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Bioremediation of coking wastewater containing carbazole, dibenzofuran, dibenzothiophene and naphthalene by a naphthalene-cultivated Arthrobacter sp. W1
Bioresource Technology 164 (2014) 28–33
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Bioremediation of coking wastewater containing carbazole, dibenzofuran, dibenzothiophene and naphthalene by a naphthalene-cultivated Arthrobacter sp. W1 Shengnan Shi a, Yuanyuan Qu b, Fang Ma a,⇑, Jiti Zhou b a
State Key Laboratory of Urban Water Resource and Environment, School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, China Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China b
h i g h l i g h t s Cometabolic degradation CA, DBF and DBT simultaneous by naphthalene-grown W1. Growing and washed cells of strain W1 degraded CA, DBF, DBT and naphthalene quickly. The degradation kinetics was described well by First-order and Michaelis–Menten kinetics. Coking wastewater with naphthalene, CA, DBF and DBT was rapidly degraded by strain W1 with zeolite. Low toxicity has been found in the effluent of coking wastewater by strain W1 with zeolite.
a r t i c l e
i n f o
Article history: Received 10 March 2014 Received in revised form 3 April 2014 Accepted 4 April 2014 Available online 15 April 2014 Keywords: Cometabolic degradation Arthrobacter sp. W1 Naphthalene Heterocyclic compound Coking wastewater
a b s t r a c t A naphthalene-utilizing bacterium, Arthrobacter sp. W1, was used to investigate the cometabolic degradation of carbazole (CA), dibenzofuran (DBF) and dibenzothiophene (DBT) using naphthalene as the primary substrate. Both the growing and washed cells of strain W1 could degrade CA, DBF, DBT, and naphthalene simultaneously and quickly. Inhibition kinetics confirmed that the presence of CA, DBF and DBT in the growing system would inhibit the cells growth and biodegradability of strain W1. The relationship between ln(C/C0) and time, and specific degradation rate and CA, DBF and DBT concentration could be described well by First-order and Michaelis–Menten kinetics. The treatment of real coking wastewater containing high concentration of phenol, naphthalene, CA, DBF, DBT and NH3-N was shown to be highly efficient by naphthalene-grown W1 coupling with activation zeolite. Toxicity assessment indicated the treatment of the coking wastewater by strain W1 coupling with activation led to less toxicity than untreated wastewater. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Carbazole (CA), dibenzofuran (DBF), and dibenzothiophene (DBT) are recognized as the predominant nitrogen-, oxygen-, and sulfur-heterocyclic compound and have often been found in coking wastewater (Lim et al., 2003; Zhang et al., 1998; Zhu et al., 2009). All of these heterocyclic compounds are known to possess toxic and mutagenic activities, and considered as the refractory organic compounds in the treatment of coking wastewater (Zhu et al., 2009). It is well known that microorganisms play a primary role in the degradation of these persistent organic compounds (Seo ⇑ Corresponding author. Tel.: +86 451 8628 2107. E-mail address: [email protected] (F. Ma). http://dx.doi.org/10.1016/j.biortech.2014.04.010 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
et al., 2006). Various bacteria utilized CA, DBF, and DBT as the sole carbon and energy have been isolated, respectively (Gai et al., 2007; Das et al., 2012; Zhang et al., 2012; Balachandran et al., 2012). Because polycyclic aromatic compounds (PACs) including CA, DBF, and DBT are widespread and usually coexist in the polluted environments, cometabolic degradation of these heterocyclic compounds by bacteria growing with some others PACs should be common (Li et al., 2009). Cometabolic bioremediation is probably the promising bioremediation strategy currently available for the degrading bacteria with various types of metabolism abilities, which has been used in the field for more than 20 years on some of the most recalcitrant contaminants, e.g., phenolic compounds, xenobiotic trichloroethylene (TCE), tetrabromobisphenol A (TBBPA), and polychlorinated
S. Shi et al. / Bioresource Technology 164 (2014) 28–33
biphenyls (PCBs) (Monsalvo et al. 2009; Aktasß 2012; Kocamemi and Ceçen, 2010; Peng and Jia, 2013; Lambo and Patel, 2006; Kim et al., 2011; Sharma et al., 2012). Recently, there have been many studies on the cometabolic degradation of CA, DBF, and DBT (each or two of them) by naphthalene-, fluorene-, biphenyl-, and phenanthreneutilizing strains (Seo et al., 2006; Grifoll et al., 1995; Gai et al., 2007; Li et al., 2009). Nevertheless, current studies are mostly focused on the pathways of such chemical metabolisms and cometabolic degradation pathways of these heterocyclic compounds are similar, in which heterocyclic compound is initially attacked at the lateral position by dioxygenase to yield a chemically unstable intermediate that is spontaneously dehydrogenated by dihydrodiol dehydrogenase, and then dioxygenolytically cleaved by extradiol dioxygenase to produce meta-cleavage product (Gai et al., 2007; Li et al., 2009). However, little is known about the cometabolic degradation of CA, DBF, and DBT simultaneous by naphthaleneutilizing strains, and even about the removal of CA, DBF, DBT, and naphthalene in the bioremediation of coking wastewater. In the present study, we characterized the versatile oxidation activities of a naphthalene-utilizing Arthrobacter sp. W1 and described the cometabolic degradation of CA, DBF, and DBT by this strain. The bioremediation of real coking wastewater containing CA, DBF, DBT, phenol, naphthalene and NH3-N was also studied by naphthalene-grown W1 coupling with activation zeolite, and ecotoxicological assessment of the treated effluent was carried out. 2. Methods 2.1. Chemicals CA, DBF, DBT, and naphthalene were purchased from J&K Scientific Ltd. (China). All other commercially available chemicals were of analytical grade.
