Microaerobic degradation of 2-Mercaptobenzothiazole present in industrial wastewater

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Microaerobic degradation of 2-Mercaptobenzothiazole present in industrial wastewater Umamaheswari B. a,∗ , Rama Rajaram (Retired Chief Scientist) b a b

Environmental Technology Division, Central Leather Research Institute, Adyar, Chennai 600 020, Tamil Nadu, India Biochemistry Laboratory, Central Leather Research Institute, Chennai, 600 020, Tamil Nadu, India

h i g h l i g h t s • • • •

The isolated strain CSMB1 utilizes benzothiazole and its derivatives as sole carbon and energy. Benzene ring opening of benzothiazoles is through meta cleavage under microaerobic condition. A microaerobic degradative pathway was proposed for 2-Mercaptobenzothiazole. The strain CSMB1 is an ideal candidate for leather industrial wastewater treatment.

a r t i c l e

i n f o

Article history: Received 27 June 2016 Received in revised form 26 September 2016 Accepted 26 September 2016 Available online xxx Keywords: Microaerobic condition 2-Mercaptobenzothiazole Alcaligenes sp. Catechol 2 3 Oxygenase Degradation pathway

a b s t r a c t Microaerobic degradation of 2-Mercaptobenzothiazole (2-MBT) was investigated using an isolated bacterial strain CSMB1. It was identified as Alcaligenes sp. MH146 by genomic analysis. The isolate degraded 50 mg/L concentration of 2-MBT which was measured in terms of Total organic carbon (TOC) (700 mg/L). A maximum degradation of 86% with a residual TOC concentration of 101 mg/L was obtained after 72 h, with the biomass growth of 290 mg/L. The presence of specific activity of catechol 2, 3 oxygenase was observed in all the tested derivatives of benzothiazoles and the benzene ring opening was observed through meta cleavage. By analyzing the 72 h incubated culture supernatant, 2-MBT, and all its biotransformed products were degraded into polar compounds. With the analytical results obtained, a possible microaerobic degradative pathway was proposed and illustrated for 2-MBT. It is concluded that microaerophilic isolate CSMB1 was able to degrade 2-MBT and its intermediates by utilizing them as sole carbon and energy. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Benzothiazoles are heterocyclic aromatic compounds, useful in multiple applications due to their biological and pharmacological properties with their bicyclic ring system. 2Mercaptobenzothiazole (2-MBT) is used as a vulcanization accelerator in the rubber processing industry [1,2] and corrosion inhibition agent in cooling systems. 2-Thiocyanomethylthiobenzothiazole (TCMTB), one of the derivatives of 2-MBT [3] is used as a preservative to prevent hides and skins from deterioration during transportation and storage [4,5]. Direct discharges of 2-MBT occur in effluents from industries producing rubber products [6] and in leather industrial wastewater [7]. 2-MBT is non-volatile and

∗ Corresponding author. E-mail address: [email protected] (U. B.).

has low water solubility. It is a toxic xenobiotic and a persistent substance in soil hence becomes, highly resistant to biological treatment [8]. Depending on soil conditions, the half-life of 2-MBT ranges from 92 to 248 days. 2-MBT and its derivatives are toxic to living forms including human beings [9]. According to the standardized Microtox test performed [10], for an exposure time of 15 min, the 50% toxic concentration was 1.5 ␮M for 2-MBT. Recently, the occurrence of benzothiazoles has been detected in exhaled human breath [11]. The advanced oxidation process (AOP) is one of the pretreatment options suggested, but the residuals cannot be decomposed biologically, and thus alternative methods need to be used [12]. In activated sludge systems, 2-MBT was resistant and degraded only at concentrations around 20 mg/L [13–15]. In a twostage anaerobic/aerobic biological treatment, 2-MBT was found to be refractory to anaerobic treatment. Brownlee and Drotar [16,17] observed that 2-MBT was transformed into methylated metabolite (MTBT) by Corynebacterium sp., Pseudomonas sp., and Escherichia

http://dx.doi.org/10.1016/j.jhazmat.2016.09.061 0304-3894/© 2016 Elsevier B.V. All rights reserved.

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coli. In recent times, there is growing interest in the bioremediation of toxic compounds under microaerobic conditions. The organisms utilize C and N sources by different metabolic strategies based on the availability of O2, switching between growth modes [18]. The microaerophilic microorganisms were found to be provided with metabolic versatility [19]. Less energy requirement for blower operation, minimum sludge production, recovery from organic shock loads are some of the possible advantages of microaerobic treatment systems [20–22]. It overcomes the major limitation of high operational costs of the aerobic process [23,24]. The possibility of the biodegradation of 2-MBT by a pure isolated strain Alcaligenes sp. MH146 strain CSMB1 has been investigated under the microaerobic condition without any external supplement of carbon and nitrogen. The mechanism of degradation was studied using UV–vis spectroscopy, enzymatic assays, HPLC and GC–MS analysis. With the results obtained a microaerobic degradative pathway for 2-MBT was proposed.

