Cometabolic biodegradation of 4-chlorophenol by sequencing batch reactors at different temperatures

Bioresource Technology 100 (2009) 4572–4578

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Cometabolic biodegradation of 4-chlorophenol by sequencing batch reactors at different temperatures V.M. Monsalvo *, A.F. Mohedano, J.A. Casas, J.J. Rodríguez Sección de Ingeniería Química, Facultad de Ciencias, Universidad Autónoma de Madrid., Campus de Cantoblanco, Crta. Colmenar km 15, Madrid 28049, Spain

a r t i c l e

i n f o

Article history: Received 13 February 2009 Received in revised form 20 April 2009 Accepted 22 April 2009 Available online 17 May 2009 Keywords: 4-Chlorophenol Cometabolism Ecotoxicity Sequencing batch reactor Temperature

a b s t r a c t The simultaneous removal of 4-chlorophenol (4-CP) and phenol in lab-scale sequencing batch reactors at different temperatures has been studied. Phenol feed concentration was fixed at 525 mg/L and 4-CP concentration was increased from 105 to 2100 mg/L at a constant hydraulic residence time (HRT) of 10.5 d. Complete phenol and 4-CP biodegradation was achieved during the aerobic stage working with 4-CP concentrations up to 1470 mg/L in the feed. Both 4-CP and phenol specific initial removal rates were strongly affected by 4-CP feed concentration and temperature. Only at the highest temperature tested (35 °C) it was possible to increase the maximum assimilative 4-CP concentration by the biological sludge up to 2100 mg/L, and a significant reduction of the ecotoxicity of the effluents was observed. 4-chlorocatechol (4-CC) was identified as the major intermediate in the aerobic cometabolic 4-CP degradation, being the ecotoxicity of that species substantially lower than that of 4-CP. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Chlorophenols represent one of the most important group of xenobiotic chemicals that enter the environment. These include useful and economically important chemicals available to industry and agriculture, being extensively used as herbicides, insecticides, fungicides, wood preservatives, resins and lubricants. Wastewaters from these activities are characterized by a variable concentration of chlorophenols which have been reported to cause inhibition on microorganisms decreasing COD, nitrogen and phosphorous uptakes (Kargi et al., 2005). Due to their persistence, phenolic and chlorophenolic compounds are frequently found in both surface and ground water, soil and sediments. Use of physical and chemical methods including adsorption, air stripping, solvent extraction and chemical oxidation to treat wastewaters containing phenolic and chlorophenolic compounds is limited by the high investment and operation costs (MatatovMeytal and Sheintuch, 1998). Therefore, despite the recalcitrant nature of chlorophenols, some efforts have been addressed towards the use of biological processes, less expensive and giving rise to less by-products. Sequencing batch reactors (SBR) have been claimed as a potentially efficient solution to remove phenolic and chlorophenolic compounds from aqueous off-streams. Mixed bacterial cultures, once the specialized members of the consortium have been selected and enriched have the ability of using chlorophenols as sole * Corresponding author. Tel.: +34 91 4973525; fax: +34 91 4972981. E-mail address: [email protected] (V.M. Monsalvo). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.04.044