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and DBT) was tested with initial concentration of naphthalene (25–450 mg L1) plus 40 mg L1 CA, 40 mg L1 DBF, and 40 mg L1 DBT, on CA (plus naphthalene, DBF, and DBT) was tested with initial concentration of CA (12.5–225 mg L1) plus 100 mg L1 naphthalene, 40 mg L1 DBF, and 40 mg L1 DBT, on DBF (plus naphthalene, CA, and DBT) was tested with initial concentration of DBF (12.5–225 mg L1) plus 100 mg L1 naphthalene, 40 mg L1 CA, and 40 mg L1 DBT, on DBT (plus naphthalene, CA, and DBF) was tested with initial concentration of DBT (12.5–225 mg L1) plus 100 mg L1 naphthalene, 40 mg L1 CA, and 40 mg L1 DBF using the naphthalene-grown W1 cell suspension with a turbidity at 660 nm of 0.1, respectively. Andrews model as a substrate inhibited model was used to describe the kinetics of degradation and the equation was as followed (Haldane 1930):
q¼
Sqmax 2
S þ K s þ SK
i
where q is the specific degradation rate (h1); qmax, the maximum specific degradation rate (h1); Ks, the affinity constant (mg L1); Ki, the inhibition constant (mg L1). Estimates of Ks and Ki were made using nonlinear regression with GraphPad Prism 5 software. The degradation of CA, DBF and DBT (0.25 mM) by naphthaleneand LB medium-grown W1 cell suspension with a turbidity at 660 nm of 2.5 (0.65 g dry cell weight L1) was investigated. Studies on CA, DBF and DBT (0.25–1.5 mM) degradation at different time by naphthalene-grown W1 cell suspension with a turbidity at 660 nm of 2.5 were also carried out. In addition to the test flasks, two types of controls were prepared as previous report (Li et al., 2009). Samples were taken at intervals to monitor the concentrations of naphthalene, CA, DBF, DBT and biomass as described below. 2.4. Coking wastewater treatment by naphthalene-grown W1
2.2. Bacterial strain and cultivation conditions Arthrobacter sp. W1 (Wang et al., 2009) has been deposited as a bacterium in China General Microorganism Culture Center with the accession number CGMCC 4376. Strain W1 was routinely grown in mineral salt medium (MSM) as previously described with a minor modification, which consisted of Na2HPO4 1.3 g L1, KH2PO4 2 g L1, (NH4)2SO4 2 g L1, FeCl3 0.25 mg L1 and naphthalene 1 mM or LB medium. CA, DBF and DBT were dissolved in dimethyl sulfoxide (100 mM) and added to the medium at a suitable concentration. All cultures or cell suspensions were incubated at 30 °C on a reciprocal shaker at 150 rpm. 2.3. Cometabolic degradation of CA, DBF and DBT by strain W1 Cell suspensions of naphthalene- and LB medium-grown W1 were prepared separately by centrifugating the cultures in late exponential phase at 10,000 g for 5 min, washing cell pallets twice with MSM, and resuspending cells in MSM to get different turbidities. The growth of strain W1 was tested with naphthalene (1 mM), naphthalene (1 mM) plus CA (0.2 mM), DBF (0.2 mM) and DBT (0.2 mM), and CA (0.2 mM), DBF (0.2 mM) and DBT (0.2 mM) using the naphthalene-grown W1 cell suspension with a turbidity at 660 nm of 0.1, respectively. The cell growth in the culture without substrate was detected as a control. Inhibition kinetic experiments were operated as previously described (Shi et al., 2013). Inhibition kinetic experiment on naphthalene was carried out with initial concentration of naphthalene (25–450 mg L1) using the naphthalene-grown W1 cell suspension with a turbidity at 660 nm of 0.1. The inhibition kinetic experiment on naphthalene (plus CA, DBF,
Raw coking wastewater was obtained from the adjusting tank of the coking wastewater treatment plant in Shanxi province of China. The wastewater quality was as follows: COD 1700 mg L1, phenol 200 mg L1, naphthalene 58.9 mg L1, CA 12.5 mg L1, DBF 11.7 mg L1, DBT 10.8 mg L1 and NH3–N 86 mg L1. The raw wastewater was diluted 1:2 (v/v) in deionized water, and final concentrations of COD, phenol, naphthalene, CA, DBF, DBT and NH3–N were 2500 mg L1, 0.7, 0.5, 0.2, 0.2, 0.2 mM and 28 mg L1, respectively. Study on the degradation of this coking wastewater by naphthalene-grown W1 cell suspensions with turbidity at 660 nm of 2.5 was carried out. Activation zeolite as the ammonium exchangers was obtained from Zhengjiang Shengshi Mining Industry Co., Ltd. (China). Samples were taken at intervals to monitor the concentrations of phenol, naphthalene, CA, DBF, and DBT, and the acute toxicity of effluent and influent samples was also tested by Microtox bioassays as described below. 2.5. Analytical methods Phenol, naphthalene, CA, DBF, and DBT concentrations were analyzed using high-performance liquid chromatography (HPLC) system (Shimadzu LC20A; Thermo Hypersil ODS-2 column, 5 lm, 250 4.6 mm) after extraction by ethyl acetate. Analytical HPLC was carried out using instruments as described by Shi et al. (2013). The COD was analyzed according to the standard method for Water and Wastewater Examination. NH3–N concentration was obtained via a Nessler’s reaction using UV/Vis spectrophotometer. The acute toxicity of effluent and influent samples was assessed by Microtox bioassays using the luminescent bacteria Vibrio fischeri (NRRL B-11177). V. fischeri suspension at 30 min
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S. Shi et al. / Bioresource Technology 164 (2014) 28–33
exposure was determined by Berthold LB960, which was used to calculate the inhibition ratio of luminescence (IR) by:
IR ð%Þ ¼
S0 C f St S0 C f
where S0 and St were the luminescence intensities of the sample at 0 and t min, respectively. Cf was the relative luminescence intensity variations of negative sample at 0–t min. All of the experiments were performed in triplicates and the average values were used in calculations. 3. Results and discussion 3.1. Cometabolic degradation of CA, DBF and DBT using naphthalene as a carbon source The growth of strain W1 on naphthalene, naphthalene plus CA, DBF and DBT, and CA, DBF and DBT is shown in Fig. 1, which indicated that strain W1 could cometabolically degrade CA, DBF and DBT simultaneously using naphthalene as the primary substrate. Few data concerning the degradation of CA, DBF, DBT and
naphthalene simultaneous by growing cells have been described. Previous study only reported that CA-growing cells could cometabolically degrade CA, DBF and DBT simultaneously (Gai et al. 2007). When naphthalene served as the sole carbon source, within 16 h, the culture turbidity at 660 nm increased from 0.1 to its maximum 0.55, and the concentration of naphthalene decreased from 1 mM to below the detection limit. With additional 0.2 mM CA, 0.2 mM DBF and 0.2 mM DBT in the culture, the rates of cell growth and naphthalene consumption, and the maximum culture turbidity decreased, which might be due to the inhibition of CA, DBF and DBT or their metabolites on the cell growth and its biodegradability. After 40 h incubation, the concentration of naphthalene, CA, and DBF decreased from 1.0 mM, 0.2 mM, and 0.2 mM to below the detection limit. Likewise, the concentration of DBT decreased from 0.2 mM to 0.037 mM. However, strain W1 cannot use CA, DBF and DBT as the sole carbon source and no significant decrease of CA, DBF and DBT is detected as shown in Fig. 1B. It might indicate that naphthalene was the real inducer of expression the degradation enzymes and metabolic pathway might be controlled by naphthalene-degrading enzymes, while CA, DBF or DBT was not. Similar regulation of degradation enzyme expression has been previously reported for DBF and DBT cometabolic degradation by naphthalene-, fluorene-, CA- and biphenyl-utilizing strains (Gai et al. 2007; Mohammadi and Sylvestre, 2005; Seeger et al. 2001). 3.2. Inhibition kinetics of the cometabolic degradation of CA, DBF and DBT In order to further elucidate the inhibition of CA, DBF and DBT on the growth and biodegradability of strain W1, inhibition kinetics was determined by incubating strain W1 with naphthalene or naphthalene plus CA, DBF and DBT at various concentrations as described in Methods. A specific growth rate versus substrate concentration plot (q–S plot) is constructed in Fig. S1 and inhibition kinetic parameters are shown in Table 1, which indicated that all q–S plots showed a good fit to Andrews model. The qmax and Ki of strain W1 on naphthalene was 1.18- and 1.04-fold higher than that of naphthalene (plus CA, DBF and DBT), respectively. Meantime, Ks of strain W1 on naphthalene was 0.67-fold lower than that of naphthalene (plus CA, DBF and DBT). It was reasonable to confirm that the presence of CA, DBF and DBT inhibited the growth and biodegradability of strain W1, which could decrease the qmax and Ki of strain W1 on naphthalene, but increase the Ks value, probably due to the toxic properties of CA, DBF and DBT and their metabolites. Such a conclusion was also reported in our previous study, which showed that the additional DBF in naphthalene degrading system by Comamonas sp. MQ could decrease the qmax and Ki of strain MQ on naphthalene, but increase the Ks value (Shi et al., 2013). Compared to DBF and DBT, qmax of strain W1 on CA (0.0192 h1) was 1.04- and 1.2-fold higher than that of strain W1 on DBF and DBT. Inhibition kinetic parameters in Table 1 are also important to prevent inhibition for future continues cultivation and application. 3.3. Degradation of CA, DBF and DBT by naphthalene-grown W1
Fig. 1. Growth of strain W1 on naphthalene (A), naphthalene plus CA, DBF and DBT, and CA, DBF and DBT (B). A: Turbidities of cultures with naphthalene (j), and concentration of naphthalene (N); B: Turbidities of cultures with naphthalene plus CA, DBF and DBT (j), and concentration of naphthalene (N), CA (), DBF (.) and DBT (d); turbidities of cultures with CA, DBF and DBT (h), and concentration of CA (}), DBF (5) and DBT (s).
