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Journal of Hazardous Materials 185 (2011) 15921595

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2-Chlorophenol consumption and its effect on the nitrifying sludge

Sergio Martnez-Hernndez, Anne-Claire Texier, Flor de Mara Cuervo-Lpez, Jorge Gmez
Departamento de Biotecnologa, Universidad Autnoma Metropolitana-Iztapalapa, Av. San Rafael Atlixco No. 186, Col. Vicentina, Del. Iztapalapa, Mxico D.F. C.P. 09340, Mexico

a r t i c l e

i n f o

a b s t r a c t
The kinetic behavior of a nitrifying sludge exposed to 2-chlorophenol (2-CP) was evaluated in batch culture. The assays were performed using a stabilized nitrifying sludge. In control assays with (mg L1 ): NH4 + -N (100) and NaHCO3 -C (250), the substrates were consumed in 8 h, the ammonium consumption efciency was 99% and the NO3 yield higher than 0.9. When 5 mg 2-CP-C L1 was added, it was transformed into an unidentied intermediate and the nitrifying efciency decreased to 10%. Ammonium specic consumption rate diminished 95%, but the NO3 yield remained higher than 0.9. The biomass previously exposed to 2-CP was newly suspended with NH4 + -N or NO2 -N in order to evaluate the ammonium and nitrite oxidizing processes. The consumption efciencies and NO3 yields were similar to those obtained in control assays. However, the total time required for ammonium and nitrite consumption increased to 120 and 42 h, respectively. Specic consumption rates for NH4 + -N and NO2 -N decreased by 95% and 83% respectively, compared to control assays. Thus, the previous contact to 2-CP had more inuence on ammonium oxidizing process than the nitrite oxidizing process. These are the rst evidences where a nitrifying sludge exposed to 2-CP are reported. 2010 Elsevier B.V. All rights reserved.

Article history: Received 12 May 2010 Received in revised form 11 September 2010 Accepted 28 September 2010 Available online 28 October 2010 Keywords: 2-Chlorophenol Inhibition Kinetics Nitrication

1. Introduction 2-CP is a halogenated phenolic compound widely used for the synthesis of many kinds of compounds such as pesticides, herbicides and dyes [1]. It is also used in the petrochemical, textile and paper industries [2]. This compound is considered as a serious pollutant because of its toxicity and signicant environmental impact on air, water, and soil [3]. Several biological processes have been tested in order to eliminate chlorophenols. Experiments under anaerobic conditions including methanogenic, sulfate, iron and denitrifying conditions have been reported [4,5]; as well as studies under aerobic conditions. Basu and Oleszkiewicz [6] observed that 30 mg L1 of 2-CP was eliminated within 8 h using a sequencing batch reactor with 2 g of biomass L1 . A similar concentration of 2-CP was evaluated using an aerobic rotating biological contact, but this compound was consumed after 215 days of acclimation [3]. In aerobic conditions, it has been observed that accumulation of 3-chlorocatecol, a toxic intermediate, might result as consequence of a meta-cleavage pathway. For a complete oxidation of chlorophenols, an ortho-pathway must be followed [7]. Studies on the effect of chlorophenols on nitrication are scarce in the literature. Nevalainen et al. [8] studied the elimination of 2,4,6-trichlorophenol (2,4,6-CP) using a nitrifying uidized-bed

reactor, reporting the removal of 2,4,6-CP, but neither the ammonia specic consumption rate nor nitrifying yield values were determined. Satoh et al. [9] evaluated the effect of 2-CP on a nitrifying biolm, observing a decrease in oxygen and ammonium consumption rate and no 2-CP consumption. The objective of this work was to determine the effects of 2-CP on the nitrifying sludge using response variables such as specic rates and yields as well as to obtain evidences of the level to which this compound acts.

