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p-Cresol biotransformation by a nitrifying consortium
Chemosphere 75 (2009) 1387–1391
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Technical Note
p-Cresol biotransformation by a nitrifying consortium C.D. Silva a, J. Gómez a, E. Houbron b, F.M. Cuervo-López a, A.-C. Texier a,* a b
Department of Biotechnology-CBS, Universidad Autónoma Metropolitana-Iztapalapa, Av. San Rafael Atlixco, No. 186-Col, Vicentina D.F., Mexico Facultad de Ciencias Químicas, Universidad Veracruzana, Prol. OTE 6, No. 1009 Orizaba, Veracruz, Mexico
a r t i c l e
i n f o
Article history: Received 6 September 2008 Received in revised form 24 February 2009 Accepted 25 February 2009 Available online 1 April 2009 Keywords: Allylthiourea p-Cresol p-Hydroxybenzaldehyde Inhibition Nitrification
a b s t r a c t The oxidizing ability of a nitrifying consortium exposed to p-cresol (25 mg C L1) was evaluated in batch cultures. Biotransformation of the phenolic compound was investigated by identifying the different intermediates formed. p-Cresol inhibited the ammonia-oxidizing process with a decrease of 83% in the specific rate of ammonium consumption. After 48 h, ammonium consumption efficiency was 1 NHþ 96 ± 9% while nitrate yield reached 0.95 ± 0.06 g NO 3 -N g 4 -N consumed. High value for nitrate production yield showed that the nitrifying metabolic pathway was only affected at the specific rate level being nitrate the main end product. The consortium was able to totally oxidize p-cresol at a specific rate of 0.17 ± 0.06 mg p-cresol-C mg1 microbial protein h1. p-Cresol was first transformed to p-hydroxybenzaldehyde and p-hydroxybenzoate, which were later completely mineralized. In the presence of allylthiourea, a specific inhibitor of ammonia monooxygenase (AMO), p-cresol was oxidized to the same intermediates and in a similar pattern as obtained without the AMO inhibitor. AMO seemed not to be involved in the p-cresol oxidation process. When p-hydroxybenzaldehyde was added (25 mg C L1), the nitrifying process was inhibited in the same way as observed with p-cresol, indicating that p-hydroxybenzaldehyde could be the main compound responsible for nitrification inhibition. p-Hydroxybenzaldehyde was accumulated during 15 h before complete consumption at a specific rate value eight times lower than the p-cresol consumption rate. Results showed that p-hydroxybenzaldehyde oxidation was the limiting step in p-cresol mineralization by the nitrifying consortium. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Ammonium is a contaminant of many municipal and industrial wastewaters. Petrochemical, chemical, steel manufacturing and resin producing industries are some examples of the diverse industries that generate wastewaters with high concentrations of phenolic compounds and ammonia (Olmos et al., 2004). These compounds cause undesirable severe effects on the environment and human health, such as eutrophication, bioaccumulation and toxicity (Veeresh et al., 2005). Nitrification and denitrification biological processes are considered economically feasible technologies for nitrogen removal from wastewaters (Kuenen and Robertson, 1994). Nitrification is commonly the rate-limiting step of the overall nitrogen removal. High sensitivity of nitrifying bacteria to the toxic or inhibitory effects of organic compounds is well-known. Most of the studies on effects of organic matter on nitrification have used axenic cultures or consortia such as activated sludge as inoculum. Works with Nitrosomonas sp. or Nitrob-
* Corresponding author. Tel.: +52 55 5804 4711; fax +52 55 5804 6407. E-mail address: [email protected] (A.-C. Texier). 0045-6535/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2009.02.059
acter sp. have been focused on the growth inhibition of bacteria (Steinmüller and Bock, 1976), others on the activity of the ammonia monooxygenase (AMO), the enzyme believed to catalyze the NH3 oxidation (McCarty, 1999). Less information is available on the effect of organic substances on the nitrifying respiratory process of microbial consortia with a stable metabolic behavior, particularly for aromatic compounds (Gomez et al., 2000; Zepeda et al., 2003, 2006). Inhibitory effects of phenolic compounds on the nitrification process have been previously reported (Yamagishi et al., 2001; Texier and Gomez, 2002; Amor et al., 2005). However, knowledge about the effects of cresols on the nitrifying respiratory process is still scarce and further work is required to understand better the underlying processes involved in the inhibition of nitrification by these recalcitrant compounds. Some studies have shown that a consortium produced in steady-state nitrification can simultaneously oxidize ammonia and aromatic compounds (Zepeda et al., 2003, 2006). For instance, Texier and Gomez (2007) observed the complete ammonium oxidation to nitrate and the total consumption of p-cresol in a nitrifying sequencing batch reactor culture. Nevertheless, the transitory intermediates were not identified. Thus, it is of interest to investigate how the oxidation of cresols proceeds by nitrifying consortia because it can be closely related to nitrification inhibition.