The degradation of CA, DBF and DBT (0.25 mM) by naphthaleneand LB medium-grown W1 is shown in Fig. 2. CA, DBF and DBT were not degraded by LB medium-grown W1, which seemed to be a common phenomenon in the case of DBF degradation by biphenyl-utilizing strains (Li et al. 2009). This may be due to the so-called catabolic repression frequently occurring during microbial degradation of xenobiotics that are not preferred growth substrates. When CA, DBF, and DBT were added as a mixture, the degradation of CA, DBF, and DBT began simultaneously and quickly by naphthalene-grown W1. Compared with the biphenyl-grown
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S. Shi et al. / Bioresource Technology 164 (2014) 28–33 Table 1 Inhibition kinetic parameters of strain W1 on substrates. Substrate
qmax (h Naphthalene Naphthalene (plus CA, DBF and DBT) CA (plus naphthalene, DBF and DBT) DBF (plus naphthalene, CA and DBT) DBT (plus naphthalene, CA and DBF)
R2
Inhibition kinetic parameters
0.0554 0.0471 0.0192 0.0184 0.0160
1
)
1
Std. error qmax (h
)
0.0593 0.03428 0.0026 0.0037 0.0017
1
Ks (mg L 125.4 85.41 13.34 11.6 10.55
)
Std. error Ks (mg L 128.6 88.09 4.111 3.891 3.004
1
)
Ki (mg L 73.6 70.64 88.6 77.5 109.3
1
)
Std. error Ki (mg L 74.71 67.30 20.9 19.8 22.57
1
) 0.8878 08676 0.9731 0.9676 0.9745
Fig. 2. Degradation of CA (.), DBF () and DBT (J) by naphthalene-grown W1 cells, and degradation of CA (N), DBF (j) and DBT (d) by LB medium-grown cells.
cell suspension of Ralstonia sp. SBUG 290 (turbidity at 600 nm of 5) and naphthalene-grown cell suspension of strain MQ (turbidity at 660 nm of 2.5) those cometabolic degradation DBF at a rate up to 0.0029 mM1 h1 and 0.08 mM1 h1, the naphthalene-grown W1 cell suspension (turbidity at 660 nm of 2.5) degraded DBF at a higher rate of 0.1 mM1 h1. Likewise, naphthalene-grown W1 cell suspension (turbidity at 660 nm of 2.5) degraded CA and DBT also at a high rate of 0.115 and 0.085 mM1 h1.
3.4. The effects of time and CA, DBF and DBT concentration on the cometabolic degradation To further study the cometabolic degradation of CA, DBF, and DBT, the effects of CA, DBF, and DBT concentrations on the cometabolic degradation at a function time were carried out. Naphthalene-grown W1 cells can degrade CA, DBF, and DBF simultaneously and quickly with the concentrations of them increasing from 0.25 to 1.5 mM in Fig. 3. As shown in Fig. 3, when naphthalene-grown W1 cell suspensions incubated CA, DBF and DBT (0.25–1.5 mM), the concentrations of CA, DBF and DBT decreased at a linear rate within 150 min. With time passed on, the concentrations of CA, DBF and DBT were stable and not decreased (180–240 min). In the sequencing batch tests, the plot of ln(C/C0) against time showed a straight line, which was consisted well with the Firstorder kinetics. The degradation kinetics parameters of cometabolic degradation CA, DBF and DBT are calculated in Table 2. As shown in Table 2, kinetics constant (k) of degradation reactions was coupling with the concentration of CA, DBF and DBT, when the concentration of CA, DBF and DBT increased from 0.25 mM to 0.5 mM, the value of k increased, indicating that with the increasing concentration of CA, DBF and DBT, the degradation rate increased.
Fig. 3. The effects of time and the concentrations of CA (A), DBF (B) and DBT (C) on the degradation of CA, DBF and DBT by naphthalene-grown W1. (j) 0.25 mM, (d) 0.50 mM, (N) 0.75 mM, (.) 1.00 mM, () 1.25 mM, (J) 1.5 mM.
