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1H-1,2,4-Triazole biodegradation by newly isolated Raoultella sp.: A novel biodegradation pathway
Bioresource Technology Reports 6 (2019) 63–69
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1H-1,2,4-Triazole biodegradation by newly isolated Raoultella sp.: A novel biodegradation pathway
T
Xiaolin Liu1, Jing Wang1, Dan Chen, Xinbai Jiang, Haobo Wu, Cheng Hou, Dejin Zhang, ⁎ Xiaodong Liu, Lianjun Wang, Jinyou Shen Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse, School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, Jiangsu Province, China
A R T I C LE I N FO
A B S T R A C T
Keywords: 1H-1,2,4-Triazole Bioaugmentation Intermediates Metabolic pathway
In this study, a novel 1H-1,2,4-triazole (TZ) degrading strain was isolated from activated sludge acclimated by TZ and was identified as Raoultella sp. The biodegradation test showed that Raoultella sp. could utilize TZ as the sole carbon and nitrogen source. TZ was completely removed at incubation temperature of 30 °C, initial pH of 7.0 and initial concentration of 100 mg L−1 within 288 h, which was accompanied by obvious TOC removal, NH4+ release, pH increase, biomass growth, biotoxicity reduction and excitation emission matrix (EEM) variation. TZ biodegradation profile by Raoultella sp. was well fitted by the first-order degradation kinetic model, confirming the inhibitory nature of TZ. Based on HPLC/MS analysis, a distinct TZ biodegradation pathway including hydroxylation, carbonylation, carboxylation and ring cleavage, was proposed for the first time. Compared with other TZ degrading species such as Shinella sp., Raoultella sp. showed relatively higher TZ removal efficiency, demonstrating great potential for the application in biological treatment of wastewater containing TZ in practice.
1. Introduction As a typical nitrogenous heterocyclic compounds (NHCs), 1H-1,2,4triazole (TZ) is widely used for the synthesis of pesticides, herbicides and fungicides, causing substantial release of wastewater containing TZ (Wang et al., 2011; Wu et al., 2016). According to Zhu et al. (2018), the fate of various contaminants in the environment is closely related to hydrolysis, biodegradation and adsorption. Since TZ is not prone to hydrolysis, biodegradation or adsorption onto soil particles or other environmental media, the half-life of TZ in the environment would be rather long (Wu et al., 2019). Therefore, the exposure of highly toxic, carcinogenic and teratogenic TZ to surface water and groundwater would cause serious ecological problems (Wu et al., 2018a; Konwick et al., 2006). Thus, in order to avoid environmental deterioration caused by TZ, wastewater containing TZ should be treated properly before discharge (Wu et al., 2018a; Wu et al., 2019). Development of efficient and economical method for TZ removal from wastewater has become an urgent need. Various treatment technologies have been developed for TZ removal from contaminated wastewater, such as adsorption (Amorim et al.,
2013), electrochemical oxidation (Han et al., 2014) and photocatalytic mineralization (Watanabe et al., 2005), etc. These physico-chemical methods have been proven to be cost ineffective and energy intensive (Wang et al., 2018; Liang et al., 2019). In addition, toxic and harmful intermediates such as cyanuric acid were often found in the effluent from these physico-chemical treatment systems, causing serious secondary pollution (Watanabe et al., 2005). Nevertheless, biological treatment, which is both cost effective and environmental friendly, has turned out to be a promising alternative (Jiang et al., 2018; Singh et al., 2008). Although the application of bioaugmentation for the treatment of wastewater containing various recalcitrant contaminants is receiving more attention, the available strains with related function are currently very limited. In order to achieve efficient biodegradation of various recalcitrant pollutants such as TZ, the inoculation of specific functional microorganisms into the biological systems will be a favorable alternative (Wu et al., 2018b; Yun et al., 2017). In our previous study, a novel TZ-degrading strain namely Shinella sp. NJUST26 was isolated from TZ-contaminated soil for the first time (Wu et al., 2016). Unfortunately, due to the extremely high toxicity and rather poor biodegradability of TZ, specific functional microorganisms capable of
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Corresponding author. E-mail address: [email protected] (J. Shen). 1 These authors contributed to the paper equally. https://doi.org/10.1016/j.biteb.2019.02.007 Received 11 January 2019; Received in revised form 10 February 2019; Accepted 11 February 2019 Available online 13 February 2019 2589-014X/ © 2019 Elsevier Ltd. All rights reserved.
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2.2. TZ biodegradation assays
mineralizing TZ are very limited (Wu et al., 2016). Up to date, no other TZ-degrading stains has been reported except for Shinella sp. NJUST26. Consequently, there is an urgent need to isolate some new strains indigenous to sites contaminated by TZ. Additionally, information on the biodegradation intermediates and metabolic pathways of TZ was very limited. More comprehensive and systematic research on TZ biodegradation mechanism deserved to be conducted in order to achieve an effective bioaugmentation strategy. In this study, in order to expand the bacterial resource for the remediation of TZ contaminant, a novel TZ biodegrading strain named as Raoultella sp. NJUST42 was isolated from activated sludge acclimated by TZ. The performance of Raoultella sp. was evaluated in terms of TZ biodegradation, TOC removal, NH4+ release, pH increase and biotoxicity reduction. Through the identification of intermediates and excitation emission matrix (EEM) analysis, a new TZ biodegradation mechanism by Raoultella sp. was proposed for the first time. In addition, the effects of various abiotic factors on TZ biodegradation were investigated, including initial TZ concentration, incubation temperature, initial pH and additional carbon source.
For the preparation of inocula, NJUST42 was inoculated into presterilized LB medium containing 100 mg L−1 TZ and then incubated at 180 rpm and 30 °C for 48 h. The resulting bacterial suspension was centrifuged at 6000 rpm for 10 min. The deposit was re-suspended and rinsed three times by 100 mL sterilized MSM to remove nutritional composition in LB medium. Finally, the bacterial deposit was diluted with sterilized MSM to an optical density of about 2.0 at wavelength of 600 nm (OD600). In the subsequent biodegradation assays, the obtained bacterial suspension was utilized as inocula at inoculation size of 5% (v/v), i.e., 5 mL bacterial suspension was inoculated into 100 mL MSM, resulting initial OD600 of 0.09 ± 0.01. TZ biodegradation performance of NJUST42 was evaluated at incubation temperature of 30 °C, pH value of 7.0 and initial TZ concentration of 100 mg L−1 using 250 mL Erlenmeyer flasks as batch reactors. In addition, a series of batch experiments were carried out to study the effect of initial TZ concentration, incubation temperature, initial pH and additional carbon source on TZ biodegradation. The initial TZ concentration varied from 25 mg L−1 to 400 mg L−1 to investigate the effect of TZ concentration at pH value of 7.0 and inoculation temperature of 30 °C. To investigate the effect of incubation temperature, TZ biodegradation was evaluated at TZ concentration of 100 mg L−1 and pH value of 7.0, with the incubation temperature varied from 20 °C to 40 °C. pH effect was explored by changing the initial pH value from 5.0 to 10.0 at initial TZ concentration of 100 mg L−1 and incubation temperature of 30 °C. The pH value was adjusted through changing the proportion of KH2PO4 and Na2HPO4·12H2O or adding HCl and NaOH solution. The effect of additional carbon source was investigated at TZ concentration of 100 mg L−1, pH value of 7.0 and incubation temperature of 30 °C, with glucose dosage varied from 500 mg L−1 to 2000 mg L−1.