2. Materials and methods 2.1. Inoculum source and culture medium The sludge used as inoculum was obtained from a 6 L continuous stirring nitrifying reactor under steady state nitrication. Under these conditions, ammonium consumption efciency and nitrate production yield were close to 100% and 1, respectively. The metabolic pattern and operating conditions have previously been reported [10]. The nitrifying inoculum was washed with a solution of NaCl (9 g L1 ) and centrifuged (4000 g, 10 min) before being used. The culture medium used for batch assays was as follows (g L1 ): (NH4 )2 SO4 (0.24), NH4 Cl (0.19), KH2 PO4 (0.28), MgSO4 (0.20), NaCl (0.20), NaHCO3 (1.75) and CaCl2 (0.01). Five millilitres of FeSO4 H2 O (5 g L1 ) were added per litre of medium. The initial concentrations of NH4 + and NaHCO3 were 100 10 mg N L1 and 250 10 mg C L1 , respectively.

Corresponding author. Tel.: +52 55 5804 4716; fax: +52 55 5804 6407. E-mail address: (J. Gmez). 0304-3894/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2010.09.100

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2.2. Culture conditions Batch assays were performed in 160 mL serologic bottles with 100 mL of work volume. Each bottle was an independent experimental unit and was discarded after sampling. The inoculum concentration was 200 20 mg protein L1 . Oxygen (99.6% of purity) was bubbled into the medium for 5 min. The bottles were sealed with rubber caps and aluminum rings. Oxygen was again bubbled for 5 min. 2-CP (5 mg C L1 ) was added after the bottles were sealed. The initial pH value was 7.7 0.1. The cultures were shaken at 200 rpm at 30 C. Four series of experiments were carried out: (1) Abiotic assays containing only mineral medium and 2-CP (5 mg C L1 ). (2) Control culture containing nitrifying sludge, NH4 + -N or NO2 -N (100 mg L1 ). (3) Assays with nitrifying sludge, NH4 + -N (100 mg L1 ) and 2-CP-C (5 mg L1 ). (4) Recovery assays with the nitrifying sludge previously exposed to 2-CP supplemented with NH4 + -N or NO2 -N (100 mg L1 ). All batch cultures were carried out by duplicate. T-test for two samples was made, where the Number Cruncher Statistical System package was used for calculation [11]. The response variables used for evaluating the behavior of the nitrifying sludge were consumption efciencies (ENH4 + or ENO , mg substrate consumed [mg substrate fed]1 100), nitrate production yield (YNO

120 100

Nitrogen (mg/l)

80 60 40 20 0

120 100

Nitrogen (mg/l)

80 60 40 20 0 0 1 2 3 4 5 6 7 8 9

N [mg substrate consumed]1 ), and specic consumption rates of NH4 + (qNH4 + ), NO2 consumption (qNO ) and NO3 production 2 (qNO ). The specic rates were calculated and expressed as mg N (mg microbial protein-d)1 .

, mg NO3

Time (h)
Fig. 1. The respiratory process of nitrifying sludge fed with (A) ammonia or (B) nitrite (control assays). NH4 + -N ( ), NO2 -N ( ) and NO3 -N ( ).

3. Results and discussion 3.1. Control assays The abiotic assays showed that after 30 days of experimentation 96% 1.2 of 2-CP remained in the culture. Thus, the loss of this chlorinated compound was negligible. Nitrifying control assays using ammonium or nitrite as nitrogen source are shown in Fig. 1. It can be seen that NH4 + -N was consumed in 6 h (Fig. 1A). A concomitant formation of NO3 -N with a transitory nitrite formation was detected. Fig. 1B shows that nitrite consumption was made within 7 h. After 8 h, for both control cultures, the substrate consumption efciency and yield were very high (Table 1). qNH4 + was 45% higher than qNO . The dissolved 2 oxygen concentration at the end of the batch assays was around 1 indicating that there was no O limitation. Nitrate 6.1 1.3 mg L 2 was the main product and the microbial growth was negligible. 3.2. Nitrifying sludge in presence of 2-CP The effect of 2-CP on nitrifying sludge is shown in Fig. 2. After 30 days, ENH4 + was of 10% while the qNH4 + decreased 95% in comparison with those calculated from ammonium control assays. Nonetheless, even when scarce ammonium consumption occurred,