C.D. Silva et al. / Chemosphere 75 (2009) 1387–1391
2. Materials and methods 2.1. Inoculum and continuous nitrifying reactor The sludge used for inoculating batch reactors was obtained from a continuous stirred nitrifying 6 L reactor. The system was kept operating continuously at 30 °C, pH 8.1 ± 0.3, 300 rpm, with a hydraulic retention time of 1.2 d and a settler for biomass recirculation. Dissolved oxygen concentration was maintained at 6.3 ± 0.6 mg L1 by continuous aeration. The reactor was fed with two media, A (as nitrogen and energy source) and B (as carbon source). Medium A contained the following nutrients (g L1): (NH4)2SO4 (1.18), NH4Cl (0.96), KH2PO4 (1.40), MgSO4 (0.60), NaCl (1.0). Medium B was composed of NaHCO3 (9.33 g L1) and CaCl2 (0.05 g L1). Media A and B were fed at 1.77 and 1.57 L d1, respec1 1 d . The inlet tively. The loading rate of NHþ 4 was 145 ± 8 mg N L C/N ratio was 2.4. 30 mL of a FeSO4 7H2O solution (5%) was added daily into the reactor. 2.2. Batch cultures All experiments were performed in 160 mL serum bottles with a working volume of 100 mL. Medium contained the following nutrients (g L1): (NH4)2SO4 (0.24), NH4Cl (0.19), KH2PO4 (0.28), MgSO4 (0.20), NaCl (0.20), NaHCO3 (1.75) and CaCl2 (0.01), resulting in ratios of: C/N, N/S, and N/P of 2.5, 1.9, and 1.6, respectively. The ini1 and tial NHþ 4 and NaHCO3 concentrations were 100 ± 10 mg N L 250 ± 25 mg C L1, respectively. p-Cresol and p-hydroxybenzaldehyde were separately added at an initial concentration of 25 mg C L1. Additional assays were conducted with p-cresol in the presence of allylthiourea at 0.2 mM (25 mg L1). Oxygen (99.6% purity) was bubbled through the medium for 2 min. Nitrifying sludge used as inoculum was drawn from the continuous reactor, centrifuged (4000 rpm for 10 min), and washed with a solution of NaCl (9 g L1) before resuspending in the medium culture. The initial microbial protein concentration was 134 ± 18 mg L1. The bottles were later sealed with rubber caps and aluminum rings. Oxygen was again injected for 2 min into the bottle’s 60 mL headspace. The initial pH value was 7.7 ± 0.2. Finally, all cultures were placed on an orbital shaker working at 300 rpm at 30 °C. Samples were withdrawn at different times, filtered (0.45 lm), and analyzed for ammonium, nitrite, nitrate, total organic carbon (TOC), p-cresol and intermediates. All batch cultures were carried out at least in duplicate. The response variables used for evaluating the physiological behavior of the nitrifying sludge in the absence and presence of p-cresol and p-hydroxybenzaldehyde were ammonium 1 NHþ consumption efficiency (ENH4 , (g NHþ 4 consumed g 4 ini 1 tial) 100), nitrate production yield (Y NO3 , g NO3 -N g NHþ 4 -N consumed), and specific rates of NHþ 4 consumption (qNH4 ) and NO 3 production (qNO3 ). Integrated Gompertz model was used to determine lag phase (k) values and fit the volumetric ammonium and organic compounds consumption rates as well as volumetric nitrate production rates (Acuña et al., 1999). The coefficients of determination were higher than 0.95 for all cases. With the microbial protein concentration known, the specific rates were calculated and expressed as mg N or C mg1 microbial protein h1. In
order to estimate the biomass formation yield, it was assumed that around 65% of the microbial biomass is protein and 50% is carbon. 2.3. Analytical methods Ammonium was analyzed by a selective electrode (Phoenix electrode company, USA). Nitrite and nitrate were measured by HPLC (Perkin–Elmer series 200) using an ion exchange column (IC-Pak Anion HC, 4.6 150 mm, Waters) and a UV detector at 214 nm. The mobile phase was composed of (mL L1): borate-gluconate solution (20), n-butanol (20) and acetonitrile (120) at 2 mL min1. The composition of borate-gluconate solution (g L1) was: sodium gluconate (16), boric acid (18) and sodium tetraborate decahydrate (25). p-Cresol and its aromatic intermediates were monitored by HPLC (Perkin–Elmer series 200) using a C18 reverse-phase column (bondclone, phenomenex, 300 3.9 mm) and a UV detector at 254 nm. The mobile phase was acetonitrile–water (60:40, v/v) at 1.5 mL min1. TOC was measured using a TOC meter (TOC-5000 Shimadzu). Dissolved oxygen and pH were measured by selective electrodes. Lowry’s method was employed to measure microbial protein concentration (Lowry et al., 1951). Standard curves were drawn in triplicates for each analytical method. In all cases, the variation coefficient was less than 10%. 3. Results and discussion 3.1. Continuous nitrifying reactor 1 1 d , the NO At a NHþ 4 loading rate of 145 ± 8 mg N L 3 production rate reached 142 ± 12 mg N L1 d1 while the output of NHþ 4 rate remained low (0.4 ± 0.3 mg N L1 d1). Nitrite was never detected. Volumetric rates of the continuous reactor remained constant with a variation coefficient lower than 10%, indicating that the nitrifying respiratory process was in steady state. Under these operating conditions, ENH4 was 99.7 ± 0.2% and Y NO3 0.98 ± 0.05. The rate of microbial protein production was 4.5 ± 0.4 mg L1 d1. These results assured that the sludge used as inoculum for batch studies carried out a stable and dissimilative nitrifying process. Therefore, variations in results due to metabolic activity fluctuations were minimized and this study was focused on the effect of p-cresol on the nitrifying respiratory process.
3.2. Effect of p-cresol on nitrification Nitrifying cultures were performed in the absence of p-cresol (Fig. 1). The initial pH of 7.7 ± 0.2 gradually changed to a final value
120 100 80
(mg N L-1)
The aim of this study was to evaluate the effect of p-cresol on the nitrifying pathway of a consortium produced in steadystate nitrification, emphasizing transformation of p-cresol to intermediates. This paper deals also with some possible reasons for nitrification inhibition by p-cresol, by studying particularly the role of the AMO in p-cresol oxidation and the inhibitory effect of phydroxybenzaldehyde, a recalcitrant intermediate of p-cresol oxidation.
Concentration
1388
NH4+
60
NO2NO3-
40 20 0 0
5
10
15
20
Time (h) Fig. 1. Kinetic profiles of the nitrifying process in batch cultures.
25
1389
C.D. Silva et al. / Chemosphere 75 (2009) 1387–1391
of 6.8 ± 0.3. It was verified that the dissolved oxygen concentration dropped to 5.3 ± 0.2 mg L1 after 24 h, ensuring that there was no limitation for oxygen. Nitrite accumulation was only transient. There was no significant microbial growth. After 24 h, ENH4 was 99.5 ± 0.1% and Y NO3 1.0 ± 0.1, indicating that the nitrification process proceeded successfully under the batch experimental conditions used. According to the Gompertz model, specific rates for ammonium oxidation and nitrate formation were 0.12 ± 0.02 and 0.03 ± 0.01 mg N mg1 microbial protein h1, respectively. Kinetic profiles were established for NHþ 4 ; NO2 , and NO3 when p-cresol was present at 25 mg C L1 (Fig. 2). The addition of p-cresol provoked a drastic decrease in the values for nitrification specific rates (Table 1). qNH4 and qNO3 decreased by 83% and 60%, respectively. Ammonium consumption was the slowest in the course of the first 16 h of incubation as described by Gompertz model. Nitrite was never detected, showing that the decrease in the specific rate of NO 3 production was mainly caused by the decrease in the specific rate of NHþ 4 consumption. After 48 h, the nitrifying process was completed with ENH4 of 96 ± 9% and Y NO3 of 0.95 ± 0.06. These results indicated that, in the presence of p-cresol, the ammoniumoxidizing process was inhibited and the nitrifying metabolic pathway was only altered at the specific rate level as nitrate was still the main end product. These results are in agreement with various studies which indicate that the ammonia-oxidizing process was the most sensitive to the presence of organic compounds (Zepeda et al., 2003; Dokianakis et al., 2006; Texier and Gomez, 2007). Some authors have proposed that this effect might be related to an enzymatic inhibition of the AMO considering its ability to oxidize a wide variety of organic compounds (Keener and Arp, 1993, 1994; McCarty, 1999; Duddleston et al., 2002).