2. Materials and methods 2.1. Isolation and identification of TZ-degrading stain Isolation and cultivation of TZ-degrading strain was performed in liquid mineral salt medium (MSM) which contained Na2HPO4·12H2O (1.529 g L−1), KH2PO4 (0.372 g L−1), MgSO4·7H2O (0.1 g L−1), CaCl2 (0.05 g L−1) and trace element solution (10 mL L−1). MSM was supplemented with TZ to provide the sole carbon source and nitrogen source at desired concentrations. Na2HPO4 and KH2PO4 served as the phosphate buffer (7 mmol L−1, pH = 7.0) to keep the pH constant. Trace element solution contained EDTA (0.5 g L−1), FeSO4·7H2O (0.2 g L−1), ZnSO4·7H2O (0.001 g L−1), MnCl2·4H2O (0.003 g L−1), H3BO3 (0.03 g L−1), CoCl2·6H2O (0.02 g L−1), CuCl2·2H2O (0.001 g L−1), NiCl2·6H2O (0.002 g L−1) and Na2MoO4·2H2O (0.003 g L−1) (Shen et al., 2009). Sludge used for the isolation of TZ-degrading strain was collected from a full-scale powdered activated carbon treatment tank (PACT) treating TZ-containing fungicide wastewater, which is located in Changzhou Fengdeng Environmental Technology Service Co. Ltd. (Jiangsu Province, China). Approximately 10 mL activated sludge with mixed liquor suspended solids (MLSS) concentration of 3.2 g L−1 was diluted with 100 mL MSM containing 20 mg L−1 TZ and then well mixed in 250 mL Erlenmeyer flasks. After mixing for 2 h, 1 mL supernatant was added into 100 mL fresh MSM supplemented with 30 mg L−1 TZ and then incubated on a rotary shaker at 30 °C and 180 rpm. After 48 h, 1 mL culture was added into the fresh MSM supplemented with 50 mg L−1 TZ for further enrichment. For the third round of enrichment, TZ concentration in fresh MSM was further increased to 100 mg L−1. After three rounds of enrichment, 20 μL suspensions diluted from 10−4–10−10 times were spread-plated onto the pre-sterilized LB plates and incubated for three days at 30 °C. Several colonies with distinct difference were purified five times and then were inoculated into pre-sterilized MSM containing 100 mg L−1 TZ to test their ability to metabolize TZ. Finally, a strain capable of degrading TZ was selected and named as NJUST42. The obtained TZ-degrading strain was identified based on cell morphology, physiological and biochemical tests, and 16S rRNA sequence analysis. The gene amplification was carried out according to our previous study (Wang et al., 2018). The nucleotide sequence was stored in the GenBank database under accession NO. MG999508 and BLAST analysis was carried out in the National Biotechnology Information Center (NCBI) sequence database. Further analysis of the sequence was performed based on Molecular Evolutionary Genetic Analysis (MEGA, version 6.0) (Cai et al., 2013).
2.3. TZ biodegradation kinetics First-order degradation and second-order degradation kinetics model were widely applied to represent the biodegradation profile of various refractory pollutants (Du et al., 2012; Wu et al., 2018b). In this study, both first-order degradation kinetics model (Eq. (1)) and secondorder degradation kinetics model (Eq. (2)) were applied to fit TZ biodegradation profile by NJUST42:
Ct = C0 × e−kt
(1) (2)
1/ Ct = 1/ C0 + k1 t −1
where Ct is the concentration of TZ (mg L ) at the incubation time t (h), C0 is the TZ initial concentration (mg L−1), e is the base of natural logarithm (2.71828), k (h−1) and k1 (L mg−1 h−1) are the rate constants of first-order and second-order, respectively. The time to reduce TZ concentration to 50% (DT50, h) was calculated based on the equation when Ct is equal to half of C0 (Tian et al., 2016). 2.4. Analytical methods TZ was identified and quantified on high performance liquid chromatography (HPLC, UltiMate 3000, Thermo, USA). HPLC analysis was carried out using a C18 column (5 μm, 4.6 × 250 mm) at column temperature of 30 °C and UV–visible wavelength of 195 nm. The mobile phase was a mixture of 30% methanol and 70% ultrapure water (v/v) pumped at flow rate of 1.0 mL min−1. The concentration of total organic carbon (TOC) was determined using Germany Elementar vario TOC analyzer. NH4+-N was analyzed by Nessler's reagent colorimetric method (Hou et al., 2018). The toxicity of MSM to the Zebrafish was assessed with 48-hour lethality tests and performed using a static procedure, according to standard method (GB/T 13267-91). The toxicity was evaluated based on the dilution ratio causing 50% lethality and 64
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described as EC50, 48h (%, v/v) (Wu et al., 2018b). Liquid chromatography-mass spectrometry (LC/MS) was used for the identification of TZ biodegradation intermediates. The chromatographic condition was consistent with HPLC analysis and the sample was pretreated according to Wang et al. (2018). The positive and negative electrospray ionization (ESI) analysis of continuous full scan from m/z 50 to 150 Da was carried out according to Cai et al. (2015). Fluorescence excitation-emission matrix (EEM) analysis was performed via a fluorescence spectrophotometer (F-7000, Hitachi, Japan) (Xue et al., 2013). The cell morphology was observed according to Ho et al. (2009) on a cold field emission scanning electron microscope (SEM, S-4800, Hitachi, Japan). The growth of bacteria was monitored by recording OD600 profile using UV–vis spectrophotometer. 3. Results and discussion 3.1. Isolation and identification of TZ-degrading strain In this study, one bacterial strain namely NJUST42, which was capable of utilizing TZ as the sole carbon and nitrogen source, was successfully isolated from the acclimatized activated sludge. The colony of NJUST42 appeared yellowish, opaque and smooth on the surface. Through SEM analysis, it could be observed that the cells were rodshaped and had a diameter of 0.47–0.60 μm (Fig. S1). Physiological and biochemical characteristics indicated that NJUST42 was negative in Gram stain and catalytic enzyme and oxidase, but positive for contact enzyme. Through the phylogenetic tree, it could be observed that NJUST42 was similar to Raoultella sp. 87A.5 (KU362618.1) and Raoultella sp. AH11 (LC342828.1) (Fig. S2). Therefore, NJUST42 was identified as Raoultella and named as Raoultella sp. NJUST42. Genus Raoultella has been reported as a versatile species for the bioremediation of various highly toxic and recalcitrant pollutants, such as atrazine (Swissa et al., 2014), di-n-butyl phthalate ester (Liu et al., 2017), pyrene and benzo[a]pyrene (Ping et al., 2017), Microcystis aeruginosa flour (Su et al., 2017) and weathered petroleum hydrocarbons (Morales-Guzmán et al., 2017). However, to the best of our knowledge, TZ biodegradation by genus Raoultella has not been revealed.
Fig. 1. Evolution profiles of TZ concentration, cell growth and pH change (a), TOC and NH4+-N concentration, toxicity reduction during TZ biodegradation (b). * indicates no toxicity observed, i.e., all Zebrafish could survive in the undiluted MSM.