carbon and energy sources (Chiavola et al., 2003; Moreno and Buitrón, 2004; Buitron et al., 2005; Sahinkaya and Dilek, 2007). The use of cosubstrates has been established as a powerful method of enhancing chlorophenols uptake rates as well as allowing to treat higher concentrations of these compounds (Tarighian et al., 2003; Sahinkaya and Dilek, 2006a). Biodegradable organic compounds can be categorised into primary substrates (growth substrates) and cometabolised species (non-growth substrates) (Saez and Rittmann, 1991). Biotransformation of the latter is generally more complex because the non-growth substrates cannot support cell growth and can only be transformed in the presence of a primary substrate. The primary substrate not only serves to sustain biomass production but also acts as an electron donor for degradation of the non-growth substrate. However, non-growth substrates have been shown to inhibit the oxidation of the primary substrate (Saez and Rittmann, 1993; Hyman et al., 1995). Thus, the rate and the efficiency of cometabolism are always dependent on a complex interaction between the primary substrate and nongrowth substrate. Several works suggest that the addition of some primary carbon source may aid in lowering the toxicity and growth inhibition of xenobiotics on cells, thus providing reducing power for degradation of recalcitrant organic compounds, or acting as inducing agents for biodegradative enzymes, thereby increasing the transformation rate of xenobiotics (Reardon et al., 2002). Sugars (Basu and Oleszkiewicz, 1995; Wang and Loh, 1999; Tarighian et al., 2003) and peptone (Sahinkaya and Dilek, 2006a,b) are common carbon sources that have been widely used in biotransformation research. Nevertheless, phenol has been presented as a better growth cosubstrate than conventional biogenic

V.M. Monsalvo et al. / Bioresource Technology 100 (2009) 4572–4578

compounds by several authors because it favours chlorophenols removal due to its closer chemical nature (Basu and Oleszkiewicz, 1995; Chiavola et al., 2004). In the present work, 4-CP and phenol have been selected as a target pollutant and growth substrate, respectively. Phenol and 4-CP can be together in effluents from hydrodechlorination (Calvo et al., 2004) or from anaerobic biological treatment of chlorophenols-bearing wastewaters (Berestovskaya et al., 1995). There is a lack of information on the effect of temperature, the analysis of intermediates and the evaluation of the ecotoxicity on the biodegradation of chlorophenols in activated sludge bioreactors. Basu and Oleszkiewicz (1995) studied the 2-chlorophenol removal in the range from 10 to 24 °C, being the last one the optimal for the SBR performance. The aerobic 4-CP metabolic pathways have been described only for pure cultures (Farrel and Quilty, 1999). Some of the intermediates identified (5-chloro-2-hydroxymuconic semialdehyde and 5-chloro-2-hydroxymuconic acid, among others) have been widely reported as dead-end metabolites, whose ecotoxicity is even higher than that of 4-CP (Admassu and Korus, 1996). Due to this fact, ecotoxicity measurements are needed to learn on the performance of the biotreatment. However, intermediates have not been measured during the 4-CP biodegradation by means of mixed culture. Kargi and Eker (2006), Kargi and Konya (2006) and Kargi and Konya (2007) reported on the reduction of ecotoxicity of 4-CP bearing synthetic wastewaters in conventional activated sludge units. The ecotoxicity measurements were carried out by the resarzurine method and no reference to possible intermediates was made. The aim of this work is to study the application of SBR for 4-CP biodegradation using phenol as growth substrate, analyzing the effect of temperature on the performance of the system, the evolution of the intermediates generated and the ecotoxicity throughout the process.

2. Methods 2.1. Sequencing batch reactors Three sequencing batch reactors of 2.1 L with a thermostatic water jacket to control the operating temperature were used. They were equipped with dissolved oxygen and pH probes. Peristaltic pumps were used for feed and effluent discharge as well as for the addition of sodium hydroxide (NaOH) solution for pH control. Air was supplied by means of a flow compressor through a ceramic diffuser and mechanical stirring was also used. An air flow rate of 9 L/min was used which ensured a sufficient dissolved oxygen concentration. The experiments were conducted in a series of stages in sequences of 12 h as follows: anoxic filling (1 h), aerated reaction (9.5 h), settling (1 h) and draw (0.5 h). The experiments were carried out at different temperatures (20, 30 and 35 °C) and, a hydraulic residence time of 10.5 days and a sludge residence time of 30 d were used in all the runs. For measurements of oxygen uptake rate (OUR), the reactor aeration was stopped for a period of 1 min. During this time dissolved oxygen measurements were recorded at short intervals, usually 5 s or less for high OUR values. The slope of the cumulative oxygen uptake curve represents the OUR. 2.2. Inoculum source The inoculum used in the bioreactors was collected from an industrial activated sludge wastewater treatment plant. The biological sludge was acclimated at 105 mg/L of 4-CP during two months in a SBR at room temperature. During the acclimation, the length of each stage in the scheme of the cycle was fixed, independent of the