intermediates obtained in the nitrifying cultures. p-Cresol was rapidly and totally consumed in less than 2 h, at a specific rate of 0.17 ± 0.06 mg p-cresol-C mg1 microbial protein h1. p-Hydroxybenzaldehyde (pOHBD) and p-hydroxybenzoate (pOHBT) were detected and quantified as transient intermediates of p-cresol metabolism. pOHBD first accumulated in the cultures for 7 h, and then was totally consumed after 18 h. pOHBT accumulation was kept low during 18 h and then it disappeared totally from the cultures after 20 h. TOC concentration remained constant for 7 h at an average value of 25 ± 1 mg L1, mainly due to the accumulation of pOHBD and pOHBT. Afterwards, TOC concentration decreased, reaching a value close to zero after 20 h. The nitrifying sludge was able to completely consume p-cresol and its intermediates. The microbial growth was negligible during the 24 h nitrifying cultures without p-cresol. In the presence of p-cresol, growth of the autotrophic nitrifying bacteria could not be significant as nitrification was inhibited. Thus, the yield for biomass formation was estimated only on p-cresol consumption discarding bicarbonate assimilation. Biomass yield was found to be of 0.15 ± 0.02 g biomass-C g1 p-cresol-C consumed, corresponding to the growth of
25
(mg N L-1)
p-Hydroxybenzoate Total Organic Carbon
15
NH4
__ __ NO 3
Convcentration (mg C L-1)
+
Concentration
p-Hydroxybenzaldehyde
10
Abiotic and sterile controls were conducted to verify that possible loss of p-cresol was not significant under the experimental conditions used. Fig. 3a shows the kinetic profiles of p-cresol and its
NO2-
80
p-Cresol
20
3.3. p-Cresol oxidation by the nitrifying sludge
100
a
30
5 0 30
b
25 20
60
15
40
10
20
5
0
0 0
10
20
30
40
50
0
5
10
Time (h)
15
20
25
Time (h)
Fig. 2. Nitrification process in the presence of p-cresol at 25 mg C L1.
Fig. 3. p-Cresol oxidation by a nitrifying consortium without (a) and with ATU (b).
Table 1 Specific rates (mg N mg1 microbial protein h1) and lag phase (k) of the nitrification cultures in the absence and presence of p-cresol or p-hydroxybenzaldehyde. NO 3 production
NHþ 4 consumption
Without aromatic compound With p-cresol (25 mg C L1) With p-hydroxybenzaldehyde (25 mg C L1)
k (h)
qNH4
k (h)
qNO3
0 16 27
0.12 ± 0.02 0.02 ± 0.002 (83%) 0.02 ± 0.005 (83%)
0 23 15
0.03 ± 0.01 0.012 ± 0.001 (60%) 0.01 ± 0.001 (67%)
C.D. Silva et al. / Chemosphere 75 (2009) 1387–1391
toluene, ethylbenzene, p-xylene, styrene) by N. europaea were prevented by acetylene, an inactivator of AMO. However, they found that p-cresol was oxidized by C2H2-treated cells, indicating that p-cresol was transformed to pOHBD by an enzyme or enzymes other than AMO in N. europaea. As in axenic cultures of N. europaea, the p-cresol oxidation by the nitrifying consortium used in the present study might not depend on AMO activity. 3.5. Effect of p-hydroxybenzaldehyde on nitrification The facts that, in the presence of p-cresol, the ammonia-oxidizing process was very slow during 16 h and in the same cultures, pcresol was totally consumed in less than 2 h while pOHBD accumulated for 7 h, indicated that p-cresol could not be the direct responsible for nitrification inhibition and pOHBD could be also involved in the nitrification inhibition processes. When p-hydroxybenzaldehyde was added to the nitrifying batch cultures, qNH4 and qNO3 decreased by 83% and 67%, respectively (Table 1). Specific rates decreased in the same ratio than when p-cresol was initially added, showing that pOHBD could be the main responsible for nitrification inhibition. As in the case with p-cresol, the nitrifying metabolic pathway was only altered in its velocity and after 75 h, ENH4 was of 98.9 ± 0.1% and Y NO3 of 1.0 ± 0.03 (Fig. 4). However, while the culture was able to totally consume p-cresol in 2 h, p-hydroxybenzaldehyde was accumulated for 15 h before to be totally oxidized (Fig. 5). The specific velocity of pOHBD consumption was 120 100
-1 (mg N L )
the heterotrophic bacteria of the consortium. These results indicate that p-cresol was mainly mineralized. In studies with axenic cultures of Nitrosomonas europaea, the transformation of aromatic compounds led to the formation of more oxidized aromatic products which remained accumulated in the cultures, and benzene ring cleavage did not occur (Keener and Arp, 1994). In contrast, when a nitrifying consortium was used, benzene, toluene, and mxylene were converted to volatile fatty acids (Zepeda et al., 2003, 2006). The authors proposed that the aromatic compounds oxidation with ring fission could have been possible due to the coexistence and participation of both, lithoautotrophic nitrifying bacteria and heterotrophic microorganisms present in the consortium. Results suggest that p-cresol oxidation followed the same initial pathway previously proposed by various authors which consists of the sequential formation of p-hydroxybenzylalcohol (pOHBOL), pOHBD, and pOHBT under different aerobic and anaerobic conditions (Hopper and Taylor, 1975; Bossert et al., 1989; Häggblom et al., 1990). Häggblom et al. (1990) have suggested that a universal strategy for the initial steps in p-cresol degradation is used whether the terminal electron acceptor is oxygen, nitrate, sulfate, or carbon dioxide. It has been reported for denitrifying and aerobic bacteria, that p-cresol can be sequentially oxidized by the p-cresol methylhydroxylase to pOHBOL and pOHBD, which is further oxidized to pOHBT by a NAD+-dependent dehydrogenase (Rudolphi et al., 1991; Yadav et al., 2005). Isotopic 18O studies by Hopper (1978) have demonstrated that the incorporated methyl oxygen by the p-cresol methylhydroxylase from Pseudomonas putida was derived from 18O-labeled water, with molecular O2 serving only as an external electron acceptor and not as a reactant in the pathway. Under the aerobic conditions used in this study, it is too likely that oxidation of the methyl group was mediated by oxygenases. Previous studies have shown that the ammonia-oxidizing bacterium N. europaea was able to oxidize a broad range of hydrocarbons, including aromatic and non-aromatic compounds, and it is believed that the AMO is participating (Hyman et al., 1985; Rasche et al., 1990; Chang et al., 2002). In particular, Keener and Arp (1994) have shown that toluene was oxidized to benzylalcohol and benzaldehyde by a pure culture of N. europaea, and this transformation was initiated only by cells with active AMO. This indicated that AMO was involved in the oxidation of the methyl (– CH3) substituent. These authors have also observed that p-cresol was quantitatively converted to pOHBD via pOHBOL formation by N. europaea. To our knowledge, the present study is the first report where pOHBD and pOHBT are identified as transient intermediates from the p-cresol oxidation pathway by a nitrifying consortium in which the diversity of enzymatic material and possible metabolic pathways is high.