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S. Shi et al. / Bioresource Technology 164 (2014) 28–33
Table 2 The kinetic parameters of CA, DBF and DBT degradation by First-order kinetics. Concentration (mM)
0.25 0.50 0.75 1.00 1.25 1.50
CA
DBF 2
Equation
k
R
lnc = 1.2398 0.0157t lnc = 0.4767 0.0161t lnc = 0.1330 0.0120t lnc = 0.1703 0.0092t lnc = 0.2551 0.0059t lnc = 0.4658 0.0049t
0.0157 0.0161 0.0120 0.0092 0.0059 0.0049
0.9645 0.9546 0.9620 0.9515 0.9943 0.9815
DBT
Equation
k
R
Equation
k
R2
lnc = 1.2558 0.0138t lnc = 0.5174 0.0145t lnc = 0.7143 0.0083t lnc = 0.1089 0.0069t lnc = 0.2646 0.0056t lnc = 0.1668 0.0047t
0.0138 0.0145 0.0083 0.0069 0.0056 0.0047
0.9524 0.9489 0.9540 0.9701 0.9903 0.9769
lnc = 1.2575 0.0101t lnc = 0.4936 0.0128t lnc = 0.1447 0.0109t lnc = 0.1312 0.0079t lnc = 0.2643 0.0057t lnc = 0.5399 0.0051t
0.0101 0.0128 0.0109 0.0079 0.0057 0.0051
0.9283 0.9446 0.9643 0.9619 0.9926 0.9898
Nevertheless, with the concentration of CA, DBF and DBT further increasing (0.75–1.5 mM), the value of k decreased, which suggested the high concentration of CA, DBF and DBT could inhibit the degradation rate. The relationships between specific degradation rate and initial CA, DBF and DBT concentration are also described well with Michaelis–Menten kinetics by GraphPad Prism 5 software in Fig. S2. The kinetic constants estimated from the experiment data are shown in Table 3. Naphthalene-grown W1 cells exhibited highest Vmax (3.435 mmol g cell1 h1) on the degradation of CA was of 1.1- and 1.11-fold higher that on DBF and DBT. The Vmax/Km ratio is also shown in Table 3, which suggested that strain W1 possessed higher efficiency for CA (5.86 L g cell1 h1) and DBF (5.82 L g cell1 h1) degradation than that for DBT (5.14 L g cell1 h1) degradation. 3.5. Coking wastewater treatment by naphthalene-grown W1 As is well known, coking wastewater is generated from coal coking, coal gas purification, and by-product recovery processes of coking, which contains considerable amounts of phenols, naphthalenes, polycyclic nitrogen-containing aromatics, oxygen- and sulfur-containing heterocyclic and inorganic pollutants (Wang et al., 2012; Fang et al., 2013; Zhu et al., 2009). In this study, the raw wastewater from Shanxi province of China with diluting 1:2 (v/v) was used, and CA, DBF, DBT and naphthalene was added to give prominence to bioremediation. Nevertheless, phenol and NH3–N were always the major organic compounds and inorganic pollutants in the coking wastewater, and 200 mg L1 phenol and 86 mg L1 NH3–N were detected in this coking wastewater. When the coking wastewater is incubated with naphthalene-grown W1, the concentration of phenol decreases from 0.8 mM to below the detection limit at 90 min in Fig. S3. Our previous study indicated that the isolated strain W1 could use phenol as a growth substrate (Wang et al. 2009). After 210 min of incubation, the concentration of naphthalene and CA decreased from 0.5 mM and 0.2 mM to under detection limit. Likewise, the concentration of DBF and DBT both gradually decreased from 0.2 mM to 0.005 mM after 300 min incubation. Despite the negative impact of phenol, naphthalene, CA, DBF, and DBT, the removal of COD was high effective, and final efficiency of COD was 95% at 300 min incubation. Because the ion-exchange adsorption of activation zeolite, the NH3–N removal was quickly, the removal efficiency was 90% at 180 min (Markou et al., 2014; Wang et al., 2011; Krishnani et al., 2012). Table 3 The kinetic parameters of CA, DBF and DBT degradation by Michaelis–Menten kinetics. Substrate
Vmax (mmol g cell1 h1)
Km (mM)
Vmax/Km (L g cell1 h1)
R2
CA DBF DBT
3.435 3.131 3.102
0.5860 0.5381 0.6031
5.86 5.82 5.14
0.9260 0.9106 0.9035
2
With the incubation time increasing further, the NH3–N removal efficiency reached a stable, might be due to the saturated adsorption ability to NH3–N by activation zeolite. It could be concluded that strain W1 that have a wide substrate range and various types of oxidation abilities coupling with activation zeolite should be a great advantage for bioremediation of the real coking wastewater. 3.6. Toxicity assessment (Microtox test) Ecotoxicology assessment of the treated effluent was performed to evaluate whether the transformation products formed from the target contaminants during treatment had higher toxicity than the parent compound (Lima et al., 2011; Cruz-Morató et al., 2013). In this study, eco-toxicity estimation was conducted by using the Microtox test (bacterium V. fischeri) to determine the change in effluent toxicity during the bioremediation of coking wastewater. The influent (0 min) contained high levels of phenol, naphthalene, CA, DBF, and DBT had highly toxicity against strain V. fischeri as indicated by IR value that exceeded 96%. During the bioremediation process, the IR value of effluent was sharply decreased from 96% (highly toxicity) to 40% (moderate toxicity) within 120 min. From 150 to 210 min, the IR value of effluent was gradually decreased from 40% to 27%. With time increased further at 240 min, the IR value of effluent was slightly decreased to 20% (low toxicity). This may be due to degradation of both the tested pollutants (phenol, naphthalene, CA, DBF, and DBT) and other pollutants in the coking wastewater. It indicated that the bioremediation of the real coking wastewater containing phenol, naphthalene, CA, DBF, and DBT by strain W1 coupling with activation zeolite led to less toxicity than the untreated coking wastewater. 4. Conclusions Arthrobacter sp. W1 could cometabolically degrade CA, DBF, and DBT by naphthalene as the primary substrate. The degradation kinetics could be described by both First-order kinetics and Michaelis–Menten equations. Naphthalene-grown W1 coupling with activation zeolite exhibited high removal efficiency for coking wastewater containing high concentration of phenol, naphthalene, CA, DBF, and DBT. Low toxicity has been detected in the effluent of coking wastewater treatment by naphthalene-grown W1 coupling with activation zeolite. According to these findings, Arthrobacter sp. W1 might be an efficient candidate for the bioremediation of the coking wastewater. Acknowledgements The authors gratefully acknowledge the financial supports from the National Natural Science Foundation of China (Nos. 51078054, 21176040, 51108120, and 51178139), and the National Creative Research Group from the National Natural Science Foundation of China (No. 51121062).