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degree of removal. Biomass concentration in the reactors was maintained between 2000 and 3000 mg VSS/L. 2.3. Wastewater composition Different compositions of the synthetic wastewaters were used maintaining always a phenol feed concentration of 525 mg/L and increasing the 4-CP concentration from 105 to 2100 mg/L according to the sequence of Fig. 1. The reactors were operated for at least 3 weeks at each combination of operating conditions investigated in order to ensure stable performance before the corresponding data collection. This strategy involving the addition of primary substrate and step increase in the influent the co-metabolite concentration has been successfully used by Wang et al. (2007). Ammonium sulphate and phosphoric acid were used as nitrogen and phosphorous sources, respectively. A COD:N:P ratio of 100:0.5:0.1 (w:w) was fixed and mineral salts were also added as micronutrients supply in a COD:micronutrients (Fe, Ca, K and Mg) ratio of 1:0.05 (w:w). The mineral solution consisted on FeCl3, CaCl2, KCl and MgSO4. 2.4. Analytical methods Different analyses were carried out to evaluate the degradation efficiency and to control the reaction system. Dissolved oxygen (DO) and pH measurements were carried out by means of selective electrodes. Analyses of total and volatile suspended solids (TSS and VSS), V30, and sludge volumetric index (SVI) were performed following the APHA standard methods (APHA-AWWA-WPCF, 1992). Aromatic compounds were analysed by HPLC/UV (Prostar, Varian) using a C18 column as stationary phase (Microsorb MW-100-5) and a mixture of acetonitrile and H2O (40:60, vol.) as mobile phase. The flow rate was maintained at 1 mL/min and a wavelength of 280 nm was used. Total organic carbon (TOC) was measured using an OI Analytic Model 1010 TOC apparatus. Ecotoxicity determinations were carried out by the Microtox Acute Toxicity Test (SCI 500 Analyzer) using a freeze-dried preparation of the marine bacterium Vibrio fischeri as described in ISO 11348-3 (1998). Contribution of abiotic processes such as volatilisation and adsorption on sludge flocs was evaluated. Adsorption of phenol and 4-CP was measured on samples after extraction with Soxhelt following the US-EPA 8041 method. Tests of volatilisation were performed under identical operating conditions to those employed in the biodegradation experiments but in absence of biomass. 2.5. Data analysis The results reported were the average values from duplicate runs. In all the cases, the standard errors were lower than 5%.

3. Results and discussion 3.1. Acclimation period The first stage of each experience conducted at a given temperature consists in a 60-days acclimation period (see Table 1) where the culture selection takes place. Buitron and co-workers (Buitron et al., 2001; Moreno and Buitrón, 2004; Moreno-Andrade and Buitron, 2004) have used the specific oxygen uptake rate (SOUR) to establish the length of each cycle which allows to reduce the acclimation period. In our case, a fixed time strategy has been used in all the experiments because one of the main objectives of our work was to analyse the effect of temperature thus maintaining constant the rest of the operating conditions. Fig. 1 shows the average phenol and 4-CP concentrations along a cycle at steady state after

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5 b)

a)

4 20 ºC 30 ºC 35 ºC abiotic test

10

4-CP (mg/L)

Phenol (mg/L)

26 25 24 15

20 ºC 30 ºC 35 ºC abiotic test

3 2

5

1

0

60

120

180

240

0

60

Time (min)

120

180

240

300

Time (min)

Fig. 1. Time-evolution of phenol (a) and 4-chlorophenol (b) concentrations at different temperatures after the acclimation period. (Feed concentration: [phenol] = 525 mg/L; [4-CP] = 105 mg/L).