2. Materials and methods 2.1. Chemicals Catechol, Benzothiazole, 2-Hydroxybenzothiazole, 2-Methyl benzothiazole, 2-Mercaptobenzothiazole 2-Methyl thiobenzothiazoles and 2-amino benzothiazole were procured from SigmaAldrich, India. 2-(thiocyanomethylthio)-benzothiazole, (TCMTB) was provided by a tannery in Chennai, India. The pesticide 2-MBT is a light yellow powder with a faint odor. The molecular weight and molecular formula are 167.2513 g/mol and C7 H5 NS2 respectively. The culture medium used for the study is a mineral salt medium containing (g/l): K2 HPO4, 1.2; KH2 PO4 , 0.3; MgCl2 ·7H2 O, 0.5; NaCl 1.0; CaCl2 ·2H2 O, 0.2; FeSO4 ·7H2 O, 0.02; and 10 ml of trace elements. All medium components of analytical grade were procured from E.Merck Mumbai (India). All the solutions were prepared in Milli-Q water.

2.2. Microorganism The bacterial strain used in this study was isolated from microaerobic reactor present in our laboratory, fed with phenol as sole carbon. For biodegradation of 2-MBT, a microaerophilic Alcaligenes sp. MH146 strain CSMB1 was selected since it is capable of utilizing 2-MBT (50 mg/L) as the sole source of carbon and nitrogen even after repeated purification. It was identified by 16S rRNA gene analysis as Alcaligenes sp. MH146 with an NCBI accession number GenBank ID: FJ626617.1. It is one among the six bacterial isolates capable of degrading a mixture of heterocyclic compounds in the consortium. The isolated bacterial strains were deposited in MTCC, IMTECH, India for patent filing [25]. For enrichment of 2-MBT and for degradation studies, a specially designed laboratory scale bioreactor (3.5 l) equipped with biosensors for automatic control to maintain microaerobic condition was used.

2.3. Analytical methods For growth determination, the cells in the cultures (50 ml) were harvested by centrifugation (12,000g) at 4 ◦ C for 10 min and centrifuged after washing with milli-Q water. The cells were then dried at 60 ◦ C overnight for 24 h until a constant weight was obtained. The analytical methods pertaining to the degradation studies were estimated as per Standard Methods [26], namely, Total Organic Carbon (TOC) (5310-B), Nitrogen Ammonia, (4500-NH3-C) and Sulfate, (4500-SO4-E).

2.4. Degradation assay Studies were conducted with 50 mg/L concentration of 2MBT. This concentration corresponds to the concentration that commonly present in the leather industrial effluents. 2-MBT biodegradation was monitored by estimating in terms of TOC and the growth of the isolated strain CSMB1 was also measured. The degradation profile of 2-MBT was observed using UV–vis spectrophotometer (Shimadzu UV2450) by wavelength scan spectra. Identification of intermediate metabolites was inferred based on the spectrum of pure compounds that was read against buffer blanks at their respective (␭max ) wavelengths (2-MBT, 310 nm 2-MeBT, 249 nm 2-MTBT, 279 nm 2-BT, 251 nm 2-ABT, 260 nm 2-OHBT 278 nm and BTSA 310 nm). The inhibitory effect on the growth of Alcaligenes sp. MH146 strain CSMB1 at different initial concentrations of 75, 100 and 125 mg/L of 2-MBT was evaluated. For tests on the metabolic versatility, the utilization of the various benzothiazole derivatives such as TCMTB, 2-BT, 2-MTBT, 2-ABT 2-OHBT and 2-MeBT by Alcaligenes sp. MH146 strain CSMB1 was evaluated. In this case, the mineral medium was substituted with the respective substrate as sole carbon and nitrogen source at 50 mg/L concentration.

2.5. Enzyme activity assays Cells of the strain Alcaligenes sp. MH146 strain CSMB1 were grown in mineral medium supplemented with 2-MBT and harvested at mid-log phase. The cells were washed twice with phosphate buffer (pH 7.0) and sonicated on ice using a Digital Sonifier Model 250 (Branson, USA). The lysate was centrifuged at 12,000 × g for 30 min. The cell-free extract thus obtained was either stored at −40 ◦ C or used immediately for enzyme assays. Catechol 1, 2 oxygenase (EC 1.13.11.2) and Catechol 2,3 Oxygenase (EC 1.14.13.1) activity was measured by monitoring the formation of either cis, cis-muconic acid or 2-HMSA at 260 or 375 nm respectively [27]. The reaction mixture (3 ml) contained a Tris-HCl buffer (50 mM, pH 8.0), catechol (1 mmol) and cell extract (100 ␮l). Readings were taken at 30 s intervals for 5 min at room temperature. Controls without substrate or cell extract were prepared for each assay. One enzyme unit is defined as the amount of enzyme that catalyzes the formation of 1 ␮mol of product per minute or consumption of the substrate. Protein concentration was determined according to the method of Bradford [28].