biodegradation systems, where the conversion of nitrogen to NH4+-N provide the key evidence for the cleavage of NHC ring (Qiao and Wang, 2010; Shen et al., 2015). In this study, more than 50% of the total nitrogen on TZ ring was converted to NH4+-N by NJUST42, which was relatively high as compared to NJUST26, indicating relatively high TZ mineralization efficiency by NJUST42 (Wu et al., 2016). In addition, during TZ biodegradation, pH value increased from the initial 7.14 ± 0.04 to the peak value of 7.96 ± 0.06 at 288 h although 7 mM phosphate buffer was used, as shown in Fig. 1a. The increase of pH in TZ biodegradation system was probably due to the generation of NH4+ and the cleavage of TZ ring. pH increase was also observed in other biodegradation system treating various NHCs such as pyridine and quinoline, providing key evidence for the cleavage of NHC ring (Zhang et al., 2017; Wang et al., 2017). Accompanied by TZ removal, obvious cell growth was observed in the system inoculated with NJUST42 after the initial lag phase, until a maximum OD600 of 0.70 ± 0.03 was observed at 288 h (Fig. 1a). Considering the fact that TZ was used as the sole carbon and nitrogen source in MSM, obvious biomass increase observed in this study indicated that TZ could be efficiently mineralized by NJUST42. In the biodegradation system inoculated with NJUST42, accompanied by complete removal of 100 mg L−1 TZ within 288 h, TOC concentration decreased from initial 30.8 ± 3.3 mg L−1 to 7.9 ± 1.3 mg L−1 at 288 h (Fig. 1b). Correspondingly, TOC removal efficiency was as high as 74.4 ± 2.9% at 288 h. In our previous study, 100 mg L−1 TZ could be completely degraded by Shinella sp. NJUST26 within 384 h, with TOC removal efficiency lower than 50% achieved (Wu et al., 2016). These results indicated that NJUST42 was superior to NJUST26 in terms of both TZ removal and mineralization efficiency. As
3.2. Biodegradation performance of NJUST42 TZ biodegradation performance of NJUST42 was evaluated at incubation temperature of 30 °C, initial pH of 7.0 and initial TZ concentration of 100 mg L−1. As shown in Fig. 1a, TZ concentration decreased slowly within the first 72 h after the inoculation of NJUST42, but decreased rapidly in the following 216 h until TZ was removed completely. However, in the abiotic control experiment, no significant TZ removal was observed during 336 hours incubation. Both first-order and second-order degradation kinetic models were used to fit TZ biodegradation profile by NJUST42. It was found that the correlation coefficient of the first-order degradation kinetics model (R2 = 0.98) was significantly higher than that of the second-order degradation kinetics model (R2 = 0.72), indicating that first-order degradation kinetics model was more accurate to fit TZ biodegradation profile. According to Du et al. (2012), fitting of the first-order degradation kinetics model confirmed the strong inhibitory nature of TZ. The inhibitory nature of TZ was also confirmed in our previous study (Wu et al., 2016), where obvious lag phase was observed at all initial TZ concentrations, and the lag phase prolonged with the increase of initial TZ concentrations. Nitrogen on the ring of NHCs such as TZ was often transformed to NH4+ during the biodegradation process (Wu et al., 2019). As indicated in Fig. 1b, significant NH4+ release was observed during TZ biodegradation. NH4+-N concentration reached a maximum value of 36.2 ± 3.3 mg L−1 when 100 mg L−1 TZ was completely removed within 288 h. Similar phenomena have been also observed in other NHC 65
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incubation time of 168 h and 336 h were compared. As demonstrated in Fig. 2, accompanied by TZ biodegradation, the characteristic fluorescence peaks in the spectrum were constantly changing during the whole TZ biodegradation process. Before incubation, a characteristic peak (peak A) appeared at Ex = 210–220 nm/Em = 290 nm in the region I, which was related to simple aromatic compounds such as TZ (Chen et al., 2003). After 168 h inoculation, peak A was shifted to Ex = 220–230 nm/Em = 325 nm. In addition, two distinct characteristic peaks at the position of Ex = 240 nm/Em = 390 nm in region III (peak B) and Ex = 280 nm/Em = 325–330 nm in Region IV (peak C) were observed at 168 h, which were related to fulvic acid and soluble microbial product (SMP), respectively (Chen et al., 2003). Peak A disappeared completely and peak C shifted significantly to position Ex = 320 nm/Em = 375–385 nm in region V after 336 h incubation, indicating the generation of humic acid-like organics (Chen et al., 2003). In addition, the strength of peak B was weakened obviously. These results confirmed that TZ could be completely removed by NJUST42, which was accompanied by the generation of some metabolites including humic acid-like organics. 3.4. TZ biodegradation pathways During TZ biodegradation, biodegradation intermediates were identified through HPLC/MS analysis. As shown in Table S1 and Fig. S3, 15 biodegradation intermediates was identified, including 5-hydroxy[1,2,4]triazolidin-3-one, N-hydrazide-formamide, [1,2,4]Triazolidine3,5-dione, N-hydrazide-carbamic acid, formic acid N′-(hydroxy-nitrosomethyl)-hydrazide, carbamic acid, hydrazinecarboxylic acid, carbamic acid aldehyde, hydroxymethyl-carbamic acid, N-methanamide carbamate, N′-(carbonyl-nitroso)-formylhydrazide, hydrazine dibasic carboxylic acid, (hydrazino-hydroxy-methyl)-carbamic acid, N′-(carbonylnitroso)-indolecarboxylic acid and N′-(hydroxy-nitroso-methyl)-hydrazinecarboxylic acid. These intermediates were different from TZ biodegradation intermediates in our previous study (Wu et al., 2016). Obviously, carbonylation and hydroxylation of TZ to form 5-hydroxy-[1,2,4]triazolidin-3-one was the first step of TZ biodegradation pathway, as shown in Fig. 3. After carbonylation and hydroxylation, three independent biodegradation pathways were proposed based on the analysis of TZ biodegradation metabolites. In pathway (I), 5-hydroxy-[1,2,4]triazolidin-3-one was oxidized to form a carbonyl derivative namely [1,2,4]triazolidine-3,5-dione, followed by CeN bond cleavage to yield N-hydrazide-formamide. This result was consistent with Wu et al. (2016), where the intermediate namely 2,4-dihydro[1,2,4]triazol-3-one (DHTO) also underwent the cleavage of the CeN bond. The ketone group was then further oxidized to form a carboxyl group, resulting into the formation of a new intermediate namely Nhydrazide-carbamic acid. The cleavage of the CeN bond at 2, 3 position resulted in the formation of intermediates namely hydrazinecarboxylic acid and carbamic acid. Cleavage of the CeN bond and release of carbamic acid was also found in pyridine biodegradation pathway by Li et al. (2017). In addition, deamination reaction could occur on N-hydrazide-carbamic acid, resulting in the formation of N-methanamide carbamate. The intermediate namely carbamic acid aldehyde could be produced through the cleavage of the CeN bond at 3,4 position. The CeN bond of 5-hydroxy-[1,2,4]triazolidin-3-one was cleaved to form the carboxylic acid derivative (hydrazino-hydroxy-methyl)-carbamic acid firstly, which was subsequently oxidized to remove the thiol group. As a result, an intermediate namely hydroxymethyl-carbamic acid was formed in pathway (II). Pathway (III) began with the cleavage of CeN bond on 5-hydroxy-[1,2,4]triazolidin-3-one, and oxidation occurred to form formic acid N′-(hydroxy-nitroso-methyl)-hydrazide, which was similar to the oxidation pathway of triazole ring in an electrochemical oxidation system (Zhong et al., 2013). After that, oxidation of the hydroxyl group resulted into the generation of N′-(carbonyl-nitroso)-formylhydrazide. A similar phenomenon regarding the oxidation of hydroxy group was observed in a pyridine oxidation system (Singh and Lo,
Fig. 2. Fluorescence EEM contours of MSM containing 100 mg L−1 TZ before incubation (a), 168 h after inoculation (b) and 336 h after inoculation (c). Horizontal and vertical lines divide EEM contours into five regions (I-V).
shown in Fig. 1b, EC50, 48h value of MSM containing 100 mg L−1 TZ was as low as 10.6 ± 2.3%, indicating the high toxicity of TZ. After incubation for 192 h, accompanied by the significant removal of TZ, EC50, 48h value of MSM obviously increased to 74.2 ± 5.5%. 288 h later, TZ was completely removed in MSM and all Zebrafish were survived in the undiluted MSM, indicating that the biotoxicity of TZ was significantly reduced by NJUST42. The reduction of toxicity observed in this study could be attributed to the cleavage of TZ ring and removal of various intermediates formed during TZ biodegradation. From the data about TZ depletion, NH4+ formation, pH increase, biomass growth, TOC decrease and toxicity reduction during TZ biodegradation, it could be inferred that TZ could be mineralized by NJUST42.