acclimation. Sludge acclimation to phenol degradation took place faster than for 4-CP and phenol removal rate was higher than 4CP one. These facts can be related to the fairly different ecotoxicity of both compounds (EC50 = 18.2 and 1.9 mg/L, for phenol and 4-CP, respectively). The increase of temperature exhibits some beneficial effect on the rate of disappearance of phenol. The average specific phenol removal rates calculated from the phenol concentration profiles (Fig. 1a), were clearly higher at 35 °C than at 20 °C (4.28 and 1.18 mg phenol/g VSS h, respectively) and sludge acclimation to phenol degradation took place faster at the highest temperature. In the case of 4-CP degradation (Fig. 1b), the temperature does not show any significant effect on the length of the acclimation period to this toxic compound as well as on the average removal rate (0.58 mg 4-CP/g VSS h). It is known that increasing temperature induces an enhanced volatilization rate of monoaromatics (Farhadian et al., 2008). The results from the abiotic tests showed negligible effects of temperature on stripping and sludge adsorption, so any decrease of the target compounds can be attributed exclusively to biological degradation (Fig. 1). As a consequence of 4-CP degradation during the acclimation phase, a yellow colour developed in the reaction medium. At 35 °C, the yellow colour did not remain in the effluent for a long time, evolving towards a straw yellow colour and finally disappearing, which suggests further metabolization of the intermediate responsible of that yellow colour. The accumulated intermediate shows the characteristics of 5-chloro-2-hydroxymuconic semialdehyde, the meta-cleavage product of 4-chlorocatechol (4-CC), as

3.2. Kinetic analysis Fig. 2 depicts the initial specific removal rates of phenol (a) and 4CP (b) at three different temperatures (20, 30 and 35 °C) using feeding mixtures of 525 mg/L of phenol and 4-CP concentrations from 210 to 2100 mg/L. Initial specific removal rates for phenol were higher than those of 4-CP at all the concentrations and temperatures tested. Biodegradation rates were clearly affected by the 4-CP concentration treated and maximum removal rates were obtained at 420 mg/L of 4-CP at 20 and 30 °C. However, at 35 °C the 4-CP concentration was double than the above mentioned value (840 mg/ L). This behaviour is related to the high anoxic phenol removal efficiencies obtained during the feeding stage when treating lower 4-CP concentrations, which lead to a limiting rate of phenol consumption. Higher 4-CP concentrations lead to increasing toxicity levels, and consequently phenol and 4-CP removal rates decreased. The temperature has a significant influence on both phenol and 4-CP removal rates, reaching the highest values at 35 °C (Fig. 2). Lowering the temperature from 35 to 30 and then to 20 °C, the maximum initial specific phenol removal rates decreased from 10.53 to 8.78 and 7.21 mg phenol/g VSS h (Fig. 2a). In general, the rate of biological

2

0 500

40 0

1500 2000

35

Tem 30 25 pera ture (ºC)

g/ L)

m

20

CF

](

35

Tem 30 25 pera ture (ºC)

[4 -

40 0

0 500 1000 1500 2000

g/ L)

2

4

(m

4

6

F]

6

8

20

-C

8

10

[4

10

Initial specific rem

oval rate (mg/g·h)

b)

Initial specific rem

oval rate (mg/g·h)

a)

described by Wieser et al. (1994). However, at 20 °C a brown colour developed probably due to the formation of 4-CC, which polymerises through autooxidation. The 4-CC accumulation was detected during the degradation process, remaining in the effluents at high concentration during this acclimation period.

Fig. 2. Initial specific uptake rates of phenol (a) and 4-CP (b) for mixtures of phenol (525 mg/L) and 4-CP (105–2100 mg/L) by SBR at different temperatures.

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consuming non-growth-associated energy instead of inhibiting cellular activity and deteriorating the biological floc structure.