2.6. HPLC analysis The 2-MBT culture supernatant was taken after every 12 h of incubation and centrifuged at 12,000 × g for 20 min. The metabolites formed after 2-MBT degradation was extracted two times using an equal volume of ethyl acetate, dried over anhydrous Na2 SO4 and concentrated in a rotary vacuum evaporator. The ethyl acetate extract in the flask was dissolved in methanol. The culture supernatant was analyzed by HPLC (Shimadzu VP series Model LC10ADVP) on Phenomenex Luna C18 reversed-phase column, 5 ␮m particle size, equipped with Photodiode Array UV–vis Spectrophotometric Detector (200–500 nm). The mobile phase was 80:20 acetonitrile/buffered aqueous solutions. The buffered aqueous solution was prepared with 4 ml phosphoric acid, 25 ml methanol in 1-l milli-Q water operated with a flow rate of 1.0 ml/min and 15 min run time at a maximum absorption wavelength of 310 nm to detect 2-MBT. Samples of 10 ␮l were injected into the Rheodyne injector port.

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350 TOC mg/L

700

Growth mg/L

300

600

250

500

200

400 150

300

100

200

50

100

Growth by dry cells (mg/L)

TOC Concentration (mg/L)

800

3

0

0 0

12

24

36 48 Time (h)

60

72

84

Fig. 1. TOC Reduction and growth of the isolate Alcaligenes sp. MH146 strain CSMB1 on utilization of 2-MBT.

2.7. GCMS analysis The ethyl acetate extracts of cell-free medium were analyzed by GCMS for mass analysis. The instrument used was a JEOL-GC-mateII benchtop double-focusing GC mass spectrometer operating in electron impact ionization (EI) mode. Helium was used as the carrier gas with a flow rate of 25 ml/min. The injector temperature was maintained at 220 ◦ C (temperature range 70–250 ◦ C), and the rate of increase in temperature was set to 15 ◦ C/min. The ethyl acetate extract of the products was dissolved in methanol and subjected to GC–MS analysis. Identification of the compounds was in accordance with retention time and mass fractions. 3. Results and discussion 3.1. Isolation A pure bacterial strain capable of utilizing 2-MBT as sole carbon and nitrogen was isolated after a month of selective enrichment. The microaerophilic bacterial strain Alcaligenes sp. MH146 strain CSMB1, when streaked on MBT mineral agar plates, grew as a thin matt with irregular colonies. It was rod-shaped, Gram-negative, non-sporing, non-capsulated and motile in nature. It gave positive reactions for catalase, oxidase, citrate utilization and nitrate reduction. It was negative for Voges-Proskauer and urease activities. 3.2. Degradation Biodegradation studies at 50 mg/L of 2-MBT, corresponding to a TOC concentration of 700 mg/L was carried out at microaerobic conditions and at an optimized pH of 7 and temperature of 30 ◦ C with the isolated strain. As can be seen from Fig. 1, growth and MBT degradation were strongly correlated. The utilization of 2-MBT by the isolated strain was confirmed by an increase in its growth with incubation time, along with a corresponding decrease in the total organic carbon content. A maximum degradation of 85–90% was observed after 72 h of incubation. The residual TOC concentration obtained was 101 mg/L with the biomass growth of 290 mg/L measured as dry cells. No significant reduction in TOC content was noticed after this period of incubation with a reduction in biomass content. Metabolism of 2-MBT by resting cells of Rhodococcus rhodochrous OBT18 was reported by Haroune [29]. In their study, 2-MBT was biotransformed into four metabolites with 30% degradation. But, under the microaerobic condition, cells of Alcaligenes sp. MH146 strain CSMB1 utilize 2-MBT with the liberation of 65 mg/L of ammonia nitrogen and 195 mg/L of sulfate in 72 h. Sulfur could not be quantified, but it was observed as white luminescence under phase contrast microscope in dark at 40 × magnifications (Fig. 2) De Wever experiments [30] on anoxic conditions showed that the iso-

Fig. 2. Phase contrast microscopic image of illuminating sulfur particles in Dark at 40 × magnification.