3.3. Fluorescence spectral analysis during TZ degradation In order to confirm TZ biodegradation by NJUST42, excitation emission matrix (EEM) fluorescence spectra of MSM sampled at 66
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Fig. 3. Proposed TZ biodegradation pathway by NJUST42.
3.5. Effects of operational condition on TZ biodegradation
2017). N′-(carbonyl-nitroso)-formylhydrazide was then further oxidized to form a new carboxylic acid derivative namely N′-(carbonylnitroso)-indolecarboxylic acid. Finally, oxidation of the CeN bond on N′-(carbonyl-nitroso)-indolecarboxylic acid resulted into the formation of hydrazine dibasic carboxylic acid. N′-(hydroxy-nitroso-methyl)-hydrazinecarboxylic acid was generated through hydroxylation reaction. In the reported photocatalytic system, electrochemical oxidation system and biodegradation system, several different TZ degradation pathways have been proposed. In a photocatalytic system, CeN bond of TZ was cleaved firstly by hydroxyl radicals to form the main intermediate N-methylene-methanehydrazonamide. After that, decarbonylation reaction occurred on N-hydrazonomethyl-fonnamide, resulting into the release of NH4+ and CO2 (Watanabe et al., 2005). A similar TZ degradation pathway was found in an electrochemical oxidation system (Han et al., 2014), where the cleavage of TZ ring by hydroxyl radicals and the formation of N-methylene-methanehydrazonamide was also observed. Through the oxidation of N-methylene-methanehydrazonamide, an intermediate namely N-amino-iminomethyl-aminomethanol, organic acids, inorganic anions and CO2 were formed. In our previous study, TZ was found to be firstly oxidized by Shinella sp. NJUST26 to form 2,4-dihydro-[1,2,4]triazol-3-one by mono-oxidation. After that, the ring was cleaved to form another intermediate namely N-hydrazonomethyl-formamide, which was further biodegraded to semicarbazide and urea (Wu et al., 2016). Based on the above analysis, it could be inferred that TZ biodegradation mechanism by Raoultella sp. NJUST42 was quite different from these previous investigations. The new TZ biodegradation pathway obtained in this study will provide a broader perspective and more possibilities for the future exploration of TZ biodegradation system.
Substrate concentration has a remarkable impact on the biodegradation of various toxic or recalcitrant contaminants (Bai et al., 2008). As the initial TZ concentration increased from 50 mg L−1 to 400 mg L−1, DT50 increased from 82.5 ± 9.1 h to 247.5 ± 20.8 h and the k value decreased from 0.0084 ± 0.0004 to 0.0028 ± 0.0004 h−1 based on the fitting of TZ removal profile to the first-order degradation kinetics (Fig. 4a). These results indicated that TZ was an inhibitory substrate, especially at high TZ concentration. Similar conclusion could be obtained in our previous study (Wu et al., 2016), where the time required for complete TZ removal increased obviously with the increase of initial TZ concentration. Incubation temperature is the main factor affecting the biodegradation of refractory compounds (Kang et al., 2012). As the incubation temperature raised from 20 °C to 30 °C, the k value increased from the initial 0.0039 ± 0.0001 h−1 to 0.0065 ± 0.0002 h−1. However, when the temperature was further increased to 40 °C, the k value decreased to 0.0033 ± 0.0002 h−1. Correspondingly, DT50 value was initially decreased from 177.7 ± 12.5 h to 106.6 ± 8.1 h, and then increased to 210.0 ± 14.1 h as the temperature continued to rise to 40 °C (Fig. 4b). The above results indicated that the optimal incubation temperature for TZ biodegradation was 30 °C, and too high or too low incubation temperature would adversely affect TZ biodegradation. Similar phenomenon has been discovered by Sun et al. (2009), where quinoline could be most efficiently biodegraded by Pseudomonas strain BW003 at 30 °C. The pH value of MSM often plays a significant role for the biodegradation of various recalcitrant compounds (Singh et al., 2003). As pH increased from 5.0 to 7.0, the k value increased from 0.0032 ± 0.0002 67
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Fig. 4. The effect of initial TZ concentration (a), temperature (b), initial pH (c) and additional glucose concentration (d) on TZ biodegradation by NJUST42.
to 0.0063 ± 0.0005 h−1, but as pH further increased to 10.0, the k value decreased to 0.0019 ± 0.0002 h−1. In contrast, when the pH value increased from 5.0 to 7.0, DT50 value decreased significantly from 216.6 ± 17.4 h to 110.0 ± 12.5 h and with pH increased to 10.0, DT50 increased to 364.7 ± 28.4 h (Fig. 4c). Actually, pH value of MSM was rather important for the activity of enzyme involved in the biodegradation process. Both acidic and alkaline condition was unfavorable for the biodegradation of various contaminants such as TZ. pH dependence was also observed in the pyridine biodegradation system inoculated by Shewanella, where pyridine biodegradation could be delayed and even inhibited under both acidic or alkaline pH conditions (Mathur et al., 2008). According to Lin et al. (2010), the effect of additional carbon sources on the biodegradation of various recalcitrant compounds was strongly dependent on the dosage of additional carbon source. When the glucose concentration was 500 mg L−1, the k value reached a maximum value of 0.0069 ± 0.0006 h−1, which was obviously higher than the control experiment without additional carbon source (Fig. 4d). However, as the glucose dosage increased to 2000 mg L−1, the k value decreased to as low as 0.0031 ± 0.0004 h−1. Correspondingly, the DT50 value reached a minimum value of 100.4 ± 8.4 h at the glucose dosage of 500 mg L−1 and a maximum value of 223.5 ± 18.4 h at the glucose dosage of 2000 mg L−1. These results indicated that low dosage of additional carbon source was positive for TZ biodegradation, while high dosage of additional carbon source was negative, probably due to the competitive inhibition exerted by additional carbon source (Wang et al., 2018).
was capable of utilizing TZ as the sole carbon source and nitrogen source, was isolated successfully from acclimated activated sludge. TZ removal was accompanied by NH4+ release, pH increase, biomass increase, TOC removal, biotoxicity reduction and EEM variation, indicating TZ mineralization by NJUST42. Based on the identification of biodegradation intermediates, a new TZ biodegradation pathway including hydroxylation, carbonylation, carboxylation and ring cleavage, was proposed for the first time. Acknowledgements This research is financed by Natural Science Foundation of Jiangsu Province for Distinguished Young Scholars (BK20170038) and National Natural Science Foundation of China (51478225). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biteb.2019.02.007. References Amorim, C.C., Bottrel, S.E., Costa, E.P., Teixeira, A.P., Leao, M.M., 2013. Removal of ethylenthiourea and 1,2,4-triazole pesticide metabolites from water by adsorption in commercial activated carbons. J. Environ. Sci. Health B 48, 183–190. Bai, Y.H., Sun, Q.H., Zhao, C., Wen, D.H., Tang, X.Y., 2008. Microbial degradation and metabolic pathway of pyridine by a Paracoccus sp. strain BW001. Biodegradation 19, 915–926. Cai, Z.Q., Shi, S., Li, S.S., Yang, B.K., Chen, Q.L., Zhao, X.Y., 2013. Microbial degradation characteristics and kinetics of novel pyrimidynyloxybenzoic herbicide ZJ0273 by a newly isolated Bacillus sp. CY. Environ. Sci. Pollut. Res. 20, 8831–8838. Cai, Z.Q., Zhang, W.J., Li, S.S., Ma, J.T., Wang, J., Zhao, X.Y., 2015. Microbial degradation mechanism and pathway of the novel insecticide paichongding by a newly isolated Sphingobacterium sp. P1-3 from Soil. J. Agric. Food Chem. 63, 3823–3829.