2.3. Analytical methods 2-CP was determined by HPLC (PerkinElmer, USA) using a C18 reverse phase column (phenomenex, USA) and a UV detector at 254 nm. The mobile phase was acetonitrile/water (60/40, v/v) at a ow of 1.5 mL min1 . Nitrate and nitrite were analyzed by HPLC (PerkinElmer) using an anion-interchange column (Waters IC-pack anion HC, Japan) and a UV detector at 214 nm. The mobile phase was a mixture of 120 mL of acetonitrile (1 M), 20 mL of butanol (1 M) and 20 mL of gluconateborate solution (16 g C6 H11 NaO7 , 18 g H3 BO3 , 25 g Na2 B4 O7 10H2 O and 250 mL of glycerol solution 1 M, all dissolved in 1 L of deionized water), all three dissolutions were dissolved in deionized water in order to obtain 1 L of solution. The ow was 2 mL min1 . Ammonium was determined by a selective electrode (Phoenix Electrode Co., USA). Dissolved oxygen (Hanna, Rumania) and pH (Cole Palmer, USA) were measured by selective electrodes. Lowrys method was employed to measure microbial protein concentration [12]. The analytical methods had a variation coefcient lower than 9%.

Table 1 Consumption efciencies of nitrogen, yields of nitrate and specic (consumption or production) rates of a nitrifying consortium with or without previous exposition to 2-CP fed with NH4 + or NO2 . Assays Nitrogen consumption efciency (E)a 99 100 100 99 1 1 1 1 NO3 yield (YNO3 )b Specic consumption rate of nitrogen (qN )c 2.36 0.11 1.62 0.27 0.10 0.01 0.09 0.01 Specic production rate of nitrate (qNO3 )d 1.48 0.12 1.65 0.26 0.02 0.002 0.04 0.001

NH4 + (control) NH4 + (exposed 2-CP) NO2 (control) NO2 (exposed 2-CP)
a b c d

0.93 1.03 0.93 0.98

0.05 0.01 0.04 0.09

mg substrate consumed (mg substrate fed)1 100. mg NO3 -N (mg substrate-N consumed)1 . mg NH4 + -N or mg NO2 -N (mg protein-d)1 . mg NO3 -N (mg protein-d)1 .

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120 100


2-chlorophenol (mg C/l)

5 4 3 2 1 0 0 5 10 15 20 25 30 35


Nitrogen (mg/l)

80 60 40 20 0

Nitrogen (mg/l)

80 60 40 20 0
120 100

Time (d)
Fig. 2. The respiratory process of nitrifying sludge in presence of 2-CP. NH4 + -N ( ), NO3 -N ( ) and 2-CP-C ( ).