Concentration
1390
80 NH4+
60
NO2
40
NO3
-
20 0 0
10
20
30
40
50
60
70
80
Time (h) Fig. 4. Nitrification process in the presence of p-hydroxybenzaldehyde at 25 mg C L1.
30
3.4. p-Cresol oxidation by allylthiourea treated cultures
p-Hydroxybenzaldehyde p-Hydroxybenzoate
20
-1
(mg C L )
Concentration
25
In order to determine if AMO was involved in p-cresol oxidation, allylthiourea was used as a selective inhibitor of AMO (Bedard and Knowles, 1989; Ginestet et al., 1998). It was first verified that the addition of allylthiourea (0.2 mM) caused a complete and stable inhibition of ammonium oxidation during the 24 h of incubation (ENH4 = 1.7 ± 1.1%). Y NO3 was 0.97 ± 0.05, showing that the little of consumed NHþ 4 was totally converted to NO3 . Therefore, allylthiourea inhibited the ammonia-oxidizing consumption but not the nitrite-oxidizing process. Fig. 3b presents the kinetic profiles of p-cresol transformation obtained in the allylthiourea treated cultures. Results show that p-cresol was oxidized to pOHBD and pOHBT in a similar pattern as obtained without the AMO inhibitor (Fig. 3a). These results suggest that the AMO was not involved in the p-cresol oxidation by the nitrifying sludge. Keener and Arp (1994) observed that most of transformations (benzene,
-
15 10 5 0 0
5
10
15
20
25
Time (h) Fig. 5. p-Hydroxybenzaldehyde oxidation by a nitrifying consortium.
C.D. Silva et al. / Chemosphere 75 (2009) 1387–1391
0.02 ± 0.01 mg pOHBD-C mg1 microbial protein h1. This value was around eight times lower than the p-cresol consumption rate, suggesting that pOHBD oxidation was the rate-limiting step in pcresol mineralization by the nitrifying consortium. These results could be related to the benzaldehydes chemical characteristics as some works have reported that these compounds might alter cell permeability and stability of some enzymes (Long and Ward, 1989; Ramos-Nino et al., 1998). 4. Conclusions Results showed that the nitrifying consortium was able to mineralize p-cresol through the sequential and transient formation of the aromatic intermediates: pOHBD and pOHBT. p-Cresol was rapidly oxidized by the sludge at a specific rate of 0.17 ± 0.06 mg p-cresol-C mg1 microbial protein h1 while pOHBD was the most recalcitrant aromatic compound formed in the cultures, with a specific consumption rate of 0.02 ± 0.01 mg pOHBD-C mg1 microbial protein h1. With p-cresol added at 25 mg C L1, the ammoniaoxidizing process was highly inhibited with a decrease of 83% in the specific rate for ammonium oxidation. However, the metabolic pathway was only altered in its velocity and nitrate kept the main end product from the nitrifying process. In spite of the presence of p-cresol, nitrification processes could successfully proceed (ENH4 = 96 ± 9%, Y NO3 = 0.95 ± 0.06), even though in a slower way. Ammonia monooxygenase was not responsible for the p-cresol oxidation. pOHBD seemed to be the main responsible for ammonium oxidation inhibition and its oxidation the limiting step in p-cresol mineralization by the nitrifying consortium. Acknowledgements This work was financially supported by the Council of Science and Technology of Mexico (Grant No. SEP-CONACYT-CO2-43144). Participation of Carlos David Silva was founded by CONACYT (164072). References Acuña, M.E., Pérez, F., Auria, R., Revah, S., 1999. Microbiological and kinetic aspects of a biofilter for the removal of toluene from waste gases. Biotechnol. Bioeng. 63, 175–184. Amor, L., Eiroa, M., Kennes, C., Veiga, M.C., 2005. Phenol biodegradation and its effect on the nitrification process. Water Res. 39, 2915–2920. Bedard, C., Knowles, R., 1989. Physiology, biochemistry, and specific inhibitors CH4, NHþ 4 , of and CO oxidation by methanotrophs and nitrifiers. Microbiol. Rev. 53, 68–84. Bossert, I.D., Whited, G., Gibson, D.T., Young, L.Y., 1989. Anaerobic oxidation of pcresol mediated partially purified methylhydroxylase from a denitrifying bacterium. J. Bacteriol. 171, 2956–2962. Chang, S.W., Hyman, M.R., Williamson, K.J., 2002. Cooxidation of naphthalene and other polycyclic aromatic hydrocarbons by the nitrifying bacterium Nitrosomonas europaea. Biodegradation 13, 373–381. Dokianakis, S.N., Kornaros, N., Lyberatos, G., 2006. Impact of five selected xenobiotics on isolated ammonium oxidizers and on nitrifying activated sludge. Environ. Toxicol. 21, 310–316.
