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Biological removal of p-cresol, phenol, p-hydroxybenzoate and ammonium using a nitrifying continuous-flow reactor
Bioresource Technology 120 (2012) 194–198
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Biological removal of p-cresol, phenol, p-hydroxybenzoate and ammonium using a nitrifying continuous-flow reactor D. Pérez-González, J. Gómez, R. Beristain-Cardoso ⇑ Universidad Autónoma Metropolitana-Iztapalapa, Department of Biotechnology, Av. San Rafael Atlixco 186, C.P. 09340, Mexico
h i g h l i g h t s " Simultaneous oxidation of phenolic compounds and ammonium. " Efficient removal in continuous-flow. " Phenolic compounds improve ammonium oxidizing activity.
a r t i c l e
i n f o
Article history: Received 26 April 2012 Received in revised form 14 June 2012 Accepted 17 June 2012 Available online 23 June 2012 Keywords: Nitrification Phenolic Removal
a b s t r a c t Phenolic compounds biodegradation and its effect on the nitrification process were studied. A continuous stirrer tank reactor was operated in four stages, and phenolic compounds were fed as sequential way. In the first stage, at loading rate of 220 mg NH4+–N/L d, the consumption efficiency was of 91%, being the product, nitrate. After that, p-cresol was fed at 53 mg C/L d, reaching removal efficiencies for both substrates higher than 90%. In the third stage, p-hydroxybenzoate was fed at 56 mg C/L d, and the removal efficiencies for all substrates remained high. In the last stage, the reactor was fed at 54 mg C/L d of phenol, and it caused a diminishing on the ammonium removal efficiency; however, all phenolic compounds were efficiently removed. Kinetic results showed that the presence of each phenolic compound improved the ammonium oxidizing activity, but the nitrite oxidizing activity was not affected. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Nowadays there is a growing interest to develop technologies for the removal of complex mixtures of organic and inorganic compounds from industrial wastewaters, such as the chemical, petrochemical, pulp and paper mill industry (Chang et al., 2003). For instance, the wastewater of petrochemical industry contains mixing of phenolic compounds (i.e. p-cresol, phenol, etc.) and ammonium (Olmos et al., 2004). The phenolic compounds may be toxic, carcinogen, mutagen and teratogen at high concentrations (Autenrieth et al., 1991). Ammonium nitrogen bioaccumulation contributes to eutrophication, and it becomes toxic for the fauna in the body waters (Veeresh et al., 2005). Nitrogen and phenolic compounds can be eliminated by physicochemical or biological methods. Biological removal of these compounds is more environmentally friendly than physico-chemical processes (Oldham and Rabinowitz, 2002).The nitrification process, besides being the pathway to oxidize ammonium, it has been ⇑ Corresponding author. Tel./fax: +52 55 58 04 4600. E-mail address: [email protected] (R. Beristain-Cardoso). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.06.052
considered as alternative to remove aromatic compounds. This biological process has been evaluated in presence of inhibitory compounds such as p-cresol (Texier and Gómez, 2007; Kim et al., 2008; Beristain-Cardoso et al., 2010) phenol (Yamaghisi et al., 2001; Amor et al., 2005; Kim et al., 2008), p-hydroxybenzaldehyde (Silva et al., 2009) and 2-chlorophenol (Martínez-Hernández et al., 2011) and peptone/glucose (Racz et al., 2010). These studies have been conducted either in batch cultures or continuous cultures using microbial nitrifying sludge. It is important to note that most of the studies that evaluated the effect of phenolic compounds on nitrification process, each were considered individually. Therefore, there are scarce studies about the performance of nitrification process in presence of mixtures of phenolic compounds in a continuous-flow reactor. It is possible that the metabolic behavior of nitrifying consortium is different when it is exposed only to one inhibitory compound than to several of them as mixture. The study of nitrifying process in presence of more than one inhibitory compound may be crucial because the industrial wastewater is highly heterogeneous and complex. The goal of this study was to evaluate the effect of a mixing of p-cresol, phenol and p-hydroxybenzoate on the behavior of a nitrifying sludge in a continuous-flow.
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2. Methods 2.1. Continuous stirrer tank reactor and culture medium composition The microbial consortium coming from laboratory-scale nitrifying activated sludge system was used for inoculating the continuous stirred tank reactor (CSTR) of 5 L (Fig. 1). The reactor was operated over a period of 6 months at a temperature of 25 ± 0.2 °C, with a hydraulic retention time of 1.8 d. The pH was automatically controlled (acid–base) at 7.0 ± 0.3.The dissolved oxygen concentration was kept inside the reactor at 4.0 ± 0.3 mg/ L. The bioreactor was fed in average at loading rates of 220 NH4+–N/L d and 55 mg C/L d of each phenolic compound. The CSTR was fed sequentially with ammonium (I stage (I-S)), ammonium and p-cresol (II-S), ammonium, p-cresol and p-hydroxybenzoate (III-S) and ammonium, p-cresol, p-hydroxybenzoate and phenol (IV-S). The bioreactor was evaluated once steady state was reached. The basal mineral medium was composed of (g/L): K2HPO4 (1.2), KH2PO4 (0.8), NaHCO3 (2.0), and trace elements solution supplied at 1 ml/L. The trace element solution contained (g/L): EDTA (0.05), CuSO45H2O (0.015), CaCl22H2O (0.07), MnCl2 (0.03), (NH4)6Mo7O244H2O (0.015), FeCl3 (0.015), MgCl2 (0.02). Sludge samples from the CSTR were washed with saline solution to remove fine particles before using as inoculum in the nitrifying batch bioassays.