4. Conclusions In this study, a novel strain namely Raoultella sp. NJUST42, which 68
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1H-1,2,4-Triazole biodegradation by newly isolated Raoultella sp.: A novel biodegradation pathway
T
Xiaolin Liu1, Jing Wang1, Dan Chen, Xinbai Jiang, Haobo Wu, Cheng Hou, Dejin Zhang, ⁎ Xiaodong Liu, Lianjun Wang, Jinyou Shen Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse, School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, Jiangsu Province, China
A R T I C LE I N FO
A B S T R A C T
Keywords: 1H-1,2,4-Triazole Bioaugmentation Intermediates Metabolic pathway
In this study, a novel 1H-1,2,4-triazole (TZ) degrading strain was isolated from activated sludge acclimated by TZ and was identified as Raoultella sp. The biodegradation test showed that Raoultella sp. could utilize TZ as the sole carbon and nitrogen source. TZ was completely removed at incubation temperature of 30 °C, initial pH of 7.0 and initial concentration of 100 mg L−1 within 288 h, which was accompanied by obvious TOC removal, NH4+ release, pH increase, biomass growth, biotoxicity reduction and excitation emission matrix (EEM) variation. TZ biodegradation profile by Raoultella sp. was well fitted by the first-order degradation kinetic model, confirming the inhibitory nature of TZ. Based on HPLC/MS analysis, a distinct TZ biodegradation pathway including hydroxylation, carbonylation, carboxylation and ring cleavage, was proposed for the first time. Compared with other TZ degrading species such as Shinella sp., Raoultella sp. showed relatively higher TZ removal efficiency, demonstrating great potential for the application in biological treatment of wastewater containing TZ in practice.
1. Introduction As a typical nitrogenous heterocyclic compounds (NHCs), 1H-1,2,4triazole (TZ) is widely used for the synthesis of pesticides, herbicides and fungicides, causing substantial release of wastewater containing TZ (Wang et al., 2011; Wu et al., 2016). According to Zhu et al. (2018), the fate of various contaminants in the environment is closely related to hydrolysis, biodegradation and adsorption. Since TZ is not prone to hydrolysis, biodegradation or adsorption onto soil particles or other environmental media, the half-life of TZ in the environment would be rather long (Wu et al., 2019). Therefore, the exposure of highly toxic, carcinogenic and teratogenic TZ to surface water and groundwater would cause serious ecological problems (Wu et al., 2018a; Konwick et al., 2006). Thus, in order to avoid environmental deterioration caused by TZ, wastewater containing TZ should be treated properly before discharge (Wu et al., 2018a; Wu et al., 2019). Development of efficient and economical method for TZ removal from wastewater has become an urgent need. Various treatment technologies have been developed for TZ removal from contaminated wastewater, such as adsorption (Amorim et al.,
2013), electrochemical oxidation (Han et al., 2014) and photocatalytic mineralization (Watanabe et al., 2005), etc. These physico-chemical methods have been proven to be cost ineffective and energy intensive (Wang et al., 2018; Liang et al., 2019). In addition, toxic and harmful intermediates such as cyanuric acid were often found in the effluent from these physico-chemical treatment systems, causing serious secondary pollution (Watanabe et al., 2005). Nevertheless, biological treatment, which is both cost effective and environmental friendly, has turned out to be a promising alternative (Jiang et al., 2018; Singh et al., 2008). Although the application of bioaugmentation for the treatment of wastewater containing various recalcitrant contaminants is receiving more attention, the available strains with related function are currently very limited. In order to achieve efficient biodegradation of various recalcitrant pollutants such as TZ, the inoculation of specific functional microorganisms into the biological systems will be a favorable alternative (Wu et al., 2018b; Yun et al., 2017). In our previous study, a novel TZ-degrading strain namely Shinella sp. NJUST26 was isolated from TZ-contaminated soil for the first time (Wu et al., 2016). Unfortunately, due to the extremely high toxicity and rather poor biodegradability of TZ, specific functional microorganisms capable of
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Corresponding author. E-mail address: [email protected] (J. Shen). 1 These authors contributed to the paper equally. https://doi.org/10.1016/j.biteb.2019.02.007 Received 11 January 2019; Received in revised form 10 February 2019; Accepted 11 February 2019 Available online 13 February 2019 2589-014X/ © 2019 Elsevier Ltd. All rights reserved.
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2.2. TZ biodegradation assays
mineralizing TZ are very limited (Wu et al., 2016). Up to date, no other TZ-degrading stains has been reported except for Shinella sp. NJUST26. Consequently, there is an urgent need to isolate some new strains indigenous to sites contaminated by TZ. Additionally, information on the biodegradation intermediates and metabolic pathways of TZ was very limited. More comprehensive and systematic research on TZ biodegradation mechanism deserved to be conducted in order to achieve an effective bioaugmentation strategy. In this study, in order to expand the bacterial resource for the remediation of TZ contaminant, a novel TZ biodegrading strain named as Raoultella sp. NJUST42 was isolated from activated sludge acclimated by TZ. The performance of Raoultella sp. was evaluated in terms of TZ biodegradation, TOC removal, NH4+ release, pH increase and biotoxicity reduction. Through the identification of intermediates and excitation emission matrix (EEM) analysis, a new TZ biodegradation mechanism by Raoultella sp. was proposed for the first time. In addition, the effects of various abiotic factors on TZ biodegradation were investigated, including initial TZ concentration, incubation temperature, initial pH and additional carbon source.
For the preparation of inocula, NJUST42 was inoculated into presterilized LB medium containing 100 mg L−1 TZ and then incubated at 180 rpm and 30 °C for 48 h. The resulting bacterial suspension was centrifuged at 6000 rpm for 10 min. The deposit was re-suspended and rinsed three times by 100 mL sterilized MSM to remove nutritional composition in LB medium. Finally, the bacterial deposit was diluted with sterilized MSM to an optical density of about 2.0 at wavelength of 600 nm (OD600). In the subsequent biodegradation assays, the obtained bacterial suspension was utilized as inocula at inoculation size of 5% (v/v), i.e., 5 mL bacterial suspension was inoculated into 100 mL MSM, resulting initial OD600 of 0.09 ± 0.01. TZ biodegradation performance of NJUST42 was evaluated at incubation temperature of 30 °C, pH value of 7.0 and initial TZ concentration of 100 mg L−1 using 250 mL Erlenmeyer flasks as batch reactors. In addition, a series of batch experiments were carried out to study the effect of initial TZ concentration, incubation temperature, initial pH and additional carbon source on TZ biodegradation. The initial TZ concentration varied from 25 mg L−1 to 400 mg L−1 to investigate the effect of TZ concentration at pH value of 7.0 and inoculation temperature of 30 °C. To investigate the effect of incubation temperature, TZ biodegradation was evaluated at TZ concentration of 100 mg L−1 and pH value of 7.0, with the incubation temperature varied from 20 °C to 40 °C. pH effect was explored by changing the initial pH value from 5.0 to 10.0 at initial TZ concentration of 100 mg L−1 and incubation temperature of 30 °C. The pH value was adjusted through changing the proportion of KH2PO4 and Na2HPO4·12H2O or adding HCl and NaOH solution. The effect of additional carbon source was investigated at TZ concentration of 100 mg L−1, pH value of 7.0 and incubation temperature of 30 °C, with glucose dosage varied from 500 mg L−1 to 2000 mg L−1.