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Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Technical Note
p-Cresol biotransformation by a nitrifying consortium C.D. Silva a, J. Gómez a, E. Houbron b, F.M. Cuervo-López a, A.-C. Texier a,* a b
Department of Biotechnology-CBS, Universidad Autónoma Metropolitana-Iztapalapa, Av. San Rafael Atlixco, No. 186-Col, Vicentina D.F., Mexico Facultad de Ciencias Químicas, Universidad Veracruzana, Prol. OTE 6, No. 1009 Orizaba, Veracruz, Mexico
a r t i c l e
i n f o
Article history: Received 6 September 2008 Received in revised form 24 February 2009 Accepted 25 February 2009 Available online 1 April 2009 Keywords: Allylthiourea p-Cresol p-Hydroxybenzaldehyde Inhibition Nitrification
a b s t r a c t The oxidizing ability of a nitrifying consortium exposed to p-cresol (25 mg C L1) was evaluated in batch cultures. Biotransformation of the phenolic compound was investigated by identifying the different intermediates formed. p-Cresol inhibited the ammonia-oxidizing process with a decrease of 83% in the specific rate of ammonium consumption. After 48 h, ammonium consumption efficiency was 1 NHþ 96 ± 9% while nitrate yield reached 0.95 ± 0.06 g NO 3 -N g 4 -N consumed. High value for nitrate production yield showed that the nitrifying metabolic pathway was only affected at the specific rate level being nitrate the main end product. The consortium was able to totally oxidize p-cresol at a specific rate of 0.17 ± 0.06 mg p-cresol-C mg1 microbial protein h1. p-Cresol was first transformed to p-hydroxybenzaldehyde and p-hydroxybenzoate, which were later completely mineralized. In the presence of allylthiourea, a specific inhibitor of ammonia monooxygenase (AMO), p-cresol was oxidized to the same intermediates and in a similar pattern as obtained without the AMO inhibitor. AMO seemed not to be involved in the p-cresol oxidation process. When p-hydroxybenzaldehyde was added (25 mg C L1), the nitrifying process was inhibited in the same way as observed with p-cresol, indicating that p-hydroxybenzaldehyde could be the main compound responsible for nitrification inhibition. p-Hydroxybenzaldehyde was accumulated during 15 h before complete consumption at a specific rate value eight times lower than the p-cresol consumption rate. Results showed that p-hydroxybenzaldehyde oxidation was the limiting step in p-cresol mineralization by the nitrifying consortium. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Ammonium is a contaminant of many municipal and industrial wastewaters. Petrochemical, chemical, steel manufacturing and resin producing industries are some examples of the diverse industries that generate wastewaters with high concentrations of phenolic compounds and ammonia (Olmos et al., 2004). These compounds cause undesirable severe effects on the environment and human health, such as eutrophication, bioaccumulation and toxicity (Veeresh et al., 2005). Nitrification and denitrification biological processes are considered economically feasible technologies for nitrogen removal from wastewaters (Kuenen and Robertson, 1994). Nitrification is commonly the rate-limiting step of the overall nitrogen removal. High sensitivity of nitrifying bacteria to the toxic or inhibitory effects of organic compounds is well-known. Most of the studies on effects of organic matter on nitrification have used axenic cultures or consortia such as activated sludge as inoculum. Works with Nitrosomonas sp. or Nitrob-
* Corresponding author. Tel.: +52 55 5804 4711; fax +52 55 5804 6407. E-mail address: [email protected] (A.-C. Texier). 0045-6535/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2009.02.059
acter sp. have been focused on the growth inhibition of bacteria (Steinmüller and Bock, 1976), others on the activity of the ammonia monooxygenase (AMO), the enzyme believed to catalyze the NH3 oxidation (McCarty, 1999). Less information is available on the effect of organic substances on the nitrifying respiratory process of microbial consortia with a stable metabolic behavior, particularly for aromatic compounds (Gomez et al., 2000; Zepeda et al., 2003, 2006). Inhibitory effects of phenolic compounds on the nitrification process have been previously reported (Yamagishi et al., 2001; Texier and Gomez, 2002; Amor et al., 2005). However, knowledge about the effects of cresols on the nitrifying respiratory process is still scarce and further work is required to understand better the underlying processes involved in the inhibition of nitrification by these recalcitrant compounds. Some studies have shown that a consortium produced in steady-state nitrification can simultaneously oxidize ammonia and aromatic compounds (Zepeda et al., 2003, 2006). For instance, Texier and Gomez (2007) observed the complete ammonium oxidation to nitrate and the total consumption of p-cresol in a nitrifying sequencing batch reactor culture. Nevertheless, the transitory intermediates were not identified. Thus, it is of interest to investigate how the oxidation of cresols proceeds by nitrifying consortia because it can be closely related to nitrification inhibition.