late Rhodococcus erythropolis BTSO31 turned the culture medium into yellow with a reddish precipitate as a dead end product due to oxygen transfer limitations in the sludge flocs. Similar to their observation, changes in color was observed in mineral agar plate amended with 2-MBT. When cells of Alcaligenes sp. MH146 strain CSMB1 was pour plated with 2-MBT, yellow color was observed on the third day, which then changed to orange-red on the fifth day, followed by a mat of bluish green color on the seventh day as depicted in Fig. 3(a) and (b). According to Czechowski and Rossmoore [31], the color change might be due to the sulfhydryl group of 2-MBT, which complexes with an essential divalent cation such as iron or copper that is presumed to be present in the enzyme dehydrogenase. It is also suggested that [26] the thiazole ring of sulfur is released as thiosulfate and bisulfite and subsequently oxidized to sulfur. It is followed by the formation of thiocyanate by the reaction of elemental sulfur at the microaerobic condition. A blood red color is formed, when thiocyanate complexes with ferrous, and produce a ligand, ferrous isothiocyanate [Fe(NCS)(H2 O)5 ]2 (stereoisomer) [32] and it gives a blue complex with Co2 +. 3.3. Effects of initial MBT concentration The effect of initial concentration of 2-MBT on growth and degradation by Alcaligenes sp. MH146 strain CSMB1 is shown in Fig. 4. The results indicated that in the presence of TOC concentration of 800 mg/L, Alcaligenes sp. MH146 strain CSMB1 was able to degrade up to 86% of TOC content after 72 h of incubation. Above this TOC concentration, substrate inhibition has been observed, where the activity of the microbes reduced to the level of about 38.5% and 14%. 3.4. Metabolic versatility To find out the metabolic versatility of benzothiazole derivatives, the isolated strain Alcaligenes sp. MH146 strain CSMB1 was inoculated in mineral medium with 50 mg/L concentrations of respective substrates as the sole sources of carbon and nitrogen (Fig. 5a & b). When the isolated strain streaked in mineral agar plates with respective benzothiazoles derivatives, it grew as white, yellow and red colonies correlating to the formation of different secondary metabolites as shown in Fig. 5(a). The isolate (10%), when inoculated in the culture medium amended with 2-ABT and TCMTB, showed a maximum growth of 232 mg/L and 210 mg/L in 72 h. The utilization of other benzothiazole derivatives by the isolate followed the order; BT < 2-OHBT < 2-BTSA < 2-MeBT < 2-MTBT. A small

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Growth by dry cells (mg/L)

TCMTB

MTBT

OHBT

BT

ABT

MeBT

BTSA

250 200 150 100 50 0 0

12

24 36 48 60 Incubation Time (h)

72

84

96

Fig. 5. Effect of metabolic diversity on Alcaligenes sp. MH146 strain CSMB1 amended with Benzothiazole (BT), 2-Hydroxybenzothiazole (2-OHBT), 2-Methyl benzothiazole (2-MeBT), 2-Mercaptobenzothiazole (2-MBT) 2-Methyl thiobenzothiazoles (2-MTBT) and 2-(thiocyanomethylthio)-benzothiazole (2-TCMTB) (a) Secondary metabolites observed in pour plate in Mineral agar and (b) Growth potential of the strain in mineral broth.

Fig. 3. Pour plate in mineral agar amended with 2-MBT showing Alcaligenes sp. MH146 strain CSMB1colonies (A) 5th day – orange red (B) 7th day – bluish green. The color represents the degraded metabolites of 2-MBT (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

increase in growth rate of the strain with 2-ABT and TCMTB may be due to the possible exposure of the strain with these chemicals, which are present in leather tannery effluents. From these results, it was confirmed that the Alcaligenes sp. MH146 strain CSMB1 was capable of utilizing all the tested benzothiazole derivatives as a sole source of carbon and nitrogen for growth. Similar to Alcaligenes sp. MH146 strain CSMB1, Rhodococcus strains were able to biotransform 2-ABT, BT, 2-OHBT, BTSA and 2-MBT [33–35]. It was reported by Bunescu [36,37] that 26% degradation of 2-ABT was observed by Rhodococcus rhodochrous OBT18 cells after 125 h. Biodegradation of 2-BTSA by activated sludge was reported by Mainprize [38]. 3.5. Biodegradation pathway

TOC degradation (%)

100 80 TOC-800mg/L TOC-1000mg/L

60

TOC-1200mg/L

40 20 0 0

20 40 60 Incubation Time (h)

80

Fig. 4. Effect of different initial concentration of 2-MBT on Alcaligenes sp. MH146 strain CSMB1.