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cultivated Pseudomonas putida strain B6–2. Environ. Sci. Technol. 43, 8635– 8642. Lim, B.R., Hu, H.Y., Fujie, K., 2003. Biological degradation and chemical oxidation characteristics of coke-oven wastewater. Water Air Soil Pollut. 146, 23–33. Lima, T.M., Procópio, L.C., Brandão, F.D., Leão, B.A., Tótola, M.R., Borges, A.C., 2011. Evaluation of bacterial surfactant toxicity towards petroleum degrading microorganisms. Bioresour. Technol. 102, 2957–2964. Markou, G., Vandamme, D., Muylaert, K., 2014. Using natural zeolite for ammonia sorption from wastewater and as nitrogen releaser for the cultivation of Arthrospira platensis. Bioresour. Technol. 7, 373–378. Mohammadi, M., Sylvestre, M., 2005. Resolving the profile of metabolites generated during oxidation of dibenzofuran and chlorodibenzofurans by the biphenyl catabolic pathway enzymes. Chem. Biol. 12, 835–846. Monsalvo, V.M., Mohedano, A.F., Casas, J.A., Rodríguez, J.J., 2009. Cometabolic biodegradation of 4-chlorophenol by sequencing batch reactors at different temperatures. Bioresour. Technol. 100, 4572–4578. Peng, X., Jia, X., 2013. Optimization of parameters for anaerobic co-metabolic degradation of TBBPA. Bioresour. Technol. 148, 386–393. Seeger, M., Camara, B., Hofer, B., 2001. Dehalogenation, denitration, dehydroxylation, and angular attack on substituted biphenyls and related compounds by a biphenyl dioxygenase. J. Bacterial. 183, 3548–3555. Seo, J.S., Keum, Y.S., Cho, I.K., Li, Q.X., 2006. Degradation of dibenzothiophene and carbazole by Arthrobacter sp. P1-1. Int. Biodeter. Biodegr. 58, 36–43. Sharma, N.K., Philip, L., Murty Bhallamudi, S., 2012. Aerobic degradation of phenolics and aromatic hydrocarbons in presence of cyanide. Bioresour. Technol. 121, 263–273. Shi, S.N., Zhang, X.W., Ma, F., Sun, T.H., Li, A., Zhou, J.T., Qu, Y.Y., 2013. Cometabolic degradation of dibenzofuran by Comamonas sp. MQ. Process Biochem. 48, 1553–1558. Wang, P., Qu, Y.Y., Zhou, J.T., 2009. Biodegradation of mixed phenolic compounds under high salt conditions and salinity fluctuations by Arthrobacter sp. W1. Appl. Biochem. Biotechnol. 159, 623–633. Wang, Q., Yang, Y., Yu, C., Huang, H., Kim, M., Feng, C., Zhang, Z., 2011. Study on a fixed zeolite bioreactor for anaerobic digestion of ammonium-rich swine wastes. Bioresour. Technol. 102, 7064–7068. Wang, F., Hu, Y., Guo, C., Huang, W., Liu, C.Z., 2012. Enhanced phenol degradation in coking wastewater by immobilized laccase on magnetic mesoporous silica nanoparticles in a magnetically stabilized fluidized bed. Bioresour. Technol. 110, 120–124. Zhang, M., Tay, J.H., Qian, Y., Gu, X.S., 1998. Coke plant wastewater treatment by fixed biofilm system for COD and NH3–N removal. Water Res. 32, 519–527. Zhang, J., Zhang, X., Liu, J., Li, R., Shen, B., 2012. Isolation of a thermophilic bacterium, Geobacillus sp. SH-1, capable of degrading aliphatic hydrocarbons and naphthalene simultaneously, and identification of its naphthalene degrading pathway. Bioresour. Technol. 124, 83–89. Zhu, X.P., Ni, J.R., Lai, P., 2009. Advanced treatment of biologically pretreated coking wastewater by electrochemical oxidation using boron-doped diamond electrodes. Water Res. 43, 4347–4355.
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Bioremediation of coking wastewater containing carbazole, dibenzofuran, dibenzothiophene and naphthalene by a naphthalene-cultivated Arthrobacter sp. W1 Shengnan Shi a, Yuanyuan Qu b, Fang Ma a,⇑, Jiti Zhou b a
State Key Laboratory of Urban Water Resource and Environment, School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, China Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China b
h i g h l i g h t s Cometabolic degradation CA, DBF and DBT simultaneous by naphthalene-grown W1. Growing and washed cells of strain W1 degraded CA, DBF, DBT and naphthalene quickly. The degradation kinetics was described well by First-order and Michaelis–Menten kinetics. Coking wastewater with naphthalene, CA, DBF and DBT was rapidly degraded by strain W1 with zeolite. Low toxicity has been found in the effluent of coking wastewater by strain W1 with zeolite.
a r t i c l e
i n f o
Article history: Received 10 March 2014 Received in revised form 3 April 2014 Accepted 4 April 2014 Available online 15 April 2014 Keywords: Cometabolic degradation Arthrobacter sp. W1 Naphthalene Heterocyclic compound Coking wastewater
a b s t r a c t A naphthalene-utilizing bacterium, Arthrobacter sp. W1, was used to investigate the cometabolic degradation of carbazole (CA), dibenzofuran (DBF) and dibenzothiophene (DBT) using naphthalene as the primary substrate. Both the growing and washed cells of strain W1 could degrade CA, DBF, DBT, and naphthalene simultaneously and quickly. Inhibition kinetics confirmed that the presence of CA, DBF and DBT in the growing system would inhibit the cells growth and biodegradability of strain W1. The relationship between ln(C/C0) and time, and specific degradation rate and CA, DBF and DBT concentration could be described well by First-order and Michaelis–Menten kinetics. The treatment of real coking wastewater containing high concentration of phenol, naphthalene, CA, DBF, DBT and NH3-N was shown to be highly efficient by naphthalene-grown W1 coupling with activation zeolite. Toxicity assessment indicated the treatment of the coking wastewater by strain W1 coupling with activation led to less toxicity than untreated wastewater. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Carbazole (CA), dibenzofuran (DBF), and dibenzothiophene (DBT) are recognized as the predominant nitrogen-, oxygen-, and sulfur-heterocyclic compound and have often been found in coking wastewater (Lim et al., 2003; Zhang et al., 1998; Zhu et al., 2009). All of these heterocyclic compounds are known to possess toxic and mutagenic activities, and considered as the refractory organic compounds in the treatment of coking wastewater (Zhu et al., 2009). It is well known that microorganisms play a primary role in the degradation of these persistent organic compounds (Seo ⇑ Corresponding author. Tel.: +86 451 8628 2107. E-mail address: [email protected] (F. Ma). http://dx.doi.org/10.1016/j.biortech.2014.04.010 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
et al., 2006). Various bacteria utilized CA, DBF, and DBT as the sole carbon and energy have been isolated, respectively (Gai et al., 2007; Das et al., 2012; Zhang et al., 2012; Balachandran et al., 2012). Because polycyclic aromatic compounds (PACs) including CA, DBF, and DBT are widespread and usually coexist in the polluted environments, cometabolic degradation of these heterocyclic compounds by bacteria growing with some others PACs should be common (Li et al., 2009). Cometabolic bioremediation is probably the promising bioremediation strategy currently available for the degrading bacteria with various types of metabolism abilities, which has been used in the field for more than 20 years on some of the most recalcitrant contaminants, e.g., phenolic compounds, xenobiotic trichloroethylene (TCE), tetrabromobisphenol A (TBBPA), and polychlorinated
S. Shi et al. / Bioresource Technology 164 (2014) 28–33
biphenyls (PCBs) (Monsalvo et al. 2009; Aktasß 2012; Kocamemi and Ceçen, 2010; Peng and Jia, 2013; Lambo and Patel, 2006; Kim et al., 2011; Sharma et al., 2012). Recently, there have been many studies on the cometabolic degradation of CA, DBF, and DBT (each or two of them) by naphthalene-, fluorene-, biphenyl-, and phenanthreneutilizing strains (Seo et al., 2006; Grifoll et al., 1995; Gai et al., 2007; Li et al., 2009). Nevertheless, current studies are mostly focused on the pathways of such chemical metabolisms and cometabolic degradation pathways of these heterocyclic compounds are similar, in which heterocyclic compound is initially attacked at the lateral position by dioxygenase to yield a chemically unstable intermediate that is spontaneously dehydrogenated by dihydrodiol dehydrogenase, and then dioxygenolytically cleaved by extradiol dioxygenase to produce meta-cleavage product (Gai et al., 2007; Li et al., 2009). However, little is known about the cometabolic degradation of CA, DBF, and DBT simultaneous by naphthaleneutilizing strains, and even about the removal of CA, DBF, DBT, and naphthalene in the bioremediation of coking wastewater. In the present study, we characterized the versatile oxidation activities of a naphthalene-utilizing Arthrobacter sp. W1 and described the cometabolic degradation of CA, DBF, and DBT by this strain. The bioremediation of real coking wastewater containing CA, DBF, DBT, phenol, naphthalene and NH3-N was also studied by naphthalene-grown W1 coupling with activation zeolite, and ecotoxicological assessment of the treated effluent was carried out. 2. Methods 2.1. Chemicals CA, DBF, DBT, and naphthalene were purchased from J&K Scientific Ltd. (China). All other commercially available chemicals were of analytical grade.