C.D. Silva et al. / Chemosphere 75 (2009) 1387–1391
2. Materials and methods 2.1. Inoculum and continuous nitrifying reactor The sludge used for inoculating batch reactors was obtained from a continuous stirred nitrifying 6 L reactor. The system was kept operating continuously at 30 °C, pH 8.1 ± 0.3, 300 rpm, with a hydraulic retention time of 1.2 d and a settler for biomass recirculation. Dissolved oxygen concentration was maintained at 6.3 ± 0.6 mg L1 by continuous aeration. The reactor was fed with two media, A (as nitrogen and energy source) and B (as carbon source). Medium A contained the following nutrients (g L1): (NH4)2SO4 (1.18), NH4Cl (0.96), KH2PO4 (1.40), MgSO4 (0.60), NaCl (1.0). Medium B was composed of NaHCO3 (9.33 g L1) and CaCl2 (0.05 g L1). Media A and B were fed at 1.77 and 1.57 L d1, respec1 1 d . The inlet tively. The loading rate of NHþ 4 was 145 ± 8 mg N L C/N ratio was 2.4. 30 mL of a FeSO4 7H2O solution (5%) was added daily into the reactor. 2.2. Batch cultures All experiments were performed in 160 mL serum bottles with a working volume of 100 mL. Medium contained the following nutrients (g L1): (NH4)2SO4 (0.24), NH4Cl (0.19), KH2PO4 (0.28), MgSO4 (0.20), NaCl (0.20), NaHCO3 (1.75) and CaCl2 (0.01), resulting in ratios of: C/N, N/S, and N/P of 2.5, 1.9, and 1.6, respectively. The ini1 and tial NHþ 4 and NaHCO3 concentrations were 100 ± 10 mg N L 250 ± 25 mg C L1, respectively. p-Cresol and p-hydroxybenzaldehyde were separately added at an initial concentration of 25 mg C L1. Additional assays were conducted with p-cresol in the presence of allylthiourea at 0.2 mM (25 mg L1). Oxygen (99.6% purity) was bubbled through the medium for 2 min. Nitrifying sludge used as inoculum was drawn from the continuous reactor, centrifuged (4000 rpm for 10 min), and washed with a solution of NaCl (9 g L1) before resuspending in the medium culture. The initial microbial protein concentration was 134 ± 18 mg L1. The bottles were later sealed with rubber caps and aluminum rings. Oxygen was again injected for 2 min into the bottle’s 60 mL headspace. The initial pH value was 7.7 ± 0.2. Finally, all cultures were placed on an orbital shaker working at 300 rpm at 30 °C. Samples were withdrawn at different times, filtered (0.45 lm), and analyzed for ammonium, nitrite, nitrate, total organic carbon (TOC), p-cresol and intermediates. All batch cultures were carried out at least in duplicate. The response variables used for evaluating the physiological behavior of the nitrifying sludge in the absence and presence of p-cresol and p-hydroxybenzaldehyde were ammonium 1 NHþ consumption efficiency (ENH4 , (g NHþ 4 consumed g 4 ini 1 tial) 100), nitrate production yield (Y NO3 , g NO3 -N g NHþ 4 -N consumed), and specific rates of NHþ 4 consumption (qNH4 ) and NO 3 production (qNO3 ). Integrated Gompertz model was used to determine lag phase (k) values and fit the volumetric ammonium and organic compounds consumption rates as well as volumetric nitrate production rates (Acuña et al., 1999). The coefficients of determination were higher than 0.95 for all cases. With the microbial protein concentration known, the specific rates were calculated and expressed as mg N or C mg1 microbial protein h1. In
order to estimate the biomass formation yield, it was assumed that around 65% of the microbial biomass is protein and 50% is carbon. 2.3. Analytical methods Ammonium was analyzed by a selective electrode (Phoenix electrode company, USA). Nitrite and nitrate were measured by HPLC (Perkin–Elmer series 200) using an ion exchange column (IC-Pak Anion HC, 4.6 150 mm, Waters) and a UV detector at 214 nm. The mobile phase was composed of (mL L1): borate-gluconate solution (20), n-butanol (20) and acetonitrile (120) at 2 mL min1. The composition of borate-gluconate solution (g L1) was: sodium gluconate (16), boric acid (18) and sodium tetraborate decahydrate (25). p-Cresol and its aromatic intermediates were monitored by HPLC (Perkin–Elmer series 200) using a C18 reverse-phase column (bondclone, phenomenex, 300 3.9 mm) and a UV detector at 254 nm. The mobile phase was acetonitrile–water (60:40, v/v) at 1.5 mL min1. TOC was measured using a TOC meter (TOC-5000 Shimadzu). Dissolved oxygen and pH were measured by selective electrodes. Lowry’s method was employed to measure microbial protein concentration (Lowry et al., 1951). Standard curves were drawn in triplicates for each analytical method. In all cases, the variation coefficient was less than 10%. 3. Results and discussion 3.1. Continuous nitrifying reactor 1 1 d , the NO At a NHþ 4 loading rate of 145 ± 8 mg N L 3 production rate reached 142 ± 12 mg N L1 d1 while the output of NHþ 4 rate remained low (0.4 ± 0.3 mg N L1 d1). Nitrite was never detected. Volumetric rates of the continuous reactor remained constant with a variation coefficient lower than 10%, indicating that the nitrifying respiratory process was in steady state. Under these operating conditions, ENH4 was 99.7 ± 0.2% and Y NO3 0.98 ± 0.05. The rate of microbial protein production was 4.5 ± 0.4 mg L1 d1. These results assured that the sludge used as inoculum for batch studies carried out a stable and dissimilative nitrifying process. Therefore, variations in results due to metabolic activity fluctuations were minimized and this study was focused on the effect of p-cresol on the nitrifying respiratory process.
3.2. Effect of p-cresol on nitrification Nitrifying cultures were performed in the absence of p-cresol (Fig. 1). The initial pH of 7.7 ± 0.2 gradually changed to a final value
120 100 80
(mg N L-1)
The aim of this study was to evaluate the effect of p-cresol on the nitrifying pathway of a consortium produced in steadystate nitrification, emphasizing transformation of p-cresol to intermediates. This paper deals also with some possible reasons for nitrification inhibition by p-cresol, by studying particularly the role of the AMO in p-cresol oxidation and the inhibitory effect of phydroxybenzaldehyde, a recalcitrant intermediate of p-cresol oxidation.
Concentration
1388
NH4+
60
NO2NO3-
40 20 0 0
5
10
15
20
Time (h) Fig. 1. Kinetic profiles of the nitrifying process in batch cultures.
25
1389
C.D. Silva et al. / Chemosphere 75 (2009) 1387–1391
of 6.8 ± 0.3. It was verified that the dissolved oxygen concentration dropped to 5.3 ± 0.2 mg L1 after 24 h, ensuring that there was no limitation for oxygen. Nitrite accumulation was only transient. There was no significant microbial growth. After 24 h, ENH4 was 99.5 ± 0.1% and Y NO3 1.0 ± 0.1, indicating that the nitrification process proceeded successfully under the batch experimental conditions used. According to the Gompertz model, specific rates for ammonium oxidation and nitrate formation were 0.12 ± 0.02 and 0.03 ± 0.01 mg N mg1 microbial protein h1, respectively. Kinetic profiles were established for NHþ 4 ; NO2 , and NO3 when p-cresol was present at 25 mg C L1 (Fig. 2). The addition of p-cresol provoked a drastic decrease in the values for nitrification specific rates (Table 1). qNH4 and qNO3 decreased by 83% and 60%, respectively. Ammonium consumption was the slowest in the course of the first 16 h of incubation as described by Gompertz model. Nitrite was never detected, showing that the decrease in the specific rate of NO 3 production was mainly caused by the decrease in the specific rate of NHþ 4 consumption. After 48 h, the nitrifying process was completed with ENH4 of 96 ± 9% and Y NO3 of 0.95 ± 0.06. These results indicated that, in the presence of p-cresol, the ammoniumoxidizing process was inhibited and the nitrifying metabolic pathway was only altered at the specific rate level as nitrate was still the main end product. These results are in agreement with various studies which indicate that the ammonia-oxidizing process was the most sensitive to the presence of organic compounds (Zepeda et al., 2003; Dokianakis et al., 2006; Texier and Gomez, 2007). Some authors have proposed that this effect might be related to an enzymatic inhibition of the AMO considering its ability to oxidize a wide variety of organic compounds (Keener and Arp, 1993, 1994; McCarty, 1999; Duddleston et al., 2002).