3.5.1. UV–vis spectroscopy The UV–vis spectra obtained with the 2-MBT amended culture supernatant are shown in Fig. 6. It was observed that maximum absorption peak (␭max ) for 2-MBT (100 mg/L) at 0 h was 2.519 at 310 nm. The spectrum observed after 24 h incubation, showed two biotransformed products with (␭max ) 249 and 251 nm with absorption peak height at 1.833 and 1.815 corresponding to 2-MeBT and 2-BT. At 48 h, two new peaks were observed at 260 and 278 nm with absorption peak height of 1.377 and 0.943 corresponding to 2-ABT and 2-OHBT. After 72 h of incubation, no prominent peaks were visible. With the results obtained, it was inferred that 2-MBT is first biotransformed into 2-MeBT and 2-BT and then further into 2-ABT and 2-OHBT. Reduction in peak heights from 24 h to 48 h confirmed the reduction in concentration of 2-MBT. Absence of significant peaks at 72 h indicated that the biotransformed products were further degraded into polar compounds of low molecular weights. But in the case of De Wever H and H. Verachtert [39] experiments

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Fig. 6. UV–Vis Spectrum scan at different incubation 0–72 h showing the degraded metabolites of 2-MBT at different nanometers. 0h-2-MBT, 24h-2-MeBT, 36h-2-BT, 48 h −2-OHBT, 72h-2-ABT. Table 1 The Specific activity of Catechol 2, 3-Oxygenase by Alcaligenes sp. MH146 strain CSMB1 cells treated with different benzothiazole compounds. Benzothiazole compounds

Protein concentration in cell extract (␮g/ml)

Catechol 2,3-Oxygenase specific activity (U/mg)

TCMTB 2-MBT 2-MTBT 2-MeBT 2-ABT 2-OHBT 2-BT 2-BTSA

150 132 110 128 144 146 144 128

2.38 2.78 2.61 2.45 2.67 2.2 2.05 2.45

with 2-MBT enriched sludge, when exposed to a concentration of 200 mg/ L of 2-MBT though resulted in polar compounds after 95 days but still no pure cultures could be obtained. 3.5.2. Enzyme activity Experiments were conducted with cell extracts of Alcaligenes sp. MH146 strain CSMB1 that were grown on 2-MBT for the presence of enzyme activities. Table 1 shows the specific activities of catechol 2, 3-dioxygenase acting on catechol as a substrate by the cells grown on 2-MBT. It was found to be 2.78 U/mg of protein. But catechol 1, 2-dioxygenase was not detectable. The presence of catechol 2, 3-dioxygenase activity confirmed the degradation of 2-MBT through catechol ring opening. Catechol 2, 3-dioxygenase transforms catechol into dihydroxy benzothiazole by the cleavage of a catechol ring. Alcaligenes sp. MH146 strain CSMB1 also exhibited the presence of catechol 2,3-dioxygenase activity when amended with different benzothiazoles on the minimal medium such as TCMTB, 2-MTBT, 2-MeBT, 2-ABT, 2-OHBT, BT and 2-BTSA. Similar to our results Rhodococcus pyridinovorans strain PA grown on OHBT and BT mineral salts medium showed catechol 2, 3-dioxygenase activity [40]. But, in the case of metabolism of styrene by Rhodococcus rhodochrous NCIMB 13259, the activity of catechol 1,2-dioxygenase was noticed [41].