2.2. Nitrifying activity The nitrifying activity of the microbial consortium was evaluated at each steady state, in order to observe whether the ammonium or nitrite oxidation was affected by nitrifying sludge exposed to phenolic compounds. Batch bioassays were conducted in 250 mL Erlenmeyer flasks containing 200 mL of mineral medium, with the same medium culture and environmental conditions above pointed out. The flask was spiked with 100 mg/L of NH4+– N. The batch assays were inoculated with 3.5 g VSS/L of nitrifying sludge. The liquid medium was continuously aerated, reaching oxygen dissolved concentration of 3.5 ± 0.3 mg/L. The initial and final pH for all batch assays was of 7.0 ± 0.5 and they were made by duplicate. To evaluate the possible effect of phenolic compounds on the nitrifying sludge, the respiratory process of nitrification was evaluated by means of consumption efficiencies, product yields and the specific rates were calculated from the kinetic data. Gompertz model was used in order to calculate the consumption and
production specific rates [(q = 0.368Ak/(g VSS/L)] (González-Blanco et al., 2012). The coefficient of determination was higher than 0.94 for all cases. 2.3. Analytical methods Nitrate and nitrite were determined by ion chromatography (Beckman Coulter, proteomeLab PA 800). The buffer was prepared with 5 mL of Na2SO4 (0.1 M), NaCl (10 mM) and a commercial solution CIA Pak OFM anion-BT (Waters) plus 35 mL of deionizer water. A microcapillar of melted silica (60 cm long and 75 mm internal diameter) was used. The absorbance was measured in the ultraviolet region using a mercury lamp at 254 nm and 25 °C. Ammonium was determined by selective ammonium electrode (pHoenix electrode Co. Mod. NH331501). The p-cresol, phenol, p-hydroxybenzoate and p-hydroxybenzaldehyde were analyzed by HPLC with a C18 reverse-phase column (Sigma–Aldrich, USA), column size 300 3.90 mm. A mixture of acetonitrile/deionized water (60:40, v/v) was used as the solvent and the flow rate was maintained at 1.5 ml/min (Perkin Elmer Series 200, USA). For soluble protein, the effluent sample was filtered through a 0.45 lm polyethersulfone membrane filter and the supernatant was determined by the Lowry Method (Lowry et al., 1951). The volatile suspended solids (VSS) were determined according to standard methods for the examination of water and wastewater (APHA/AWWA/WEF, 2005). Microbial activity was evaluated in terms of consumption efficiency (E, (g of N or C consumed/g of N or C fed) 100), production yield (Y, g of N produced/g of N consumed), a specific consumption rates (mg of N consumed/g VSS d) and specific production rates (mg of N produced/g VSS d). 3. Results and discussion 3.1. Effect of phenolic compounds on nitrifying process in continuousflow Initially, the CSTR was operated for 65 days with a feeding of 220 mg NH4+–N/L d (Fig. 2, I-S). Under the steady state (days 25–65), the ammonium consumption efficiency was of 91 ± 1.3%. Under this experimental period, ammonium conversion to nitrate was higher, with a nitrifying yield of 0.94 ± 0.04 g NO3 –N produced/g NH4+-N consumed (Table 1). Under these operational conditions the reactor was fed with p-cresol at 53.42 ± 4.63 mg C/L d (Fig. 3, II-S). The phenolic compound was consumed and the nitrifying steady state was reached after 10 d. Under the steady state
S-II
S-I
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50
0 0
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220
Time (h) Fig. 1. Diagram of the nitrifying reactor (1) aeration basin; (2) settler; (3) pH and temperature controller; (4) feed solution; (5) aeration line; (6) recycle line and (7) effluent.
Fig. 2. Evolution of nitrogen compounds in the nitrifying reactor: NH4+–N influent (j); NH4+–N effluent (h); NO3 –N effluent (s); NO2 –N effluent (N).