Table 1 Biomass stabilization procedure by increasing 4-CP feed concentration. [4-CP] (mg/L)

[Phenol] (mg/L)

105 420 840 1050 1470 2100

525

T (°C)

Time (days) 0–60 61–101 102–139 140–169 170–200 201–240

20 30 35

reactions in mesophilic conditions increases by a factor of 2 for each 10 °C rise in temperature (Farhadian et al., 2008). In contrast with the described for the acclimation period, the temperature affects significantly the kinetics of 4-CP degradation, reaching initial specific 4-CP removal rates of 9.90 to 5.96 and 3.13 mg 4-CP/g VSS h at 35, 30 and 20 °C, respectively (Fig. 2b). As can be observed in Fig. 3, during the biodegradation period, simultaneous degradation of both substrates occurred. Once phenol concentration is reduced to about 10 mg/L, the 4-CP removal rate slowed sharply, indicating that biodegradation of both phenol and 4-CP is closely related (Fig. 3b). The results obtained show also that even though 4-CP is triggered by the presence of phenol, it continues after phenol depletion, proving that the properly acclimated mixed culture is able to use 4-CP as sole carbon and energy source. A possible explanation is that phenol supplies the electrons required for the initial monooxygenase step of 4-CP removal (Chiavola et al., 2003). In Fig. 3a can be observed that the time required to achieve complete phenol removal depends on the 4-CP concentration. It is known that phenol uptake rate depends on the maximum concentration reached inside the reactor (Jiang et al., 2004). However, in this case, even reaching the same concentration (25 mg/L), the initial aerobic phenol uptake rate is reduced from 2.88 to 1.33 and then to 1.21 mg phenol/g VSS h when the feed 4-CP concentration is increased from 1050 to 1470 and then to 2100 mg/L. This fact is due to the cumulative inhibitory effect derived from the presence of both compounds, which resulted in the occurrence of inhibition profiles in the evolution of phenol concentration when treating 2100 mg/L of 4-CP. Only in the reactor operated at 35 °C was possible to increase the 4-CP feed concentration from 1470 to 2100 mg/L whereas at 20 and 30 °C accumulation of 4-CP was detected, reaching high concentrations which led to destabilization of the system. At 20 and 30 °C with 1470 mg/L of 4-CP the microbial community could not regulate its metabolic pathways to adapt to the environmental conditions by

The removal of both phenol and 4-CP is mainly accomplished during the aerobic stage. However, as can be observed in Fig. 3, a phenol concentration detected after the filling stage was lower than the expected (25 mg/L). This fact is related with the anoxic phenol removal during this stage. There are a number of references on phenol removal under denitrifying conditions where NO 3 is presented as the electron acceptor. However, the degradation of phenol by sulphate-reducing bacteria has been documented in the literature by the isolation of pure strains growing in media with phenol as sole carbon source (Bak and Widdel, 1986; Boopathy, 1995). In the present work the typical light brown colour of granules and the absence of H2S characteristic odour also suggest that sulphate reduction must be negligible. Previous works have concluded that although sulphate was available was not used as electron acceptor when treating phenol in presence of nitrates (Sarfaraz et al., 2004; Chakraborty and Veeramani, 2006). The phenol uptake efficiency is enhanced by increasing temperature for very low 4-CP feed concentrations (less than 420 mg/L). Moreover, due to the inhibition of the anoxic phenol degraders, anoxic biodegradation of phenol was not detected when 4-CP was fed at 1470 mg/L (Fig. 4). Taking into account these results, for low 4CP concentration the use of a extended anoxic filling phase is interesting not only to improve settleability and denitrification, but also to reduce the energetic cost due to the aeration of the system. Within the last years, several strains of denitrifying bacteria have been isolated capable of oxidize phenol in the absence of molecular oxygen with NO 3 as terminal electron acceptor. Denitrifying phenol-degrading bacteria that have been isolated so far are Thiobacillus sp., Thaurea sp., Dehalobacterium sp. (Beristain-Cardoso et al., 2009), Azoarcus sp. (Zhou et al., 1995; Van Schie and Young, 1998; Shinoda et al., 2000), Magnetospirillum sp. (Shinoda et al., 2000), Pseudomonas sp. (Tschech and Fuchs, 1987; Son et al., 1998; Tong et al., 1998; Khoury et al., 1992), Alcaligenes sp. (Son et al., 1998; Tong et al., 1998; Thomas et al., 2002), Acinetobacter sp. (Khoury et al., 1992) and Enterobacter sp. (Thomas et al., 2002). Biodegradation of phenolic compounds under anoxic conditions by SBR has been scarcely reported in the literature. Sarfaraz et al. (2004) reported high phenol removal efficiencies using granular denitrifying sludge. In the present work, 41% of phenol was removed when treating 525 mg/L of phenol in 1 h of anoxic filling phase at 35 °C, which in comparison with the 93% phenol removal