29
and DBT) was tested with initial concentration of naphthalene (25–450 mg L1) plus 40 mg L1 CA, 40 mg L1 DBF, and 40 mg L1 DBT, on CA (plus naphthalene, DBF, and DBT) was tested with initial concentration of CA (12.5–225 mg L1) plus 100 mg L1 naphthalene, 40 mg L1 DBF, and 40 mg L1 DBT, on DBF (plus naphthalene, CA, and DBT) was tested with initial concentration of DBF (12.5–225 mg L1) plus 100 mg L1 naphthalene, 40 mg L1 CA, and 40 mg L1 DBT, on DBT (plus naphthalene, CA, and DBF) was tested with initial concentration of DBT (12.5–225 mg L1) plus 100 mg L1 naphthalene, 40 mg L1 CA, and 40 mg L1 DBF using the naphthalene-grown W1 cell suspension with a turbidity at 660 nm of 0.1, respectively. Andrews model as a substrate inhibited model was used to describe the kinetics of degradation and the equation was as followed (Haldane 1930):
q¼
Sqmax 2
S þ K s þ SK
i
where q is the specific degradation rate (h1); qmax, the maximum specific degradation rate (h1); Ks, the affinity constant (mg L1); Ki, the inhibition constant (mg L1). Estimates of Ks and Ki were made using nonlinear regression with GraphPad Prism 5 software. The degradation of CA, DBF and DBT (0.25 mM) by naphthaleneand LB medium-grown W1 cell suspension with a turbidity at 660 nm of 2.5 (0.65 g dry cell weight L1) was investigated. Studies on CA, DBF and DBT (0.25–1.5 mM) degradation at different time by naphthalene-grown W1 cell suspension with a turbidity at 660 nm of 2.5 were also carried out. In addition to the test flasks, two types of controls were prepared as previous report (Li et al., 2009). Samples were taken at intervals to monitor the concentrations of naphthalene, CA, DBF, DBT and biomass as described below. 2.4. Coking wastewater treatment by naphthalene-grown W1
2.2. Bacterial strain and cultivation conditions Arthrobacter sp. W1 (Wang et al., 2009) has been deposited as a bacterium in China General Microorganism Culture Center with the accession number CGMCC 4376. Strain W1 was routinely grown in mineral salt medium (MSM) as previously described with a minor modification, which consisted of Na2HPO4 1.3 g L1, KH2PO4 2 g L1, (NH4)2SO4 2 g L1, FeCl3 0.25 mg L1 and naphthalene 1 mM or LB medium. CA, DBF and DBT were dissolved in dimethyl sulfoxide (100 mM) and added to the medium at a suitable concentration. All cultures or cell suspensions were incubated at 30 °C on a reciprocal shaker at 150 rpm. 2.3. Cometabolic degradation of CA, DBF and DBT by strain W1 Cell suspensions of naphthalene- and LB medium-grown W1 were prepared separately by centrifugating the cultures in late exponential phase at 10,000 g for 5 min, washing cell pallets twice with MSM, and resuspending cells in MSM to get different turbidities. The growth of strain W1 was tested with naphthalene (1 mM), naphthalene (1 mM) plus CA (0.2 mM), DBF (0.2 mM) and DBT (0.2 mM), and CA (0.2 mM), DBF (0.2 mM) and DBT (0.2 mM) using the naphthalene-grown W1 cell suspension with a turbidity at 660 nm of 0.1, respectively. The cell growth in the culture without substrate was detected as a control. Inhibition kinetic experiments were operated as previously described (Shi et al., 2013). Inhibition kinetic experiment on naphthalene was carried out with initial concentration of naphthalene (25–450 mg L1) using the naphthalene-grown W1 cell suspension with a turbidity at 660 nm of 0.1. The inhibition kinetic experiment on naphthalene (plus CA, DBF,
Raw coking wastewater was obtained from the adjusting tank of the coking wastewater treatment plant in Shanxi province of China. The wastewater quality was as follows: COD 1700 mg L1, phenol 200 mg L1, naphthalene 58.9 mg L1, CA 12.5 mg L1, DBF 11.7 mg L1, DBT 10.8 mg L1 and NH3–N 86 mg L1. The raw wastewater was diluted 1:2 (v/v) in deionized water, and final concentrations of COD, phenol, naphthalene, CA, DBF, DBT and NH3–N were 2500 mg L1, 0.7, 0.5, 0.2, 0.2, 0.2 mM and 28 mg L1, respectively. Study on the degradation of this coking wastewater by naphthalene-grown W1 cell suspensions with turbidity at 660 nm of 2.5 was carried out. Activation zeolite as the ammonium exchangers was obtained from Zhengjiang Shengshi Mining Industry Co., Ltd. (China). Samples were taken at intervals to monitor the concentrations of phenol, naphthalene, CA, DBF, and DBT, and the acute toxicity of effluent and influent samples was also tested by Microtox bioassays as described below. 2.5. Analytical methods Phenol, naphthalene, CA, DBF, and DBT concentrations were analyzed using high-performance liquid chromatography (HPLC) system (Shimadzu LC20A; Thermo Hypersil ODS-2 column, 5 lm, 250 4.6 mm) after extraction by ethyl acetate. Analytical HPLC was carried out using instruments as described by Shi et al. (2013). The COD was analyzed according to the standard method for Water and Wastewater Examination. NH3–N concentration was obtained via a Nessler’s reaction using UV/Vis spectrophotometer. The acute toxicity of effluent and influent samples was assessed by Microtox bioassays using the luminescent bacteria Vibrio fischeri (NRRL B-11177). V. fischeri suspension at 30 min
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S. Shi et al. / Bioresource Technology 164 (2014) 28–33
exposure was determined by Berthold LB960, which was used to calculate the inhibition ratio of luminescence (IR) by:
IR ð%Þ ¼
S0 C f St S0 C f
where S0 and St were the luminescence intensities of the sample at 0 and t min, respectively. Cf was the relative luminescence intensity variations of negative sample at 0–t min. All of the experiments were performed in triplicates and the average values were used in calculations. 3. Results and discussion 3.1. Cometabolic degradation of CA, DBF and DBT using naphthalene as a carbon source The growth of strain W1 on naphthalene, naphthalene plus CA, DBF and DBT, and CA, DBF and DBT is shown in Fig. 1, which indicated that strain W1 could cometabolically degrade CA, DBF and DBT simultaneously using naphthalene as the primary substrate. Few data concerning the degradation of CA, DBF, DBT and