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Table 1 Nitrogen and phenolic compounds removal under nitrifying steady state in the continuous-flow reactor. Stage
E% NH4+
E% p-cresol
E% p-OH
E% phenol
Y N–NO3
I II III IV
91 ± 1.3 97 ± 4 98 ± 4 77 ± 3
– 99 ± 5 95 ± 4 99 ± 0.5
– – 97 ± 7 99 ± 0.5
– – – 95 ± 16
0.94 ± 0.04 0.97 ± 0.03 1.0 ± 0.08 0.97 ± 0.05
140
II-S
III-S
IV-S
120
mg C / L d
100 80 60 40 20 0 60
80
100
120
140
160
180
200
220
Time (h) Fig. 3. Evolution of carbon compounds in the nitrifying reactor: p-cresol-C influent (N); p-cresol-C effluent (D); p-OH-benzaldehyde effluent (e); p-OH-benzoate influent (h); p-OH-benzoate effluent (j); phenol-C influent (d); phenol-C effluent (s).
(days 75–95), ammonium consumption efficiency slightly increased to 97.16 ± 4.0%, with a yield of 0.97 ± 0.03. Thus, the nitrification was improved by the feeding with p-cresol. These experimental results suggested that p-cresol fed to the reactor did not affect the respiratory process of nitrification, since the ammonium consumption and nitrifying yield were high (Table 1). Silva et al. (2011) working with batch cultures also found that the presence of p-cresol improved the kinetics of nitrification. It is well known that in biological systems fed with ammonium and organic matter, heterotrophs and nitrifiers can compete for the same electron acceptor (oxygen), affecting in some cases the catabolism or anabolism of the nitrifying process (Hanaki et al., 1990). However, either heterotrophs or nitrifiers might be involved on organic matter oxidation. For instance, in nitrifiers the ammonium monooxygenase (AMO) enzyme has been reported to oxidize either organic compounds or ammonium (McCarty, 1999). On the other hand, Moir et al. (1996) reported that the heterotroph (i.e. Paraccocus) also contains the AMO enzyme, thus contributing to the organic matter and ammonium oxidation. This enzymatic information might partially explain why the ammonium consumption efficiency can improve. When 58.33 ± 7.20 mg C/L d of p-hydroxybenzoate was fed to the reactor (Fig. 3, III-S) the phenolic compound did not affect the nitrate production. Nonetheless, the microbial consortium took 10 days for totally removing the p-hydroxybenzoate. Throughout the experimental period under steady state (days 105–145), the consumption efficiencies of ammonium, p-cresol and p-hydroxybenzoate were higher than 94%. Ammonium was recovery mainly as nitrate, with a yield of 1.0 ± 0.08. Soluble organic carbon in the effluent was not detected, indicating that the phenolic compounds were mineralized. These experimental results indicated that p-hydroxybenzoate fed to the CSTR did not affect the respiratory process of nitrification in presence of p-cresol, since the ammonium consumption and nitrifying yield values did not significantly change.
In IV-S, 53 mg phenol-C/L d significantly disturbed the system taking 20 days to reach the nitrifying steady state (Fig. 3), after this time, phenol was consumed. This experimental remark suggested that the metabolic process affected at the beginning was the nitrification, but with the time course, the nitrifying activity improved, reaching an ammonium consumption efficiency of 77%, while the consumption efficiency for all phenolic compounds was higher than 95%. Similar behavior was observed by Amor et al. (2005), whom evaluated the phenol consumption in presence of ammonium, showing high consumption efficiencies for phenol and ammonium in continuous culture. Phenol is known to have a significant impact at the membrane level. However, there are reports of microorganisms that have developed mechanisms to consume high phenol concentrations. For instance, the chains of trans-fatty acids molecules can align closer together in a biological membrane than those in the cis-configuration and time after, a more rigid membrane is formed (van Schie and Young, 2000). When phenol was added, the nitrifying yield decreased to 0.83 ± 0.04 and nitrite intermediary was not detected, indicating that 17% of ammonium nitrogen consumed followed another biochemical pathway. In fact, soluble protein was detected in the output of the nitrifying reactor, indicating that part of nitrogen fed was used by biosynthetic pathways. Regarding this, Mortberg et al. (1988) suggested that the synthesis of proteins might be associated with the high affinity transport system for phenol. These authors argued that the highaffinity system seems to be induced by the presence of phenol and dependent on de novo protein synthesis. The experimental results showed that using nitrifying consortium metabolically stable was possible to mineralize concomitantly ammonium, p-cresol, p-hydroxybenzoate and phenol that are present in industrial wastewater. It must be remarked that in reactors with cell high density the hydraulic retention time might significantly be shortened. However, with the stirred continuous tank reactor was shown that the simultaneous ammonium and recalcitrant organic matter consumption was possible.