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3.5.3. HPLC chromatogram The biodegradation of 2-MBT (50 mg/L) by the cells of Alcaligenes sp. MH146 strain CSMB1 was analysed using HPLC. As observed in Fig. 7(a) and (b), the concentration of 2-MBT (retention time, 8.2 min) was found to decrease with incubation times, and it completely disappeared after 48 h of incubation. Two new metabolites appeared along with 2-MBT after 12 h of incubation (Fig. 7a). The retention time of these compounds (2.5 and 5.7 min) was similar to that of 2-BT and 2-MeBT and its identity was confirmed by coinjection with the pure product of 2-BT and 2-MeBT. After 24 h incubation, (Fig. 7b) a metabolite was observed (retention time 4.1 min) similar to 2-MTBT, with the disappearance of 2-MeBT. In addition the 36 h culture sample (Fig. 7c), exhibited two new peaks and the retention times were shorter (1.8 and 2.9 min) indicating greater polarity and these are probably bisulfite and thiosulfate. Similar to our studies, De Wever [42] had reported on the simultaneous elimination of the sulfonate group and the introduction of the hydroxyl group in their experiments on BTSO3 degradation by Rhodococcus erythropolis strain. With the disappearance of above intermediates, two new metabolites appeared after 48 h of incubation (Fig. 7d). The retention times of these compounds (4.9 and 5.8 min) were similar to that of 2-OHBT and 2-ABT, and their identities were confirmed by co-injection with the pure product of 2-OHBT and 2-ABT onto the HPLC column. Peak height was also reduced considerably, indicating degradation of 2-MBT within 48 h. Chromatogram results of the 60 h incubated sample (Fig. 7e) indicated 75–80% degradation in 2-ABT and a single peak corresponding to 2-OHBT was observed. No prominent peaks were observed with supernatant tested after 72 h of incubations. 3.5.4. GC–MS spectrum The 2-MBT amended mineral culture supernatant samples incubated with Alcaligenes sp. MH146 strain CSMB1 were analyzed by gas chromatography. The gas chromatogram of the biodegraded 2MBT culture exhibited peaks between 12 and 22 min. It was further analyzed by mass spectroscopy for the presence of the corresponding peak that was observed in the gas chromatogram at 20.05 min. Fig. 8(a) shows that the peaks that appeared with mass/charge ratios of 54 and 80 correspond to the solvent fragments. The mass/charge ratios of 137 appear to be the protonated BT-molecule (m/z 136); it was found in the daughter ion spectra of all benzothiazoles bearing a sulfur in the 2-position. The mass/charge ratios m/z 165 is due to the formation of 2-MBT radical cation from which CS is eliminated and m/z 123 or m/z 95 is formed. The mass spectrum of the 48 h sample (Fig. 8b) shows m/z 54 of high intensity (in the gas chromatogram at 21.75 min), which might be due to thiocyanate ion, m/z 106 of low intensity due to loss of HCN (Fig. 8b). In the mass spectrum of 48 h culture, the molecular ion MH1, MH2, had a mass/charge ratio of 163 and 176 since the initial fragment of high intensity related to 2-OHBT (151) and 2-ABT (150) combined with the allyl fragments from the solvent ethyl acetate and methane. In the 72 h sample (Fig. 8c), only 2-OHBT (151) appeared (in the gas chromatogram at 12.97 min) with a high intensity and with the mass/charge ratios m/z 201 by combining with the allyl fragments was obtained. The result is similar to the study by Junker [43] on desulfonation of 2-aminobenzenesulphonic, benzene sulphonic and 4-toluene sulphonic acids by Alcaligenes sp. strain O-1. He suggested that desulfonation occurs either by an NADH-linked dioxygenation, or monooxygenation, or hydrolytic desulfonation and is associated with meta ring cleavage. 3.5.5. Proposed degradative pathway Based on the studies conducted, a proposed degradative pathway of 2-MBT by Alcaligenes sp. MH146 strain CSMB1 is shown in Fig. 9. 2-MBT is oxidized to 2-BT, and in parallel, methylated to 2-MeBT. The disappearance of 2-MeBT resulted in the production

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Fig. 7. HPLC Chromatograms observed with supernatant of 2-MBT culture medium collected at different incubating hours such as (a) 12 h, (b) 24 h, (c) 36 h, (d) 48 h, (e) 60 h.

of 2-MTBT due to cleavage of functional group-thiol. By desulfurylation, 2-MTBT is biotransformed into 2-ABT. The degradation of 2-ABT led to the liberation of sulfite and ammonia. The sulfite was subsequently oxidized to sulfur which was followed by the formation of thiocyanate. BT was then hydroxylated into 2-OHBT and further hydroxylated into catechol. Through meta cleavage of catechol ring opening, 2-MBT may enter the Krebs cycle.

4. Conclusion Biodegradation of 2-Mercaptobenzothiazole (2-MBT) was investigated using an isolated Alcaligenes sp. MH146 strain CSMB1, after enrichment with 2-MBT. The results obtained herein revealed that the pure strain Alcaligenes sp. MH146 strain CSMB1 was able to utilize the recalcitrant pollutant 2-MBT, as sole carbon and nitrogen

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Fig. 8. GC–Mass spectrum of mass/charge ratios observed with supernatant of 2-MBT culture medium collected at different incubating hours such as (a) 24 h, BT (m/z136) 2-MBT (m/z 167) (b) 48 h −2-OHBT (m/z 163), 2-ABT (m/z 176), (c) 72 h 2-OHBT (m/z201).

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2 - MBT N n tio ida Ox

Me thy lat ion

S SH

BT

2 - MeBT N

S H3C N

S Hydroxylation

Thiol cleavage

2 - MTBT

2 - OHBT N

N

S OH

S

Hydroxylation

S

Desulfurylation

Catechol

2 - ABT

OH

N

OH

2,3 Oxygenase enzyme activity

S NH2

Ammonia

O

N H

H

H

Sulfite

bi sulfite O S

2-

S H

O

O

O

O

Meta cleavage

Sulfur

Thiocynate

Kreb cycle

S¯

C

N

Fig. 9. Postulated microaerobic degradative pathway of 2-MBT by the isolated strain Alcaligenes sp. MH146 strain CSMB1.

for its growth under microaerobic condition. The isolate utilized 50 mg/L concentration of 2-MBT (TOC–700 mg/L) with 89% degradation at 72 h which is measured as TOC content and showed exponential growth with biomass production of 290 mg/L. The presence of the catechol 2, 3 oxygenase activities was observed in all the other tested intermediates and confirmed that the benzene ring opening was through meta cleavage. The analyses of culture supernatant collected after 72 h of incubation with 2-MBT confirmed that all the biotransformed products were degraded into polar compounds. With the results obtained, a degradative pathway was derived. The results obtained confirmed that Alcaligenes sp. MH146 strain CSMB1 may be an ideal candidate for commercial application.