100

25

b)

a)

20

80

[4-CP] (mg/L) 210 420 840 1050 1470 2100

15 10

4-CP (mg/L)

Phenol (mg/L)

3.3. Anoxic phenol removal

[4-CP] (mg/L) 210 420 840 1050 1470 2100

60 40 20

5 0 0

60

120

180

240

300

Time (min)

360

420

480

540

0

60

120

180

240

300

360

420

480

540

Time (min)

Fig. 3. Time-evolution of phenol (a) and 4-CP concentration (b) along a complete cycle at different 4-CP feed concentrations. (Feed: [phenol] = 525 mg/L; T = 35 °C).

V.M. Monsalvo et al. / Bioresource Technology 100 (2009) 4572–4578

70

70

20 ºC 30 ºC 35 ºC

Phenol 4-CP 4-CC TOC measured TOC calculated

60

Concentration (mg/L)

Phenol removal efficiency (%)

40

30

20

10

50

60 50

40

40

30

30

20

20

10

10

TOC (mg/L)

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0 400

600

800

1000

1200

1400

1600

0

4-CP concentration treated (mg/L)

Some authors conclude that temperature affects to the sludge settleability (Lee et al., 1975; Del Pino and Zirk, 1982). However, these and other studies are controversial about the relation between both parameters. 4-CP has been previously presented as an undesirable agent in continuous activated sludge units because its presence impairs the sludge settleability (Kargi and Konya, 2006). The SVI values (mL/g) in our SBR experiments increased with the feed concentration of 4-CP from around 87 at 105 mg/L to about 277 at 1470 mg/L, respectively. Sedimentation was improved at low temperatures during the acclimation period treating 4-CP concentrations bellow 105 mg/L. However, higher temperatures allowed better SVI values at 4-CP concentrations higher than about 200 mg/L. This result is in agreement with the reported by Basu and Oleszkiewicz (1995). The effect of temperature on the sludge settleability is related with changes in extracellular polymers of bacteria, made of proteins, lipids and polysaccharides, which are susceptible to temperature variations (Gulerman and Dilek, 1990).

4 3 2 0.04

5 0.02

0.00 120 180 240 300 360 420 480 540 600

Concentration (mg/L)

a)

100

DO (mg/L) and OUR (mg DO/L·h)

5

15

Concentration (mg/L)

600

Fig. 6 shows the time-evolution of initial compounds and 4-CC as well as measured and calculated TOC. The calculated TOC represents the amount of carbon computed from the identified intermediate (4-CC) and the residual amounts of 4-CP and phenol. In all the experiments a fraction of TOC remains at the end of each cycle and because of that a residual TOC is detected at the beginning of the cycle. Discounting that fraction, the measured and calculated TOC are practically identical, except at the end of the cycle, where there may be some unidentified refractory species with certain toxicity. The ecotoxicity was also calculated from the values of the individual compounds (Table 2) and their existing concentra-

DO and the oxygen uptake were monitored throughout the cycles, and their relation with the concentrations of the fed com-

Time (min)