3.2. Nitrifying activities The kinetic behavior of nitrifying sludge produced in steady state taken of I, II, III and IV stages was kinetically evaluated by 10 h, in batch cultures. In the S-I, ammonium was oxidized up to nitrate, with consumption efficiency of 91% and nitrifying yield of 1.0 ± 0.01. Nitrite was transitory formed, but it was totally oxidized to nitrate at the end of batch cultures (Fig. 4, S-I). The ammonium consumption specific rate was of 115 mg NH4+–N/g VSS d, while the nitrate production specific rate was of 117 mg NO3 –N/ g VSS d (Fig. 5). In the S-II, the ammonium consumption efficiency and nitrifying yield did not significantly change with regards to the previous stage. Nonetheless, the nitrate production specific rate increased 8.5%, while the ammonium consumption specific rate was detained. In S-III, the ammonium consumption and nitrifying yield were high. Regarding to the kinetics of nitrification, nitrate production specific rate did not change. However, ammonium consumption specific rate of the nitrifying sludge increased around 16% regarding to the first stage. In last stage, the ammonium consumption efficiency and nitrate production were high. The specific nitrate production rate did not significantly change. However, the ammonium consumption specific rate was improved, increasing 4.8 times regarding to first stage. A possible explanation might be related with the AMO enzyme, perhaps under this culture conditions its activity improved. But, further studies are required in order to quantify and evaluate this enzymatic behavior. Nitrite accumulation was notable, recovering it at the end of batch cultures as nitrate. This metabolic behavior suggested that the step more affected was the nitrite oxidation. It is worthy to note that
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120
S-II
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100
mg N/ L
80
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0
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mg N / L
80
60
40
20
0 0
2
4
6
8
10
0
2
Time (h)
4
6
8
10
Time (h)
Fig. 4. Time course of nitrogen compounds in batch cultures: NH4+-N (j); NO3 -N (s); NO2 -N (N). The bar errors represent the standard deviation.
cantly improved when phenol was fed into the continuous reactor. These results give novel information in order to oxidize concomitantly ammonium, p-cresol, p-hydroxybenzoate and phenol using a steady state nitrifying bioreactor.
500
mg N / g VSS d
400
Acknowledgements
300
This work was financed by CONACyT-repatriation project I0007-2009-01. Pérez-González received fellowship from ICyT, D.F, México.
200
100
References 0 I
II
III
IV
Stages Fig. 5. Nitrifying activity of the consortium. Black bars represent specific nitrate production rate, and the white bars represent the specific ammonium oxidation rate.
when the reactor was fed with p-hydroxybenzoate and/or phenol, the ammonium oxidizing activity increased notably.
4. Conclusions The microbial nitrifying consortium showed the metabolic capability for oxidizing ammonium, p-cresol, p-hydroxybenzoate and phenol, in continuous-flow. The kinetic study showed that nitrite oxidation activity was not influenced by feeding of phenolic compounds to the nitrifying sludge. Nonetheless, in batch cultures was observed that the ammonium oxidation activity was signifi-
Amor, L., Eiroa, M., Kennes, C., Veiga, M.C., 2005. Phenol biodegradation and its effect on the nitrification process. Water Res. 39, 2915–2920. APHA/AWWA/WEF, 2005. Standard Methods for the Examination of Water and Wastewater, Washington DC, USA. Autenrieth, R.L., Bonner, J.S., Akgerman, A., Okaygum, M., McCreary, E.M., 1991. Biodegradation of phenolic wastes. J. Hazard Mater. 28, 29–53. Beristain-Cardoso, R., Gómez, J., Méndez-Pampín, Ramón, 2010. The behavior of nitrifying sludge in presence of sulfur compounds using a floating biofilm reactor. Bioresour. Technol. 101, 8593–8598. Chang, Chao.-Chien., Tseng, Szu.-Kung., Chang, Chih.-Cheng., Ho, Chun.-Ming., 2003. Reductive dechlorination of 2-chlorophenol in a hydrogenotrophic, gaspermeable, silicone membrane bioreactor. Bioresour. Technol. 90, 323–328. González-Blanco, G., Beristain-Cardoso, R., Cuervo-López, F., Cervantes, F.J., Gómez, J., 2012. Simultaneous oxidation of ammonium and p-cresol linked to nitrite reduction by denitrifying sludge. Bioresour. Technol. 103 (1), 48–55. Hanaki, K., Wantawin, C., Ohgaki, S., 1990. Effects of the activity of heterotrophs on nitrification in a suspended-growth reactor. Water Res. 24 (3), 289–296. Kim, Y.M., Park, D., Lee, D.S., Park, J.M., 2008. Inhibitory effects of toxic compounds on nitrification. J. Hazard. Mater. 152, 915–921. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275. Martínez-Hernández, S., Texier, A.-C., Cuervo-López, F.M., Gómez, J., 2011. 2Chlorophenol consumption and its effect on the nitrifying sludge. J. Hazard. Mater. 185, 1592–1595.