naphthalene simultaneous by growing cells have been described. Previous study only reported that CA-growing cells could cometabolically degrade CA, DBF and DBT simultaneously (Gai et al. 2007). When naphthalene served as the sole carbon source, within 16 h, the culture turbidity at 660 nm increased from 0.1 to its maximum 0.55, and the concentration of naphthalene decreased from 1 mM to below the detection limit. With additional 0.2 mM CA, 0.2 mM DBF and 0.2 mM DBT in the culture, the rates of cell growth and naphthalene consumption, and the maximum culture turbidity decreased, which might be due to the inhibition of CA, DBF and DBT or their metabolites on the cell growth and its biodegradability. After 40 h incubation, the concentration of naphthalene, CA, and DBF decreased from 1.0 mM, 0.2 mM, and 0.2 mM to below the detection limit. Likewise, the concentration of DBT decreased from 0.2 mM to 0.037 mM. However, strain W1 cannot use CA, DBF and DBT as the sole carbon source and no significant decrease of CA, DBF and DBT is detected as shown in Fig. 1B. It might indicate that naphthalene was the real inducer of expression the degradation enzymes and metabolic pathway might be controlled by naphthalene-degrading enzymes, while CA, DBF or DBT was not. Similar regulation of degradation enzyme expression has been previously reported for DBF and DBT cometabolic degradation by naphthalene-, fluorene-, CA- and biphenyl-utilizing strains (Gai et al. 2007; Mohammadi and Sylvestre, 2005; Seeger et al. 2001). 3.2. Inhibition kinetics of the cometabolic degradation of CA, DBF and DBT In order to further elucidate the inhibition of CA, DBF and DBT on the growth and biodegradability of strain W1, inhibition kinetics was determined by incubating strain W1 with naphthalene or naphthalene plus CA, DBF and DBT at various concentrations as described in Methods. A specific growth rate versus substrate concentration plot (q–S plot) is constructed in Fig. S1 and inhibition kinetic parameters are shown in Table 1, which indicated that all q–S plots showed a good fit to Andrews model. The qmax and Ki of strain W1 on naphthalene was 1.18- and 1.04-fold higher than that of naphthalene (plus CA, DBF and DBT), respectively. Meantime, Ks of strain W1 on naphthalene was 0.67-fold lower than that of naphthalene (plus CA, DBF and DBT). It was reasonable to confirm that the presence of CA, DBF and DBT inhibited the growth and biodegradability of strain W1, which could decrease the qmax and Ki of strain W1 on naphthalene, but increase the Ks value, probably due to the toxic properties of CA, DBF and DBT and their metabolites. Such a conclusion was also reported in our previous study, which showed that the additional DBF in naphthalene degrading system by Comamonas sp. MQ could decrease the qmax and Ki of strain MQ on naphthalene, but increase the Ks value (Shi et al., 2013). Compared to DBF and DBT, qmax of strain W1 on CA (0.0192 h1) was 1.04- and 1.2-fold higher than that of strain W1 on DBF and DBT. Inhibition kinetic parameters in Table 1 are also important to prevent inhibition for future continues cultivation and application. 3.3. Degradation of CA, DBF and DBT by naphthalene-grown W1
Fig. 1. Growth of strain W1 on naphthalene (A), naphthalene plus CA, DBF and DBT, and CA, DBF and DBT (B). A: Turbidities of cultures with naphthalene (j), and concentration of naphthalene (N); B: Turbidities of cultures with naphthalene plus CA, DBF and DBT (j), and concentration of naphthalene (N), CA (), DBF (.) and DBT (d); turbidities of cultures with CA, DBF and DBT (h), and concentration of CA (}), DBF (5) and DBT (s).
The degradation of CA, DBF and DBT (0.25 mM) by naphthaleneand LB medium-grown W1 is shown in Fig. 2. CA, DBF and DBT were not degraded by LB medium-grown W1, which seemed to be a common phenomenon in the case of DBF degradation by biphenyl-utilizing strains (Li et al. 2009). This may be due to the so-called catabolic repression frequently occurring during microbial degradation of xenobiotics that are not preferred growth substrates. When CA, DBF, and DBT were added as a mixture, the degradation of CA, DBF, and DBT began simultaneously and quickly by naphthalene-grown W1. Compared with the biphenyl-grown
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S. Shi et al. / Bioresource Technology 164 (2014) 28–33 Table 1 Inhibition kinetic parameters of strain W1 on substrates. Substrate
qmax (h Naphthalene Naphthalene (plus CA, DBF and DBT) CA (plus naphthalene, DBF and DBT) DBF (plus naphthalene, CA and DBT) DBT (plus naphthalene, CA and DBF)
R2
Inhibition kinetic parameters
0.0554 0.0471 0.0192 0.0184 0.0160
1
)
1
Std. error qmax (h
)
0.0593 0.03428 0.0026 0.0037 0.0017
1
Ks (mg L 125.4 85.41 13.34 11.6 10.55
)
Std. error Ks (mg L 128.6 88.09 4.111 3.891 3.004
1
)
Ki (mg L 73.6 70.64 88.6 77.5 109.3
1
)
Std. error Ki (mg L 74.71 67.30 20.9 19.8 22.57
1
) 0.8878 08676 0.9731 0.9676 0.9745
Fig. 2. Degradation of CA (.), DBF () and DBT (J) by naphthalene-grown W1 cells, and degradation of CA (N), DBF (j) and DBT (d) by LB medium-grown cells.
cell suspension of Ralstonia sp. SBUG 290 (turbidity at 600 nm of 5) and naphthalene-grown cell suspension of strain MQ (turbidity at 660 nm of 2.5) those cometabolic degradation DBF at a rate up to 0.0029 mM1 h1 and 0.08 mM1 h1, the naphthalene-grown W1 cell suspension (turbidity at 660 nm of 2.5) degraded DBF at a higher rate of 0.1 mM1 h1. Likewise, naphthalene-grown W1 cell suspension (turbidity at 660 nm of 2.5) degraded CA and DBT also at a high rate of 0.115 and 0.085 mM1 h1.
3.4. The effects of time and CA, DBF and DBT concentration on the cometabolic degradation To further study the cometabolic degradation of CA, DBF, and DBT, the effects of CA, DBF, and DBT concentrations on the cometabolic degradation at a function time were carried out. Naphthalene-grown W1 cells can degrade CA, DBF, and DBF simultaneously and quickly with the concentrations of them increasing from 0.25 to 1.5 mM in Fig. 3. As shown in Fig. 3, when naphthalene-grown W1 cell suspensions incubated CA, DBF and DBT (0.25–1.5 mM), the concentrations of CA, DBF and DBT decreased at a linear rate within 150 min. With time passed on, the concentrations of CA, DBF and DBT were stable and not decreased (180–240 min). In the sequencing batch tests, the plot of ln(C/C0) against time showed a straight line, which was consisted well with the Firstorder kinetics. The degradation kinetics parameters of cometabolic degradation CA, DBF and DBT are calculated in Table 2. As shown in Table 2, kinetics constant (k) of degradation reactions was coupling with the concentration of CA, DBF and DBT, when the concentration of CA, DBF and DBT increased from 0.25 mM to 0.5 mM, the value of k increased, indicating that with the increasing concentration of CA, DBF and DBT, the degradation rate increased.