Acknowledgements The authors wish to thank Council of Scientific & Industrial Research (CSIR), India to undertake the study under the STRAIT – XII Five Year Plan Network project. The authors would also like to thank the Director, Central Leather Research Institute (CLRI) India for permitting to publish this work.

References [1] S.T. Nawrocki, K.D. Drake, C.F. Watson, G.D. Foster, K.J. Maier, Comparative aquatic toxicity evaluation of 2-(thiocyanomethylthio)benzothiazole and selected degradation products using Ceriodaphnia dubia, Arch. Environ. Contam. Toxicol. 48 (2005) 344–350. [2] E. Fries, Determination of benzothiazole in untreated wastewater using polar-phase stir bar sorptive extraction and gas chromatography?mass spectrometry, Anal. Chim. Acta 689 (2011) 65–68. [3] L. Muthusubramanian, V.S. Rao, R.B. Mitra, Efficient synthesis of 2-(thiocyanomethylthio) benzothiazole, J. Clean. Prod. 9 (2001) 65–67. [4] R.L. Hinojosa, K. Wróbel, Indirect extraction-spectrophotometric determination of 2-(thiocyanomethylthiol) benzothiazole in chrome tanning liquors after its breakdown to 2-mercaptobenzothiazole, Talanta 56 (2002) 515–521. [5] T. Reemtsma, B. Zywicki, M. Stueber, A. Kloepfer, M. Jekel, Removal of sulfur organic polar micropollutants in a membrane bioreactor treating industrial wastewater, Environ. Sci. Technol. 36 (2002) 1102–1106. [6] C.M. Reddy, J.G. Quinn, Environmental chemistry of benzothiazoles derived from rubber, Environ. Sci. Technol. 31 (1997) 2847–2853. [7] T. Reemtsma, O. Fiehn, G. Kalnowski, M. Jekel, Microbial transformations and biological effects of fungicide-derived benzothiazoles determined in industrial wastewater, Environ. Sci. Technol. 29 (1995) 478–485. [8] J. Chudoba, F. Tucek, K. Zeis, Biochemischer abbau von benzthiazolderivaten, Acta Hydrochim. Hydrobiol. 5 (1977) 495–498. [9] FDA Approved Labeling Text for NDA 022421, U.S. Patent No. 4, 886, 812 (2009).

Please cite this article in press as: U. B., R. Rajaram, Microaerobic degradation of 2-Mercaptobenzothiazole present in industrial wastewater, J. Hazard. Mater. (2016), http://dx.doi.org/10.1016/j.jhazmat.2016.09.061

G Model HAZMAT-18067; No. of Pages 9

ARTICLE IN PRESS U. B., R. Rajaram / Journal of Hazardous Materials xxx (2016) xxx–xxx