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Mccarty, G.W., 1999. Modes of action of nitrification inhibitors. Biol. Fertil Soils. 29, 1–9. Moir, J.W.B., Crossman, L.C., Spiro, S., Richardson, D.J., 1996. The purification of ammonia monooxygenase from Paracoccus denitrificans. FEBS Letters 387, 71– 74. Mortberg, M., Spanning, A., Neujahr, A.H., 1988. Induction of high-affinity phenol uptake in glycerol-grown Trichosporon cutaneum. J. Bacteriol. 170 (5), 2383– 2384. Oldham, W.K., Rabinowitz, B., 2002. Development of biological nutrient removal technology in western Canada. J. Environ. Eng. Sci. 1, 33–43. Olmos, A., Olguin, P., Fajardo, C., Razo-Flores, E., Monroy, O., 2004. Physicochemical characterization of spent caustic from the OXIMER process and source waters from Mexican oil refineries. Energy Fuels. 18, 302–304. Racz, L.A., Datta, T., Goel, R., 2010. Effect of organic carbon on ammonia oxidizing bacteria in a mixed culture. Bioresour. Technol. 101, 6454–6460.
Silva, C.D., Gómez, J., Houbron, E., Cuervo-López, F.M., Texier, A.-C., 2009. p-Cresol biotransformation by a nitrifying consortium. Chemosphere 75, 1387–1391. Silva, C.D., Gómez, J., Beristain-Cardoso, R., 2011. Simultaneous removal of 2chlorophenol, phenol, p-cresol and p-hydroxybenzaldehyde under nitrifying conditions: Kinetic study. Biores. Technol. 102, 6464–6468. Texier, A.-C., Gómez, J., 2007. Simultaneous nitrification and p-cresol oxidation in a nitrifying sequencing batch reactor. Water Res. 41, 315–322. van Schie, P.M., Young, L.Y., 2000. Biodegradation of phenol: mechanisms and applications. Bioremed. J. 4, 1–18. Veeresh, G.S., Kumar, P., Mehrotra, I., 2005. Treatment of phenol and cresol in upflow anaerobic sludge blanket (UASB) process: a review. Water Res. 39, 154– 170. Yamaghisi, T., Leite, J., Ueda, S., Yamaguchi, F., Suwa, Y., 2001. Simultaneous removal of phenol and ammonia by an activated sludge process with cross-flow filtration. Water Res. 35, 3089–3096.
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Biological removal of p-cresol, phenol, p-hydroxybenzoate and ammonium using a nitrifying continuous-flow reactor D. Pérez-González, J. Gómez, R. Beristain-Cardoso ⇑ Universidad Autónoma Metropolitana-Iztapalapa, Department of Biotechnology, Av. San Rafael Atlixco 186, C.P. 09340, Mexico
h i g h l i g h t s " Simultaneous oxidation of phenolic compounds and ammonium. " Efficient removal in continuous-flow. " Phenolic compounds improve ammonium oxidizing activity.
a r t i c l e
i n f o
Article history: Received 26 April 2012 Received in revised form 14 June 2012 Accepted 17 June 2012 Available online 23 June 2012 Keywords: Nitrification Phenolic Removal
a b s t r a c t Phenolic compounds biodegradation and its effect on the nitrification process were studied. A continuous stirrer tank reactor was operated in four stages, and phenolic compounds were fed as sequential way. In the first stage, at loading rate of 220 mg NH4+–N/L d, the consumption efficiency was of 91%, being the product, nitrate. After that, p-cresol was fed at 53 mg C/L d, reaching removal efficiencies for both substrates higher than 90%. In the third stage, p-hydroxybenzoate was fed at 56 mg C/L d, and the removal efficiencies for all substrates remained high. In the last stage, the reactor was fed at 54 mg C/L d of phenol, and it caused a diminishing on the ammonium removal efficiency; however, all phenolic compounds were efficiently removed. Kinetic results showed that the presence of each phenolic compound improved the ammonium oxidizing activity, but the nitrite oxidizing activity was not affected. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Nowadays there is a growing interest to develop technologies for the removal of complex mixtures of organic and inorganic compounds from industrial wastewaters, such as the chemical, petrochemical, pulp and paper mill industry (Chang et al., 2003). For instance, the wastewater of petrochemical industry contains mixing of phenolic compounds (i.e. p-cresol, phenol, etc.) and ammonium (Olmos et al., 2004). The phenolic compounds may be toxic, carcinogen, mutagen and teratogen at high concentrations (Autenrieth et al., 1991). Ammonium nitrogen bioaccumulation contributes to eutrophication, and it becomes toxic for the fauna in the body waters (Veeresh et al., 2005). Nitrogen and phenolic compounds can be eliminated by physicochemical or biological methods. Biological removal of these compounds is more environmentally friendly than physico-chemical processes (Oldham and Rabinowitz, 2002).The nitrification process, besides being the pathway to oxidize ammonium, it has been ⇑ Corresponding author. Tel./fax: +52 55 58 04 4600. E-mail address: [email protected] (R. Beristain-Cardoso). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.06.052
considered as alternative to remove aromatic compounds. This biological process has been evaluated in presence of inhibitory compounds such as p-cresol (Texier and Gómez, 2007; Kim et al., 2008; Beristain-Cardoso et al., 2010) phenol (Yamaghisi et al., 2001; Amor et al., 2005; Kim et al., 2008), p-hydroxybenzaldehyde (Silva et al., 2009) and 2-chlorophenol (Martínez-Hernández et al., 2011) and peptone/glucose (Racz et al., 2010). These studies have been conducted either in batch cultures or continuous cultures using microbial nitrifying sludge. It is important to note that most of the studies that evaluated the effect of phenolic compounds on nitrification process, each were considered individually. Therefore, there are scarce studies about the performance of nitrification process in presence of mixtures of phenolic compounds in a continuous-flow reactor. It is possible that the metabolic behavior of nitrifying consortium is different when it is exposed only to one inhibitory compound than to several of them as mixture. The study of nitrifying process in presence of more than one inhibitory compound may be crucial because the industrial wastewater is highly heterogeneous and complex. The goal of this study was to evaluate the effect of a mixing of p-cresol, phenol and p-hydroxybenzoate on the behavior of a nitrifying sludge in a continuous-flow.
