorganisms but also heterotrophic microorganisms are inhibited by micropollutants. Indeed, COD removal rates by microorganisms grown without micropollutants are immeditely reduced by approximately 20% by the addition of micropollutants. Moreover, the continuous exposure of the process to micropollutants seems to select for microorganisms with maximal substrate uptake rates which are lower than those of microorganisms grown in the absence of micropollutants. Even though this reduced substrate uptake rate had not any practical effect on COD removal in our reactors, it may have an adverse effect on COD removal in systems operated at higher organic loads. The selective effect of the micropollutants on the selected biomasses was also suggested by microscopic observation that showed a reduction of morphological biodiversity in SBR2, SBR3, and SBR4 in comparison with SBR1. Copper and zinc concentrations lower than 10 mg/L caused a decrease in COD removal;13 however, the conditions used in that study, a chemostat operated at sludge age