Nitrogen (mg/l)
80 60 40 20 0 0 10 20 30 40 50 60

the NH4 conversion to NO3 (YNO ) was similar to the con3 trol culture. Therefore, the ammonium consumption efciency and consumption rate were the response variables mainly affected. Several authors [13,10] have observed adverse effects of aromatic substances such as phenol and p-cresol on nitrication process. The effect of these substances might be at different levels such as enzymatic rate, cell transport or by toxicity, which mainly inuence the efciency and rate [14,15]. The nitrifying sludge required long time for 2-CP depletion (30 days). The 2-CP specic consumption rate was 0.001 0.0001 mg 2-CP-C (mg protein-d)1 . Satoh et al. [9] evaluated the effect of 2-CP (10 mg L1 ) on a nitrifying biolm, nding that both O2 and nitrifying consumption rates were inhibited and the 2-CP was not consumed by the biolm. These results are contrasting with our assays, as the nitrifying sludge was able to consume the 2CP. The aromatic compound was transformed into an unidentied intermediate detected by HPLC from the seventh day of experimentation. It has been proposed that incomplete oxidation of 2-CP is noticed when the aerobic oxidation is conducted by means of meta-cleavage pathway, as 3-chlorocatechol is formed [16]. This compound seems to be more toxic than 2-CP [16], as it has been observed that 3-chlorocatechol accumulation results in the inactivation of catechol 2,3-dioxygenase [17]. Thus, it is possible that the negative effect found in our results might be caused by the production of 3-chlorocatecol or a similar compound. 3.3. Recovery assays with nitrifying sludge previously exposed to 2-CP In order to investigate which was the action site of 2-CP, the sludge previously exposed to the chlorinated compound was washed and newly suspended in mineral medium supplemented with NH4 + or NO2 . The ammonium was consumed in 120 h, corresponding to 20 times more than the assays with sludge not exposed to 2-CP (Fig. 3A). Nevertheless, the YNO values remained close to 3 1 (Table 1), even though a transitory accumulation of nitrite was detected. The exposition to the chlorinated compound decreased the qNH4 + 21 times compared with control assays (Table 1). A similar behavior was observed on the qNO as the values decreased 12 3 times. Fig. 3B shows the behavior of the nitrifying sludge exposed to 2-CP and resuspended in NO2 . The time to completely consume the nitrogen substrate was longer than control assays (5 times). Likewise, the qNO decreased 6 times and the qNO was 2 3 also affected (Table 1). Nonetheless, the sludge exposed to 2-CP recovered its nitrifying activity as the efciencies and nitrate yields were high. These results show that 2-CP had rather inhibitory than toxic effects on the nitrifying sludge. Moreover, the ammonium oxidizing process was more affected to 2-CP exposition than the nitrite oxidizing one. Many works have indicated a negative effect of organic compounds on ammonia and nitrite oxidizing processes,

+ -N


Time (h)
Fig. 3. The respiratory process of the nitrifying sludge previously exposed to 2-CP and resuspended in (A) ammonia or (B) nitrite media. NH4 + -N ( ), NO2 -N ( ) and NO3 -N ( ).

being more affected the former process. Blum and Speece [18] suggested that the higher sensibility of Nitrosomonas species is due to the ammonia monooxygenase enzyme (AMO). McCarty [19] observed that diverse compounds might be partially oxidized by AMO to highly reactive products which covalently bind the enzyme causing an irreversible inhibition. According to our results, the 2-CP effect on the ammonia oxidation might also be at transport level. If the transport is affected, the substrate concentration inside cell will be low and therefore the metabolic rate will decrease. The nitrifying process is a rst order kinetic reaction (r = dS/dt = qs S, where S is the substrate concentration, and qs is the specic consumption rate), therefore, if S is low, the qs will also be low. Finally, the results indicated that both ammonia and nitrite specic consumption rates were affected by the presence of 2-CP, but the ammonia oxidation rate was more sensitive to the 2-CP. 4. Conclusions The nitrifying microbial consortium consumed the 2-CP after long time. The 2-CP consumption was related with a concomitant accumulation of a carbon intermediate. The presence of 2-CP decreased signicantly the ENH4 + and qNH4 + of the nitrifying sludge. Nonetheless, the low fraction of ammonia consumed was converted to nitrate as the yield for this was close to one. Studies using the nitrifying sludge previously exposed to 2-CP showed that the ammonia oxidizing process was more affected than the nitrite oxidizing process, as the qNH4 + decreased 21 times, while the nitrite specic consumption rate decreased 6 times. These results show that 2-CP had rather inhibitory than toxic effects on the nitrifying sludge. Acknowledgements This work was nancially supported by the Council of Science and Technology of Mexico (grant no. CONACYT-CB-2005C01-49748-Z). Participation of Sergio Martnez Hernndez as postdoctoral researcher was founded by CONACYT.

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