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2. Methods 2.1. Continuous stirrer tank reactor and culture medium composition The microbial consortium coming from laboratory-scale nitrifying activated sludge system was used for inoculating the continuous stirred tank reactor (CSTR) of 5 L (Fig. 1). The reactor was operated over a period of 6 months at a temperature of 25 ± 0.2 °C, with a hydraulic retention time of 1.8 d. The pH was automatically controlled (acid–base) at 7.0 ± 0.3.The dissolved oxygen concentration was kept inside the reactor at 4.0 ± 0.3 mg/ L. The bioreactor was fed in average at loading rates of 220 NH4+–N/L d and 55 mg C/L d of each phenolic compound. The CSTR was fed sequentially with ammonium (I stage (I-S)), ammonium and p-cresol (II-S), ammonium, p-cresol and p-hydroxybenzoate (III-S) and ammonium, p-cresol, p-hydroxybenzoate and phenol (IV-S). The bioreactor was evaluated once steady state was reached. The basal mineral medium was composed of (g/L): K2HPO4 (1.2), KH2PO4 (0.8), NaHCO3 (2.0), and trace elements solution supplied at 1 ml/L. The trace element solution contained (g/L): EDTA (0.05), CuSO45H2O (0.015), CaCl22H2O (0.07), MnCl2 (0.03), (NH4)6Mo7O244H2O (0.015), FeCl3 (0.015), MgCl2 (0.02). Sludge samples from the CSTR were washed with saline solution to remove fine particles before using as inoculum in the nitrifying batch bioassays.
2.2. Nitrifying activity The nitrifying activity of the microbial consortium was evaluated at each steady state, in order to observe whether the ammonium or nitrite oxidation was affected by nitrifying sludge exposed to phenolic compounds. Batch bioassays were conducted in 250 mL Erlenmeyer flasks containing 200 mL of mineral medium, with the same medium culture and environmental conditions above pointed out. The flask was spiked with 100 mg/L of NH4+– N. The batch assays were inoculated with 3.5 g VSS/L of nitrifying sludge. The liquid medium was continuously aerated, reaching oxygen dissolved concentration of 3.5 ± 0.3 mg/L. The initial and final pH for all batch assays was of 7.0 ± 0.5 and they were made by duplicate. To evaluate the possible effect of phenolic compounds on the nitrifying sludge, the respiratory process of nitrification was evaluated by means of consumption efficiencies, product yields and the specific rates were calculated from the kinetic data. Gompertz model was used in order to calculate the consumption and
production specific rates [(q = 0.368Ak/(g VSS/L)] (González-Blanco et al., 2012). The coefficient of determination was higher than 0.94 for all cases. 2.3. Analytical methods Nitrate and nitrite were determined by ion chromatography (Beckman Coulter, proteomeLab PA 800). The buffer was prepared with 5 mL of Na2SO4 (0.1 M), NaCl (10 mM) and a commercial solution CIA Pak OFM anion-BT (Waters) plus 35 mL of deionizer water. A microcapillar of melted silica (60 cm long and 75 mm internal diameter) was used. The absorbance was measured in the ultraviolet region using a mercury lamp at 254 nm and 25 °C. Ammonium was determined by selective ammonium electrode (pHoenix electrode Co. Mod. NH331501). The p-cresol, phenol, p-hydroxybenzoate and p-hydroxybenzaldehyde were analyzed by HPLC with a C18 reverse-phase column (Sigma–Aldrich, USA), column size 300 3.90 mm. A mixture of acetonitrile/deionized water (60:40, v/v) was used as the solvent and the flow rate was maintained at 1.5 ml/min (Perkin Elmer Series 200, USA). For soluble protein, the effluent sample was filtered through a 0.45 lm polyethersulfone membrane filter and the supernatant was determined by the Lowry Method (Lowry et al., 1951). The volatile suspended solids (VSS) were determined according to standard methods for the examination of water and wastewater (APHA/AWWA/WEF, 2005). Microbial activity was evaluated in terms of consumption efficiency (E, (g of N or C consumed/g of N or C fed) 100), production yield (Y, g of N produced/g of N consumed), a specific consumption rates (mg of N consumed/g VSS d) and specific production rates (mg of N produced/g VSS d). 