500

3.6. TOC and ecotoxicity evolution

3.5. Oxygen consumption and intermediates

60

400

pounds and the intermediates produced, was investigated. As can be seen in Fig. 5, at the beginning of the aerobic phase DO increases slightly until reaching saturation. The time required to reach that level depends on the organic loading rate applied. Cumulative oxygen uptake is strongly related with the initial 4-CP concentration, reaching the values of 134 and 886 mg/L, when 210 and 2100 mg/L of 4-CP were respectively treated at 35 °C (Fig. 5a and b). In the experiments at 35 °C (Fig. 5) three OUR peaks were obtained; two of them (centered at 217 and 312 min) are related to maximum phenol and 4-CP removal rates, respectively. 4-CC was found to be the major intermediate of aerobic 4-CP biodegradation by meta-cleavage. The OUR shows also a peak, centered at 420 min, corresponding to the degradation of that species.

3.4. Sludge settleability

0

300

Fig. 6. Time-evolution of 4-CP, phenol, 4-CC, measured and calculated TOC at 30 °C. (Feed: [phenol] = 525 mg/L; [4-CP] = 1050 mg/L).

after 8 h reported by Sarfaraz et al. (2004) for similar phenol concentration seems to be a satisfactorily good result.

10

200

Time (min)

Fig. 4. Anoxic phenol removal during the filling stage for different 4-CP inlet concentrations at different temperatures.

Phenol 4-CP OUR DO

100

5

80

b)

Phenol 4-CP 4-CC OUR DO

60

4 3

40

2

20

1

0 0

60

0 120 180 240 300 360 420 480 540 600

DO (mg/L) and OUR (mg DO/L·h)

200

Time (min)

Fig. 5. Time-evolution of 4-CP, phenol, 4-CC, DO and OUR at 35 °C. (Feed: [phenol] = 525 mg/L; [4-CP] = 210 mg/L (a) and 2100 mg/L (b)).

V.M. Monsalvo et al. / Bioresource Technology 100 (2009) 4572–4578 Table 2 Ecotoxicity values for phenol, 4-CP and 4-CC. Compound

EC50 (mg/L)

TU (1000 mg/L solution)

Phenol 4-CP 4-CC

18.2 1.9 15.3

55 526 65

tions, according to the following expression based on the concept of concentration addition on the toxicity units (Chen and Lu, 2002):

T:U: mixture ¼

X Ci EC50i i

The toxicity levels measured in the effluents depend on the working temperature. In spite of the high effectiveness (almost 100%) achieved in each cycle, a residual ecotoxicity of 3.5 T.U. at 35 °C and 8.7 T.U. at 20 °C was measured when treating phenol and 4-CP concentrations of 525 and 1470 mg/L, respectively. This result is consistent with the improvement of reactors performance observed when increasing the temperature. The calculated ecotoxicity values are significantly higher than the measured ones. This effect is not observed during the filling phase, suggesting the existence of antagonistic effects related with the presence of intermediate species generated during the aerobic phase. Hoffman et al. (2003) tested mixtures of 10 chemicals in water to investigate possible synergistic, additive or antagonistic toxicity effects. The results indicated that most of the mixtures showed lower values than the expected from the pure toxicants, concluding that synergistic effects are rather unusual in combinations of toxicants. 4. Conclusions The results of this study show that both 4-CP and phenol can be completely removed in SBR within a wide range of 4-CP influent concentrations (phenol and 4-CP feed concentrations: 525 and 105–1470 mg/L, respectively) at temperatures between 20 and 35 °C. At 35 °C a 4-CP feed concentration up to 2100 mg/L can be treated. A significant phenol removal was observed during the anoxic filling stage at low 4-CP concentrations. However, 4-CP removal was only possible during the aerobic reaction phase. Maximum initial specific rates were found treating 840 mg/L of 4-CP at 35 °C. Acknowledgements The authors greatly appreciate financial support from the Spanish MCI through the projects CTM2004–00337 and CTM2007– 60959. V. Monsalvo wishes to thank the MCI and the ESF for a research grant.

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