2. Materials and methods 2.1. Isolation and identification of TZ-degrading stain Isolation and cultivation of TZ-degrading strain was performed in liquid mineral salt medium (MSM) which contained Na2HPO4·12H2O (1.529 g L−1), KH2PO4 (0.372 g L−1), MgSO4·7H2O (0.1 g L−1), CaCl2 (0.05 g L−1) and trace element solution (10 mL L−1). MSM was supplemented with TZ to provide the sole carbon source and nitrogen source at desired concentrations. Na2HPO4 and KH2PO4 served as the phosphate buffer (7 mmol L−1, pH = 7.0) to keep the pH constant. Trace element solution contained EDTA (0.5 g L−1), FeSO4·7H2O (0.2 g L−1), ZnSO4·7H2O (0.001 g L−1), MnCl2·4H2O (0.003 g L−1), H3BO3 (0.03 g L−1), CoCl2·6H2O (0.02 g L−1), CuCl2·2H2O (0.001 g L−1), NiCl2·6H2O (0.002 g L−1) and Na2MoO4·2H2O (0.003 g L−1) (Shen et al., 2009). Sludge used for the isolation of TZ-degrading strain was collected from a full-scale powdered activated carbon treatment tank (PACT) treating TZ-containing fungicide wastewater, which is located in Changzhou Fengdeng Environmental Technology Service Co. Ltd. (Jiangsu Province, China). Approximately 10 mL activated sludge with mixed liquor suspended solids (MLSS) concentration of 3.2 g L−1 was diluted with 100 mL MSM containing 20 mg L−1 TZ and then well mixed in 250 mL Erlenmeyer flasks. After mixing for 2 h, 1 mL supernatant was added into 100 mL fresh MSM supplemented with 30 mg L−1 TZ and then incubated on a rotary shaker at 30 °C and 180 rpm. After 48 h, 1 mL culture was added into the fresh MSM supplemented with 50 mg L−1 TZ for further enrichment. For the third round of enrichment, TZ concentration in fresh MSM was further increased to 100 mg L−1. After three rounds of enrichment, 20 μL suspensions diluted from 10−4–10−10 times were spread-plated onto the pre-sterilized LB plates and incubated for three days at 30 °C. Several colonies with distinct difference were purified five times and then were inoculated into pre-sterilized MSM containing 100 mg L−1 TZ to test their ability to metabolize TZ. Finally, a strain capable of degrading TZ was selected and named as NJUST42. The obtained TZ-degrading strain was identified based on cell morphology, physiological and biochemical tests, and 16S rRNA sequence analysis. The gene amplification was carried out according to our previous study (Wang et al., 2018). The nucleotide sequence was stored in the GenBank database under accession NO. MG999508 and BLAST analysis was carried out in the National Biotechnology Information Center (NCBI) sequence database. Further analysis of the sequence was performed based on Molecular Evolutionary Genetic Analysis (MEGA, version 6.0) (Cai et al., 2013).
2.3. TZ biodegradation kinetics First-order degradation and second-order degradation kinetics model were widely applied to represent the biodegradation profile of various refractory pollutants (Du et al., 2012; Wu et al., 2018b). In this study, both first-order degradation kinetics model (Eq. (1)) and secondorder degradation kinetics model (Eq. (2)) were applied to fit TZ biodegradation profile by NJUST42:
Ct = C0 × e−kt
(1) (2)
1/ Ct = 1/ C0 + k1 t −1
where Ct is the concentration of TZ (mg L ) at the incubation time t (h), C0 is the TZ initial concentration (mg L−1), e is the base of natural logarithm (2.71828), k (h−1) and k1 (L mg−1 h−1) are the rate constants of first-order and second-order, respectively. The time to reduce TZ concentration to 50% (DT50, h) was calculated based on the equation when Ct is equal to half of C0 (Tian et al., 2016). 2.4. Analytical methods TZ was identified and quantified on high performance liquid chromatography (HPLC, UltiMate 3000, Thermo, USA). HPLC analysis was carried out using a C18 column (5 μm, 4.6 × 250 mm) at column temperature of 30 °C and UV–visible wavelength of 195 nm. The mobile phase was a mixture of 30% methanol and 70% ultrapure water (v/v) pumped at flow rate of 1.0 mL min−1. The concentration of total organic carbon (TOC) was determined using Germany Elementar vario TOC analyzer. NH4+-N was analyzed by Nessler's reagent colorimetric method (Hou et al., 2018). The toxicity of MSM to the Zebrafish was assessed with 48-hour lethality tests and performed using a static procedure, according to standard method (GB/T 13267-91). The toxicity was evaluated based on the dilution ratio causing 50% lethality and 64
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described as EC50, 48h (%, v/v) (Wu et al., 2018b). Liquid chromatography-mass spectrometry (LC/MS) was used for the identification of TZ biodegradation intermediates. The chromatographic condition was consistent with HPLC analysis and the sample was pretreated according to Wang et al. (2018). The positive and negative electrospray ionization (ESI) analysis of continuous full scan from m/z 50 to 150 Da was carried out according to Cai et al. (2015). Fluorescence excitation-emission matrix (EEM) analysis was performed via a fluorescence spectrophotometer (F-7000, Hitachi, Japan) (Xue et al., 2013). The cell morphology was observed according to Ho et al. (2009) on a cold field emission scanning electron microscope (SEM, S-4800, Hitachi, Japan). The growth of bacteria was monitored by recording OD600 profile using UV–vis spectrophotometer. 3. Results and discussion 3.1. Isolation and identification of TZ-degrading strain In this study, one bacterial strain namely NJUST42, which was capable of utilizing TZ as the sole carbon and nitrogen source, was successfully isolated from the acclimatized activated sludge. The colony of NJUST42 appeared yellowish, opaque and smooth on the surface. Through SEM analysis, it could be observed that the cells were rodshaped and had a diameter of 0.47–0.60 μm (Fig. S1). Physiological and biochemical characteristics indicated that NJUST42 was negative in Gram stain and catalytic enzyme and oxidase, but positive for contact enzyme. Through the phylogenetic tree, it could be observed that NJUST42 was similar to Raoultella sp. 87A.5 (KU362618.1) and Raoultella sp. AH11 (LC342828.1) (Fig. S2). Therefore, NJUST42 was identified as Raoultella and named as Raoultella sp. NJUST42. Genus Raoultella has been reported as a versatile species for the bioremediation of various highly toxic and recalcitrant pollutants, such as atrazine (Swissa et al., 2014), di-n-butyl phthalate ester (Liu et al., 2017), pyrene and benzo[a]pyrene (Ping et al., 2017), Microcystis aeruginosa flour (Su et al., 2017) and weathered petroleum hydrocarbons (Morales-Guzmán et al., 2017). However, to the best of our knowledge, TZ biodegradation by genus Raoultella has not been revealed.
Fig. 1. Evolution profiles of TZ concentration, cell growth and pH change (a), TOC and NH4+-N concentration, toxicity reduction during TZ biodegradation (b). * indicates no toxicity observed, i.e., all Zebrafish could survive in the undiluted MSM.