3. Results and discussion 3.1. Effect of phenolic compounds on nitrifying process in continuousflow Initially, the CSTR was operated for 65 days with a feeding of 220 mg NH4+–N/L d (Fig. 2, I-S). Under the steady state (days 25–65), the ammonium consumption efficiency was of 91 ± 1.3%. Under this experimental period, ammonium conversion to nitrate was higher, with a nitrifying yield of 0.94 ± 0.04 g NO3 –N produced/g NH4+-N consumed (Table 1). Under these operational conditions the reactor was fed with p-cresol at 53.42 ± 4.63 mg C/L d (Fig. 3, II-S). The phenolic compound was consumed and the nitrifying steady state was reached after 10 d. Under the steady state
S-II
S-I
250
S-III
S-IV
mg N / L d
200
150
100
50
0 0
20
40
60
80
100
120
140
160
180
200
220
Time (h) Fig. 1. Diagram of the nitrifying reactor (1) aeration basin; (2) settler; (3) pH and temperature controller; (4) feed solution; (5) aeration line; (6) recycle line and (7) effluent.
Fig. 2. Evolution of nitrogen compounds in the nitrifying reactor: NH4+–N influent (j); NH4+–N effluent (h); NO3 –N effluent (s); NO2 –N effluent (N).
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Table 1 Nitrogen and phenolic compounds removal under nitrifying steady state in the continuous-flow reactor. Stage
E% NH4+
E% p-cresol
E% p-OH
E% phenol
Y N–NO3
I II III IV
91 ± 1.3 97 ± 4 98 ± 4 77 ± 3
– 99 ± 5 95 ± 4 99 ± 0.5
– – 97 ± 7 99 ± 0.5
– – – 95 ± 16
0.94 ± 0.04 0.97 ± 0.03 1.0 ± 0.08 0.97 ± 0.05
140
II-S
III-S
IV-S
120
mg C / L d
100 80 60 40 20 0 60
80
100
120
140
160
180
200
220
Time (h) Fig. 3. Evolution of carbon compounds in the nitrifying reactor: p-cresol-C influent (N); p-cresol-C effluent (D); p-OH-benzaldehyde effluent (e); p-OH-benzoate influent (h); p-OH-benzoate effluent (j); phenol-C influent (d); phenol-C effluent (s).
(days 75–95), ammonium consumption efficiency slightly increased to 97.16 ± 4.0%, with a yield of 0.97 ± 0.03. Thus, the nitrification was improved by the feeding with p-cresol. These experimental results suggested that p-cresol fed to the reactor did not affect the respiratory process of nitrification, since the ammonium consumption and nitrifying yield were high (Table 1). Silva et al. (2011) working with batch cultures also found that the presence of p-cresol improved the kinetics of nitrification. It is well known that in biological systems fed with ammonium and organic matter, heterotrophs and nitrifiers can compete for the same electron acceptor (oxygen), affecting in some cases the catabolism or anabolism of the nitrifying process (Hanaki et al., 1990). However, either heterotrophs or nitrifiers might be involved on organic matter oxidation. For instance, in nitrifiers the ammonium monooxygenase (AMO) enzyme has been reported to oxidize either organic compounds or ammonium (McCarty, 1999). On the other hand, Moir et al. (1996) reported that the heterotroph (i.e. Paraccocus) also contains the AMO enzyme, thus contributing to the organic matter and ammonium oxidation. This enzymatic information might partially explain why the ammonium consumption efficiency can improve. When 58.33 ± 7.20 mg C/L d of p-hydroxybenzoate was fed to the reactor (Fig. 3, III-S) the phenolic compound did not affect the nitrate production. Nonetheless, the microbial consortium took 10 days for totally removing the p-hydroxybenzoate. Throughout the experimental period under steady state (days 105–145), the consumption efficiencies of ammonium, p-cresol and p-hydroxybenzoate were higher than 94%. Ammonium was recovery mainly as nitrate, with a yield of 1.0 ± 0.08. Soluble organic carbon in the effluent was not detected, indicating that the phenolic compounds were mineralized. These experimental results indicated that p-hydroxybenzoate fed to the CSTR did not affect the respiratory process of nitrification in presence of p-cresol, since the ammonium consumption and nitrifying yield values did not significantly change.