Fig. 3. The effects of time and the concentrations of CA (A), DBF (B) and DBT (C) on the degradation of CA, DBF and DBT by naphthalene-grown W1. (j) 0.25 mM, (d) 0.50 mM, (N) 0.75 mM, (.) 1.00 mM, () 1.25 mM, (J) 1.5 mM.
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S. Shi et al. / Bioresource Technology 164 (2014) 28–33
Table 2 The kinetic parameters of CA, DBF and DBT degradation by First-order kinetics. Concentration (mM)
0.25 0.50 0.75 1.00 1.25 1.50
CA
DBF 2
Equation
k
R
lnc = 1.2398 0.0157t lnc = 0.4767 0.0161t lnc = 0.1330 0.0120t lnc = 0.1703 0.0092t lnc = 0.2551 0.0059t lnc = 0.4658 0.0049t
0.0157 0.0161 0.0120 0.0092 0.0059 0.0049
0.9645 0.9546 0.9620 0.9515 0.9943 0.9815
DBT
Equation
k
R
Equation
k
R2
lnc = 1.2558 0.0138t lnc = 0.5174 0.0145t lnc = 0.7143 0.0083t lnc = 0.1089 0.0069t lnc = 0.2646 0.0056t lnc = 0.1668 0.0047t
0.0138 0.0145 0.0083 0.0069 0.0056 0.0047
0.9524 0.9489 0.9540 0.9701 0.9903 0.9769
lnc = 1.2575 0.0101t lnc = 0.4936 0.0128t lnc = 0.1447 0.0109t lnc = 0.1312 0.0079t lnc = 0.2643 0.0057t lnc = 0.5399 0.0051t
0.0101 0.0128 0.0109 0.0079 0.0057 0.0051
0.9283 0.9446 0.9643 0.9619 0.9926 0.9898
Nevertheless, with the concentration of CA, DBF and DBT further increasing (0.75–1.5 mM), the value of k decreased, which suggested the high concentration of CA, DBF and DBT could inhibit the degradation rate. The relationships between specific degradation rate and initial CA, DBF and DBT concentration are also described well with Michaelis–Menten kinetics by GraphPad Prism 5 software in Fig. S2. The kinetic constants estimated from the experiment data are shown in Table 3. Naphthalene-grown W1 cells exhibited highest Vmax (3.435 mmol g cell1 h1) on the degradation of CA was of 1.1- and 1.11-fold higher that on DBF and DBT. The Vmax/Km ratio is also shown in Table 3, which suggested that strain W1 possessed higher efficiency for CA (5.86 L g cell1 h1) and DBF (5.82 L g cell1 h1) degradation than that for DBT (5.14 L g cell1 h1) degradation. 3.5. Coking wastewater treatment by naphthalene-grown W1 As is well known, coking wastewater is generated from coal coking, coal gas purification, and by-product recovery processes of coking, which contains considerable amounts of phenols, naphthalenes, polycyclic nitrogen-containing aromatics, oxygen- and sulfur-containing heterocyclic and inorganic pollutants (Wang et al., 2012; Fang et al., 2013; Zhu et al., 2009). In this study, the raw wastewater from Shanxi province of China with diluting 1:2 (v/v) was used, and CA, DBF, DBT and naphthalene was added to give prominence to bioremediation. Nevertheless, phenol and NH3–N were always the major organic compounds and inorganic pollutants in the coking wastewater, and 200 mg L1 phenol and 86 mg L1 NH3–N were detected in this coking wastewater. When the coking wastewater is incubated with naphthalene-grown W1, the concentration of phenol decreases from 0.8 mM to below the detection limit at 90 min in Fig. S3. Our previous study indicated that the isolated strain W1 could use phenol as a growth substrate (Wang et al. 2009). After 210 min of incubation, the concentration of naphthalene and CA decreased from 0.5 mM and 0.2 mM to under detection limit. Likewise, the concentration of DBF and DBT both gradually decreased from 0.2 mM to 0.005 mM after 300 min incubation. Despite the negative impact of phenol, naphthalene, CA, DBF, and DBT, the removal of COD was high effective, and final efficiency of COD was 95% at 300 min incubation. Because the ion-exchange adsorption of activation zeolite, the NH3–N removal was quickly, the removal efficiency was 90% at 180 min (Markou et al., 2014; Wang et al., 2011; Krishnani et al., 2012). Table 3 The kinetic parameters of CA, DBF and DBT degradation by Michaelis–Menten kinetics. Substrate
Vmax (mmol g cell1 h1)
Km (mM)
Vmax/Km (L g cell1 h1)
R2
CA DBF DBT
3.435 3.131 3.102
0.5860 0.5381 0.6031
5.86 5.82 5.14
0.9260 0.9106 0.9035
2
With the incubation time increasing further, the NH3–N removal efficiency reached a stable, might be due to the saturated adsorption ability to NH3–N by activation zeolite. It could be concluded that strain W1 that have a wide substrate range and various types of oxidation abilities coupling with activation zeolite should be a great advantage for bioremediation of the real coking wastewater. 3.6. Toxicity assessment (Microtox test) Ecotoxicology assessment of the treated effluent was performed to evaluate whether the transformation products formed from the target contaminants during treatment had higher toxicity than the parent compound (Lima et al., 2011; Cruz-Morató et al., 2013). In this study, eco-toxicity estimation was conducted by using the Microtox test (bacterium V. fischeri) to determine the change in effluent toxicity during the bioremediation of coking wastewater. The influent (0 min) contained high levels of phenol, naphthalene, CA, DBF, and DBT had highly toxicity against strain V. fischeri as indicated by IR value that exceeded 96%. During the bioremediation process, the IR value of effluent was sharply decreased from 96% (highly toxicity) to 40% (moderate toxicity) within 120 min. From 150 to 210 min, the IR value of effluent was gradually decreased from 40% to 27%. With time increased further at 240 min, the IR value of effluent was slightly decreased to 20% (low toxicity). This may be due to degradation of both the tested pollutants (phenol, naphthalene, CA, DBF, and DBT) and other pollutants in the coking wastewater. It indicated that the bioremediation of the real coking wastewater containing phenol, naphthalene, CA, DBF, and DBT by strain W1 coupling with activation zeolite led to less toxicity than the untreated coking wastewater. 4. Conclusions Arthrobacter sp. W1 could cometabolically degrade CA, DBF, and DBT by naphthalene as the primary substrate. The degradation kinetics could be described by both First-order kinetics and Michaelis–Menten equations. Naphthalene-grown W1 coupling with activation zeolite exhibited high removal efficiency for coking wastewater containing high concentration of phenol, naphthalene, CA, DBF, and DBT. Low toxicity has been detected in the effluent of coking wastewater treatment by naphthalene-grown W1 coupling with activation zeolite. According to these findings, Arthrobacter sp. W1 might be an efficient candidate for the bioremediation of the coking wastewater. Acknowledgements The authors gratefully acknowledge the financial supports from the National Natural Science Foundation of China (Nos. 51078054, 21176040, 51108120, and 51178139), and the National Creative Research Group from the National Natural Science Foundation of China (No. 51121062).
S. Shi et al. / Bioresource Technology 164 (2014) 28–33
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