biodegradation systems, where the conversion of nitrogen to NH4+-N provide the key evidence for the cleavage of NHC ring (Qiao and Wang, 2010; Shen et al., 2015). In this study, more than 50% of the total nitrogen on TZ ring was converted to NH4+-N by NJUST42, which was relatively high as compared to NJUST26, indicating relatively high TZ mineralization efficiency by NJUST42 (Wu et al., 2016). In addition, during TZ biodegradation, pH value increased from the initial 7.14 ± 0.04 to the peak value of 7.96 ± 0.06 at 288 h although 7 mM phosphate buffer was used, as shown in Fig. 1a. The increase of pH in TZ biodegradation system was probably due to the generation of NH4+ and the cleavage of TZ ring. pH increase was also observed in other biodegradation system treating various NHCs such as pyridine and quinoline, providing key evidence for the cleavage of NHC ring (Zhang et al., 2017; Wang et al., 2017). Accompanied by TZ removal, obvious cell growth was observed in the system inoculated with NJUST42 after the initial lag phase, until a maximum OD600 of 0.70 ± 0.03 was observed at 288 h (Fig. 1a). Considering the fact that TZ was used as the sole carbon and nitrogen source in MSM, obvious biomass increase observed in this study indicated that TZ could be efficiently mineralized by NJUST42. In the biodegradation system inoculated with NJUST42, accompanied by complete removal of 100 mg L−1 TZ within 288 h, TOC concentration decreased from initial 30.8 ± 3.3 mg L−1 to 7.9 ± 1.3 mg L−1 at 288 h (Fig. 1b). Correspondingly, TOC removal efficiency was as high as 74.4 ± 2.9% at 288 h. In our previous study, 100 mg L−1 TZ could be completely degraded by Shinella sp. NJUST26 within 384 h, with TOC removal efficiency lower than 50% achieved (Wu et al., 2016). These results indicated that NJUST42 was superior to NJUST26 in terms of both TZ removal and mineralization efficiency. As
3.2. Biodegradation performance of NJUST42 TZ biodegradation performance of NJUST42 was evaluated at incubation temperature of 30 °C, initial pH of 7.0 and initial TZ concentration of 100 mg L−1. As shown in Fig. 1a, TZ concentration decreased slowly within the first 72 h after the inoculation of NJUST42, but decreased rapidly in the following 216 h until TZ was removed completely. However, in the abiotic control experiment, no significant TZ removal was observed during 336 hours incubation. Both first-order and second-order degradation kinetic models were used to fit TZ biodegradation profile by NJUST42. It was found that the correlation coefficient of the first-order degradation kinetics model (R2 = 0.98) was significantly higher than that of the second-order degradation kinetics model (R2 = 0.72), indicating that first-order degradation kinetics model was more accurate to fit TZ biodegradation profile. According to Du et al. (2012), fitting of the first-order degradation kinetics model confirmed the strong inhibitory nature of TZ. The inhibitory nature of TZ was also confirmed in our previous study (Wu et al., 2016), where obvious lag phase was observed at all initial TZ concentrations, and the lag phase prolonged with the increase of initial TZ concentrations. Nitrogen on the ring of NHCs such as TZ was often transformed to NH4+ during the biodegradation process (Wu et al., 2019). As indicated in Fig. 1b, significant NH4+ release was observed during TZ biodegradation. NH4+-N concentration reached a maximum value of 36.2 ± 3.3 mg L−1 when 100 mg L−1 TZ was completely removed within 288 h. Similar phenomena have been also observed in other NHC 65
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incubation time of 168 h and 336 h were compared. As demonstrated in Fig. 2, accompanied by TZ biodegradation, the characteristic fluorescence peaks in the spectrum were constantly changing during the whole TZ biodegradation process. Before incubation, a characteristic peak (peak A) appeared at Ex = 210–220 nm/Em = 290 nm in the region I, which was related to simple aromatic compounds such as TZ (Chen et al., 2003). After 168 h inoculation, peak A was shifted to Ex = 220–230 nm/Em = 325 nm. In addition, two distinct characteristic peaks at the position of Ex = 240 nm/Em = 390 nm in region III (peak B) and Ex = 280 nm/Em = 325–330 nm in Region IV (peak C) were observed at 168 h, which were related to fulvic acid and soluble microbial product (SMP), respectively (Chen et al., 2003). Peak A disappeared completely and peak C shifted significantly to position Ex = 320 nm/Em = 375–385 nm in region V after 336 h incubation, indicating the generation of humic acid-like organics (Chen et al., 2003). In addition, the strength of peak B was weakened obviously. These results confirmed that TZ could be completely removed by NJUST42, which was accompanied by the generation of some metabolites including humic acid-like organics. 3.4. TZ biodegradation pathways During TZ biodegradation, biodegradation intermediates were identified through HPLC/MS analysis. As shown in Table S1 and Fig. S3, 15 biodegradation intermediates was identified, including 5-hydroxy[1,2,4]triazolidin-3-one, N-hydrazide-formamide, [1,2,4]Triazolidine3,5-dione, N-hydrazide-carbamic acid, formic acid N′-(hydroxy-nitrosomethyl)-hydrazide, carbamic acid, hydrazinecarboxylic acid, carbamic acid aldehyde, hydroxymethyl-carbamic acid, N-methanamide carbamate, N′-(carbonyl-nitroso)-formylhydrazide, hydrazine dibasic carboxylic acid, (hydrazino-hydroxy-methyl)-carbamic acid, N′-(carbonylnitroso)-indolecarboxylic acid and N′-(hydroxy-nitroso-methyl)-hydrazinecarboxylic acid. These intermediates were different from TZ biodegradation intermediates in our previous study (Wu et al., 2016). Obviously, carbonylation and hydroxylation of TZ to form 5-hydroxy-[1,2,4]triazolidin-3-one was the first step of TZ biodegradation pathway, as shown in Fig. 3. After carbonylation and hydroxylation, three independent biodegradation pathways were proposed based on the analysis of TZ biodegradation metabolites. In pathway (I), 5-hydroxy-[1,2,4]triazolidin-3-one was oxidized to form a carbonyl derivative namely [1,2,4]triazolidine-3,5-dione, followed by CeN bond cleavage to yield N-hydrazide-formamide. This result was consistent with Wu et al. (2016), where the intermediate namely 2,4-dihydro[1,2,4]triazol-3-one (DHTO) also underwent the cleavage of the CeN bond. The ketone group was then further oxidized to form a carboxyl group, resulting into the formation of a new intermediate namely Nhydrazide-carbamic acid. The cleavage of the CeN bond at 2, 3 position resulted in the formation of intermediates namely hydrazinecarboxylic acid and carbamic acid. Cleavage of the CeN bond and release of carbamic acid was also found in pyridine biodegradation pathway by Li et al. (2017). In addition, deamination reaction could occur on N-hydrazide-carbamic acid, resulting in the formation of N-methanamide carbamate. The intermediate namely carbamic acid aldehyde could be produced through the cleavage of the CeN bond at 3,4 position. The CeN bond of 5-hydroxy-[1,2,4]triazolidin-3-one was cleaved to form the carboxylic acid derivative (hydrazino-hydroxy-methyl)-carbamic acid firstly, which was subsequently oxidized to remove the thiol group. As a result, an intermediate namely hydroxymethyl-carbamic acid was formed in pathway (II). Pathway (III) began with the cleavage of CeN bond on 5-hydroxy-[1,2,4]triazolidin-3-one, and oxidation occurred to form formic acid N′-(hydroxy-nitroso-methyl)-hydrazide, which was similar to the oxidation pathway of triazole ring in an electrochemical oxidation system (Zhong et al., 2013). After that, oxidation of the hydroxyl group resulted into the generation of N′-(carbonyl-nitroso)-formylhydrazide. A similar phenomenon regarding the oxidation of hydroxy group was observed in a pyridine oxidation system (Singh and Lo,
Fig. 2. Fluorescence EEM contours of MSM containing 100 mg L−1 TZ before incubation (a), 168 h after inoculation (b) and 336 h after inoculation (c). Horizontal and vertical lines divide EEM contours into five regions (I-V).
shown in Fig. 1b, EC50, 48h value of MSM containing 100 mg L−1 TZ was as low as 10.6 ± 2.3%, indicating the high toxicity of TZ. After incubation for 192 h, accompanied by the significant removal of TZ, EC50, 48h value of MSM obviously increased to 74.2 ± 5.5%. 288 h later, TZ was completely removed in MSM and all Zebrafish were survived in the undiluted MSM, indicating that the biotoxicity of TZ was significantly reduced by NJUST42. The reduction of toxicity observed in this study could be attributed to the cleavage of TZ ring and removal of various intermediates formed during TZ biodegradation. From the data about TZ depletion, NH4+ formation, pH increase, biomass growth, TOC decrease and toxicity reduction during TZ biodegradation, it could be inferred that TZ could be mineralized by NJUST42.