In IV-S, 53 mg phenol-C/L d significantly disturbed the system taking 20 days to reach the nitrifying steady state (Fig. 3), after this time, phenol was consumed. This experimental remark suggested that the metabolic process affected at the beginning was the nitrification, but with the time course, the nitrifying activity improved, reaching an ammonium consumption efficiency of 77%, while the consumption efficiency for all phenolic compounds was higher than 95%. Similar behavior was observed by Amor et al. (2005), whom evaluated the phenol consumption in presence of ammonium, showing high consumption efficiencies for phenol and ammonium in continuous culture. Phenol is known to have a significant impact at the membrane level. However, there are reports of microorganisms that have developed mechanisms to consume high phenol concentrations. For instance, the chains of trans-fatty acids molecules can align closer together in a biological membrane than those in the cis-configuration and time after, a more rigid membrane is formed (van Schie and Young, 2000). When phenol was added, the nitrifying yield decreased to 0.83 ± 0.04 and nitrite intermediary was not detected, indicating that 17% of ammonium nitrogen consumed followed another biochemical pathway. In fact, soluble protein was detected in the output of the nitrifying reactor, indicating that part of nitrogen fed was used by biosynthetic pathways. Regarding this, Mortberg et al. (1988) suggested that the synthesis of proteins might be associated with the high affinity transport system for phenol. These authors argued that the highaffinity system seems to be induced by the presence of phenol and dependent on de novo protein synthesis. The experimental results showed that using nitrifying consortium metabolically stable was possible to mineralize concomitantly ammonium, p-cresol, p-hydroxybenzoate and phenol that are present in industrial wastewater. It must be remarked that in reactors with cell high density the hydraulic retention time might significantly be shortened. However, with the stirred continuous tank reactor was shown that the simultaneous ammonium and recalcitrant organic matter consumption was possible.
3.2. Nitrifying activities The kinetic behavior of nitrifying sludge produced in steady state taken of I, II, III and IV stages was kinetically evaluated by 10 h, in batch cultures. In the S-I, ammonium was oxidized up to nitrate, with consumption efficiency of 91% and nitrifying yield of 1.0 ± 0.01. Nitrite was transitory formed, but it was totally oxidized to nitrate at the end of batch cultures (Fig. 4, S-I). The ammonium consumption specific rate was of 115 mg NH4+–N/g VSS d, while the nitrate production specific rate was of 117 mg NO3 –N/ g VSS d (Fig. 5). In the S-II, the ammonium consumption efficiency and nitrifying yield did not significantly change with regards to the previous stage. Nonetheless, the nitrate production specific rate increased 8.5%, while the ammonium consumption specific rate was detained. In S-III, the ammonium consumption and nitrifying yield were high. Regarding to the kinetics of nitrification, nitrate production specific rate did not change. However, ammonium consumption specific rate of the nitrifying sludge increased around 16% regarding to the first stage. In last stage, the ammonium consumption efficiency and nitrate production were high. The specific nitrate production rate did not significantly change. However, the ammonium consumption specific rate was improved, increasing 4.8 times regarding to first stage. A possible explanation might be related with the AMO enzyme, perhaps under this culture conditions its activity improved. But, further studies are required in order to quantify and evaluate this enzymatic behavior. Nitrite accumulation was notable, recovering it at the end of batch cultures as nitrate. This metabolic behavior suggested that the step more affected was the nitrite oxidation. It is worthy to note that
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120
S-II
S-I
100
mg N/ L
80
60
40
20
0
100
S-IV
S-III
mg N / L
80
60
40
20
0 0
2
4
6
8
10
0
2
Time (h)
4
6
8
10
Time (h)
Fig. 4. Time course of nitrogen compounds in batch cultures: NH4+-N (j); NO3 -N (s); NO2 -N (N). The bar errors represent the standard deviation.
cantly improved when phenol was fed into the continuous reactor. These results give novel information in order to oxidize concomitantly ammonium, p-cresol, p-hydroxybenzoate and phenol using a steady state nitrifying bioreactor.
500
mg N / g VSS d
400
Acknowledgements
300
This work was financed by CONACyT-repatriation project I0007-2009-01. Pérez-González received fellowship from ICyT, D.F, México.
200
100
References 0 I
II
III
IV
Stages Fig. 5. Nitrifying activity of the consortium. Black bars represent specific nitrate production rate, and the white bars represent the specific ammonium oxidation rate.
when the reactor was fed with p-hydroxybenzoate and/or phenol, the ammonium oxidizing activity increased notably.
4. Conclusions The microbial nitrifying consortium showed the metabolic capability for oxidizing ammonium, p-cresol, p-hydroxybenzoate and phenol, in continuous-flow. The kinetic study showed that nitrite oxidation activity was not influenced by feeding of phenolic compounds to the nitrifying sludge. Nonetheless, in batch cultures was observed that the ammonium oxidation activity was signifi-
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