intermediates obtained in the nitrifying cultures. p-Cresol was rapidly and totally consumed in less than 2 h, at a specific rate of 0.17 ± 0.06 mg p-cresol-C mg1 microbial protein h1. p-Hydroxybenzaldehyde (pOHBD) and p-hydroxybenzoate (pOHBT) were detected and quantified as transient intermediates of p-cresol metabolism. pOHBD first accumulated in the cultures for 7 h, and then was totally consumed after 18 h. pOHBT accumulation was kept low during 18 h and then it disappeared totally from the cultures after 20 h. TOC concentration remained constant for 7 h at an average value of 25 ± 1 mg L1, mainly due to the accumulation of pOHBD and pOHBT. Afterwards, TOC concentration decreased, reaching a value close to zero after 20 h. The nitrifying sludge was able to completely consume p-cresol and its intermediates. The microbial growth was negligible during the 24 h nitrifying cultures without p-cresol. In the presence of p-cresol, growth of the autotrophic nitrifying bacteria could not be significant as nitrification was inhibited. Thus, the yield for biomass formation was estimated only on p-cresol consumption discarding bicarbonate assimilation. Biomass yield was found to be of 0.15 ± 0.02 g biomass-C g1 p-cresol-C consumed, corresponding to the growth of
25
(mg N L-1)
p-Hydroxybenzoate Total Organic Carbon
15
NH4
__ __ NO 3
Convcentration (mg C L-1)
+
Concentration
p-Hydroxybenzaldehyde
10
Abiotic and sterile controls were conducted to verify that possible loss of p-cresol was not significant under the experimental conditions used. Fig. 3a shows the kinetic profiles of p-cresol and its
NO2-
80
p-Cresol
20
3.3. p-Cresol oxidation by the nitrifying sludge
100
a
30
5 0 30
b
25 20
60
15
40
10
20
5
0
0 0
10
20
30
40
50
0
5
10
Time (h)
15
20
25
Time (h)
Fig. 2. Nitrification process in the presence of p-cresol at 25 mg C L1.
Fig. 3. p-Cresol oxidation by a nitrifying consortium without (a) and with ATU (b).
Table 1 Specific rates (mg N mg1 microbial protein h1) and lag phase (k) of the nitrification cultures in the absence and presence of p-cresol or p-hydroxybenzaldehyde. NO 3 production
NHþ 4 consumption
Without aromatic compound With p-cresol (25 mg C L1) With p-hydroxybenzaldehyde (25 mg C L1)
k (h)
qNH4
k (h)
qNO3
0 16 27
0.12 ± 0.02 0.02 ± 0.002 (83%) 0.02 ± 0.005 (83%)
0 23 15
0.03 ± 0.01 0.012 ± 0.001 (60%) 0.01 ± 0.001 (67%)
C.D. Silva et al. / Chemosphere 75 (2009) 1387–1391
toluene, ethylbenzene, p-xylene, styrene) by N. europaea were prevented by acetylene, an inactivator of AMO. However, they found that p-cresol was oxidized by C2H2-treated cells, indicating that p-cresol was transformed to pOHBD by an enzyme or enzymes other than AMO in N. europaea. As in axenic cultures of N. europaea, the p-cresol oxidation by the nitrifying consortium used in the present study might not depend on AMO activity. 3.5. Effect of p-hydroxybenzaldehyde on nitrification The facts that, in the presence of p-cresol, the ammonia-oxidizing process was very slow during 16 h and in the same cultures, pcresol was totally consumed in less than 2 h while pOHBD accumulated for 7 h, indicated that p-cresol could not be the direct responsible for nitrification inhibition and pOHBD could be also involved in the nitrification inhibition processes. When p-hydroxybenzaldehyde was added to the nitrifying batch cultures, qNH4 and qNO3 decreased by 83% and 67%, respectively (Table 1). Specific rates decreased in the same ratio than when p-cresol was initially added, showing that pOHBD could be the main responsible for nitrification inhibition. As in the case with p-cresol, the nitrifying metabolic pathway was only altered in its velocity and after 75 h, ENH4 was of 98.9 ± 0.1% and Y NO3 of 1.0 ± 0.03 (Fig. 4). However, while the culture was able to totally consume p-cresol in 2 h, p-hydroxybenzaldehyde was accumulated for 15 h before to be totally oxidized (Fig. 5). The specific velocity of pOHBD consumption was 120 100
-1 (mg N L )
the heterotrophic bacteria of the consortium. These results indicate that p-cresol was mainly mineralized. In studies with axenic cultures of Nitrosomonas europaea, the transformation of aromatic compounds led to the formation of more oxidized aromatic products which remained accumulated in the cultures, and benzene ring cleavage did not occur (Keener and Arp, 1994). In contrast, when a nitrifying consortium was used, benzene, toluene, and mxylene were converted to volatile fatty acids (Zepeda et al., 2003, 2006). The authors proposed that the aromatic compounds oxidation with ring fission could have been possible due to the coexistence and participation of both, lithoautotrophic nitrifying bacteria and heterotrophic microorganisms present in the consortium. Results suggest that p-cresol oxidation followed the same initial pathway previously proposed by various authors which consists of the sequential formation of p-hydroxybenzylalcohol (pOHBOL), pOHBD, and pOHBT under different aerobic and anaerobic conditions (Hopper and Taylor, 1975; Bossert et al., 1989; Häggblom et al., 1990). Häggblom et al. (1990) have suggested that a universal strategy for the initial steps in p-cresol degradation is used whether the terminal electron acceptor is oxygen, nitrate, sulfate, or carbon dioxide. It has been reported for denitrifying and aerobic bacteria, that p-cresol can be sequentially oxidized by the p-cresol methylhydroxylase to pOHBOL and pOHBD, which is further oxidized to pOHBT by a NAD+-dependent dehydrogenase (Rudolphi et al., 1991; Yadav et al., 2005). Isotopic 18O studies by Hopper (1978) have demonstrated that the incorporated methyl oxygen by the p-cresol methylhydroxylase from Pseudomonas putida was derived from 18O-labeled water, with molecular O2 serving only as an external electron acceptor and not as a reactant in the pathway. Under the aerobic conditions used in this study, it is too likely that oxidation of the methyl group was mediated by oxygenases. Previous studies have shown that the ammonia-oxidizing bacterium N. europaea was able to oxidize a broad range of hydrocarbons, including aromatic and non-aromatic compounds, and it is believed that the AMO is participating (Hyman et al., 1985; Rasche et al., 1990; Chang et al., 2002). In particular, Keener and Arp (1994) have shown that toluene was oxidized to benzylalcohol and benzaldehyde by a pure culture of N. europaea, and this transformation was initiated only by cells with active AMO. This indicated that AMO was involved in the oxidation of the methyl (– CH3) substituent. These authors have also observed that p-cresol was quantitatively converted to pOHBD via pOHBOL formation by N. europaea. To our knowledge, the present study is the first report where pOHBD and pOHBT are identified as transient intermediates from the p-cresol oxidation pathway by a nitrifying consortium in which the diversity of enzymatic material and possible metabolic pathways is high.