3.3. Fluorescence spectral analysis during TZ degradation In order to confirm TZ biodegradation by NJUST42, excitation emission matrix (EEM) fluorescence spectra of MSM sampled at 66
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Fig. 3. Proposed TZ biodegradation pathway by NJUST42.
3.5. Effects of operational condition on TZ biodegradation
2017). N′-(carbonyl-nitroso)-formylhydrazide was then further oxidized to form a new carboxylic acid derivative namely N′-(carbonylnitroso)-indolecarboxylic acid. Finally, oxidation of the CeN bond on N′-(carbonyl-nitroso)-indolecarboxylic acid resulted into the formation of hydrazine dibasic carboxylic acid. N′-(hydroxy-nitroso-methyl)-hydrazinecarboxylic acid was generated through hydroxylation reaction. In the reported photocatalytic system, electrochemical oxidation system and biodegradation system, several different TZ degradation pathways have been proposed. In a photocatalytic system, CeN bond of TZ was cleaved firstly by hydroxyl radicals to form the main intermediate N-methylene-methanehydrazonamide. After that, decarbonylation reaction occurred on N-hydrazonomethyl-fonnamide, resulting into the release of NH4+ and CO2 (Watanabe et al., 2005). A similar TZ degradation pathway was found in an electrochemical oxidation system (Han et al., 2014), where the cleavage of TZ ring by hydroxyl radicals and the formation of N-methylene-methanehydrazonamide was also observed. Through the oxidation of N-methylene-methanehydrazonamide, an intermediate namely N-amino-iminomethyl-aminomethanol, organic acids, inorganic anions and CO2 were formed. In our previous study, TZ was found to be firstly oxidized by Shinella sp. NJUST26 to form 2,4-dihydro-[1,2,4]triazol-3-one by mono-oxidation. After that, the ring was cleaved to form another intermediate namely N-hydrazonomethyl-formamide, which was further biodegraded to semicarbazide and urea (Wu et al., 2016). Based on the above analysis, it could be inferred that TZ biodegradation mechanism by Raoultella sp. NJUST42 was quite different from these previous investigations. The new TZ biodegradation pathway obtained in this study will provide a broader perspective and more possibilities for the future exploration of TZ biodegradation system.
Substrate concentration has a remarkable impact on the biodegradation of various toxic or recalcitrant contaminants (Bai et al., 2008). As the initial TZ concentration increased from 50 mg L−1 to 400 mg L−1, DT50 increased from 82.5 ± 9.1 h to 247.5 ± 20.8 h and the k value decreased from 0.0084 ± 0.0004 to 0.0028 ± 0.0004 h−1 based on the fitting of TZ removal profile to the first-order degradation kinetics (Fig. 4a). These results indicated that TZ was an inhibitory substrate, especially at high TZ concentration. Similar conclusion could be obtained in our previous study (Wu et al., 2016), where the time required for complete TZ removal increased obviously with the increase of initial TZ concentration. Incubation temperature is the main factor affecting the biodegradation of refractory compounds (Kang et al., 2012). As the incubation temperature raised from 20 °C to 30 °C, the k value increased from the initial 0.0039 ± 0.0001 h−1 to 0.0065 ± 0.0002 h−1. However, when the temperature was further increased to 40 °C, the k value decreased to 0.0033 ± 0.0002 h−1. Correspondingly, DT50 value was initially decreased from 177.7 ± 12.5 h to 106.6 ± 8.1 h, and then increased to 210.0 ± 14.1 h as the temperature continued to rise to 40 °C (Fig. 4b). The above results indicated that the optimal incubation temperature for TZ biodegradation was 30 °C, and too high or too low incubation temperature would adversely affect TZ biodegradation. Similar phenomenon has been discovered by Sun et al. (2009), where quinoline could be most efficiently biodegraded by Pseudomonas strain BW003 at 30 °C. The pH value of MSM often plays a significant role for the biodegradation of various recalcitrant compounds (Singh et al., 2003). As pH increased from 5.0 to 7.0, the k value increased from 0.0032 ± 0.0002 67
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Fig. 4. The effect of initial TZ concentration (a), temperature (b), initial pH (c) and additional glucose concentration (d) on TZ biodegradation by NJUST42.
to 0.0063 ± 0.0005 h−1, but as pH further increased to 10.0, the k value decreased to 0.0019 ± 0.0002 h−1. In contrast, when the pH value increased from 5.0 to 7.0, DT50 value decreased significantly from 216.6 ± 17.4 h to 110.0 ± 12.5 h and with pH increased to 10.0, DT50 increased to 364.7 ± 28.4 h (Fig. 4c). Actually, pH value of MSM was rather important for the activity of enzyme involved in the biodegradation process. Both acidic and alkaline condition was unfavorable for the biodegradation of various contaminants such as TZ. pH dependence was also observed in the pyridine biodegradation system inoculated by Shewanella, where pyridine biodegradation could be delayed and even inhibited under both acidic or alkaline pH conditions (Mathur et al., 2008). According to Lin et al. (2010), the effect of additional carbon sources on the biodegradation of various recalcitrant compounds was strongly dependent on the dosage of additional carbon source. When the glucose concentration was 500 mg L−1, the k value reached a maximum value of 0.0069 ± 0.0006 h−1, which was obviously higher than the control experiment without additional carbon source (Fig. 4d). However, as the glucose dosage increased to 2000 mg L−1, the k value decreased to as low as 0.0031 ± 0.0004 h−1. Correspondingly, the DT50 value reached a minimum value of 100.4 ± 8.4 h at the glucose dosage of 500 mg L−1 and a maximum value of 223.5 ± 18.4 h at the glucose dosage of 2000 mg L−1. These results indicated that low dosage of additional carbon source was positive for TZ biodegradation, while high dosage of additional carbon source was negative, probably due to the competitive inhibition exerted by additional carbon source (Wang et al., 2018).
was capable of utilizing TZ as the sole carbon source and nitrogen source, was isolated successfully from acclimated activated sludge. TZ removal was accompanied by NH4+ release, pH increase, biomass increase, TOC removal, biotoxicity reduction and EEM variation, indicating TZ mineralization by NJUST42. Based on the identification of biodegradation intermediates, a new TZ biodegradation pathway including hydroxylation, carbonylation, carboxylation and ring cleavage, was proposed for the first time. Acknowledgements This research is financed by Natural Science Foundation of Jiangsu Province for Distinguished Young Scholars (BK20170038) and National Natural Science Foundation of China (51478225). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biteb.2019.02.007. References Amorim, C.C., Bottrel, S.E., Costa, E.P., Teixeira, A.P., Leao, M.M., 2013. Removal of ethylenthiourea and 1,2,4-triazole pesticide metabolites from water by adsorption in commercial activated carbons. J. Environ. Sci. Health B 48, 183–190. Bai, Y.H., Sun, Q.H., Zhao, C., Wen, D.H., Tang, X.Y., 2008. Microbial degradation and metabolic pathway of pyridine by a Paracoccus sp. strain BW001. Biodegradation 19, 915–926. Cai, Z.Q., Shi, S., Li, S.S., Yang, B.K., Chen, Q.L., Zhao, X.Y., 2013. Microbial degradation characteristics and kinetics of novel pyrimidynyloxybenzoic herbicide ZJ0273 by a newly isolated Bacillus sp. CY. Environ. Sci. Pollut. Res. 20, 8831–8838. Cai, Z.Q., Zhang, W.J., Li, S.S., Ma, J.T., Wang, J., Zhao, X.Y., 2015. Microbial degradation mechanism and pathway of the novel insecticide paichongding by a newly isolated Sphingobacterium sp. P1-3 from Soil. J. Agric. Food Chem. 63, 3823–3829.
4. Conclusions In this study, a novel strain namely Raoultella sp. NJUST42, which 68
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