Concentration
1390
80 NH4+
60
NO2
40
NO3
-
20 0 0
10
20
30
40
50
60
70
80
Time (h) Fig. 4. Nitrification process in the presence of p-hydroxybenzaldehyde at 25 mg C L1.
30
3.4. p-Cresol oxidation by allylthiourea treated cultures
p-Hydroxybenzaldehyde p-Hydroxybenzoate
20
-1
(mg C L )
Concentration
25
In order to determine if AMO was involved in p-cresol oxidation, allylthiourea was used as a selective inhibitor of AMO (Bedard and Knowles, 1989; Ginestet et al., 1998). It was first verified that the addition of allylthiourea (0.2 mM) caused a complete and stable inhibition of ammonium oxidation during the 24 h of incubation (ENH4 = 1.7 ± 1.1%). Y NO3 was 0.97 ± 0.05, showing that the little of consumed NHþ 4 was totally converted to NO3 . Therefore, allylthiourea inhibited the ammonia-oxidizing consumption but not the nitrite-oxidizing process. Fig. 3b presents the kinetic profiles of p-cresol transformation obtained in the allylthiourea treated cultures. Results show that p-cresol was oxidized to pOHBD and pOHBT in a similar pattern as obtained without the AMO inhibitor (Fig. 3a). These results suggest that the AMO was not involved in the p-cresol oxidation by the nitrifying sludge. Keener and Arp (1994) observed that most of transformations (benzene,
-
15 10 5 0 0
5
10
15
20
25
Time (h) Fig. 5. p-Hydroxybenzaldehyde oxidation by a nitrifying consortium.
C.D. Silva et al. / Chemosphere 75 (2009) 1387–1391
0.02 ± 0.01 mg pOHBD-C mg1 microbial protein h1. This value was around eight times lower than the p-cresol consumption rate, suggesting that pOHBD oxidation was the rate-limiting step in pcresol mineralization by the nitrifying consortium. These results could be related to the benzaldehydes chemical characteristics as some works have reported that these compounds might alter cell permeability and stability of some enzymes (Long and Ward, 1989; Ramos-Nino et al., 1998). 4. Conclusions Results showed that the nitrifying consortium was able to mineralize p-cresol through the sequential and transient formation of the aromatic intermediates: pOHBD and pOHBT. p-Cresol was rapidly oxidized by the sludge at a specific rate of 0.17 ± 0.06 mg p-cresol-C mg1 microbial protein h1 while pOHBD was the most recalcitrant aromatic compound formed in the cultures, with a specific consumption rate of 0.02 ± 0.01 mg pOHBD-C mg1 microbial protein h1. With p-cresol added at 25 mg C L1, the ammoniaoxidizing process was highly inhibited with a decrease of 83% in the specific rate for ammonium oxidation. However, the metabolic pathway was only altered in its velocity and nitrate kept the main end product from the nitrifying process. In spite of the presence of p-cresol, nitrification processes could successfully proceed (ENH4 = 96 ± 9%, Y NO3 = 0.95 ± 0.06), even though in a slower way. Ammonia monooxygenase was not responsible for the p-cresol oxidation. pOHBD seemed to be the main responsible for ammonium oxidation inhibition and its oxidation the limiting step in p-cresol mineralization by the nitrifying consortium. Acknowledgements This work was financially supported by the Council of Science and Technology of Mexico (Grant No. SEP-CONACYT-CO2-43144). Participation of Carlos David Silva was founded by CONACYT (164072). References Acuña, M.E., Pérez, F., Auria, R., Revah, S., 1999. Microbiological and kinetic aspects of a biofilter for the removal of toluene from waste gases. Biotechnol. Bioeng. 63, 175–184. Amor, L., Eiroa, M., Kennes, C., Veiga, M.C., 2005. Phenol biodegradation and its effect on the nitrification process. Water Res. 39, 2915–2920. Bedard, C., Knowles, R., 1989. Physiology, biochemistry, and specific inhibitors CH4, NHþ 4 , of and CO oxidation by methanotrophs and nitrifiers. Microbiol. Rev. 53, 68–84. Bossert, I.D., Whited, G., Gibson, D.T., Young, L.Y., 1989. Anaerobic oxidation of pcresol mediated partially purified methylhydroxylase from a denitrifying bacterium. J. Bacteriol. 171, 2956–2962. Chang, S.W., Hyman, M.R., Williamson, K.J., 2002. Cooxidation of naphthalene and other polycyclic aromatic hydrocarbons by the nitrifying bacterium Nitrosomonas europaea. Biodegradation 13, 373–381. Dokianakis, S.N., Kornaros, N., Lyberatos, G., 2006. Impact of five selected xenobiotics on isolated ammonium oxidizers and on nitrifying activated sludge. Environ. Toxicol. 21, 310–316.
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