[10] M.A. Malouki, G. Giry, P. Besse, B. Combourieu, M. Sancelme, F. Bonnemoy, A.M. Delort, Sequential bio-and phototransformation of the herbicide methabenzthiazuron in water, Environ. Toxicol. Chem. 22 (2003) 2013–2019. [11] D. García-Go´ımez, P. Martínez-Lozano Sinues, C. Barrios-Collado, G. Vidal-de-Miguel, M. Gaugg, R. Zenobi, Identification of 2-Alkenals, 4-Hydroxy-2-Alkenals, and 4-Hydroxy-2 6-Alkadienals in exhaled breath condensate by UHPLC-HRMS and in breath by real-time HRMS, Anal. Chem. 87 (2015) 3087–3093. [12] M. Melicher, J. Derco, L. Sumegová, M. Drtil, Ozone utilisation in decreasing water sources load with resistant and toxic substances, Acta Chim. Slov. 8 (2015) 65–69. [13] A. Kloepfer, M. Jekel, T. Reemtsma, Occurrence, sources, and fate of benzothiazoles in municipal wastewater treatment plants, Environ. Sci. Technol. 39 (2005) 3792–3798. [14] T. Reemtsma, S. Weiss, J. Mueller, M. Petrovic, S. Gonzalez, D. Barcelo, F. Ventura, T.P. Knepper, Polar pollutants entry into the water cycle by municipal wastewater: a European perspective, Environ. Sci. Technol. 40 (2006) 5451–5458. [15] H. De Wever, P. Besse, H. Verachtert, Microbial transformations of 2-substituted benzothiazoles, mini review, Appl. Microbiol. Biotechnol. 57 (2001) 620–625. [16] B.G. Brownlee, J.H. Carey, G.A. MacInnis, I.T. Pellizzari, Aquatic environmental chemistry of 2-(thiocyanomethylthio)-benzothiazole and related benzothiazoles, Environ. Toxicol. Chem. 11 (1992) 1153–1168. [17] A. Drotar, G.A. Burton, J.E. Tavernier, R. Fall, Wide spread occurrence of bacterial thiol methyltransferases and the biogenic emission of methylated sulfur gases, Appl. Environ. Microbiol. 53 (1987) 1626–1631. [18] G. Fuchs, M. Boll, J. Heider, Microbial degradation of aromatic compounds—from one strategy to four, Nat. Rev. Microbiol. 9 (2011) 803–816. [19] S. Zheng, H. Li, C. Cui, An upflow microaerobic sludge blanket reactor operating at high organic loading and low dissolved oxygen levels, Biotechnol. Lett. 33 (2011) 693–697. [20] D.H. Zitomer, J.D. Shrout, High-sulfate, high-chemical oxygen demand wastewater treatment using aerated methanogenic fluidized beds, Water Environ. Res. 72 (2000) 90–97. [21] Z. Zhou, W. Qiao, C. Xing, X. Shen, D. Hu, L. Wang, A micro-aerobic hydrolysis process for sludge in situ reduction: performance and microbial community structure, Bioresour. Technol. 173 (2014) 452–456. [22] T. Niu, Z. Zhou, X. Shen, W. Qiao, L.M. Jiang, W. Pan, J. Zhou, Effects of dissolved oxygen on performance and microbial community structure in a micro-aerobic hydrolysis sludge in situ reduction process, Water Res. 90 (2016) 369–377. [23] M. Daumer, F. Beline, F. Guiziou, M. Sperandio, Influence of pH and biological metabolism on dissolved phosphorus during biological treatment of piggery wastewater, Biosyst. Eng. 96 (2007) 379–386. [24] J.H. Guo, Y.Z. Peng, C.Y. Peng, S.Y. Wang, Y. Chen, H.J. Huang, Z.R. Sun, Energy saving achieved by limited filamentous bulking sludge under low dissolved oxygen, Bioresour. Technol. 101 (2010) 1120–1126. [25] B. Umamaheswari, R. Rama, K. Chitra, E. Ravindranath, K. Thirumaran, A microaerophilic bacterial consortium and use thereof for the simultaneous biodegradation of mixture of recalcitrants present in water, (2012) Indian Patent application No.3437. DEL2012. [26] L.S. Cleceri, A.E. Greenberg, A.D. Eaton, Standard Methods for the Examination of Water and Wastewater, twentieth ed., American Public Health Association American Water Works Association and Water Environment Association, Washington DC, USA, 1998.

9

[27] H.Y. Neujahr, J.M. Varga, Degradation of phenols by intact cells and cell free preparations of Trichosporon cutaneum, Eur. J. Biochem. 13 (1970) 37–44. [28] M.M. Bradford, Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [29] N. Haroune, B. Combourieu, P. Besse, M. Sancelme, A. Kloepfer, T. Reemtsma, H. De Wever, A.M. Delort, Metabolism of 2-mercaptobenzothiazole by Rhodococcus rhodochrous, Appl. Environ. Microbiol. 70 (2004) 6315–6319. [30] H. De Wever, K. Vereecken, A. Stolz, H. Verachtert, Initial transformations in the biodegradation of benzothiazoles by Rhodococcus isolates, Appl. Environ. Microbiol. 64 (1998) 3270–3274. [31] M. Czechowski, H.W. Rossmoore, The effect of selected industrial biocides on lactate metabolism in Desulfovibrio desulfuricans, Dev. Ind. Microbiol. 22 (1981) 794–804. [32] I. BERTIN, Inorganic and bio-inorganic chemistry Encyclopedia of Life Support Systems, Vol. I, 2009, pp. 136–137. [33] M.A. Gaja, J.S. Knapp, The microbial degradation of benzothiazoles, J. Appl. Microbiol. 83 (1997) 327–334. [34] H. De Wever, H. Verachtert, Biodegradation and toxicity of benzothiazoles, Water Res. 31 (1997) 2673–2684. [35] H. De Wever, S. Van Der Neste, H. Verachtert, Inhibitory effect of 2-mercaptobenzothiazole on microbial growth in a variety of trophic conditions, Environ. Toxicol. Chem. 16 (1997) 843–848. [36] A. Bunescu, P. Besse-Hoggan, M. Sancelme, G. Mailhot, A.M. Delort, Comparison of microbial and photochemical processes and their combination for degradation of 2-aminobenzothiazole, Appl. Environ. Microbiol. 74 (2008) 2976–2984. [37] A. Bunescu, P. Besse-Hoggan, M. Sancelme, G. Mailhot, A.M. Delort, Fate of the Nitrilotriacetic acid Fe(III) complex (FeNTA) during phototransformation and biodegradation by Rhodococcus rhodochrous, Appl. Environ. Microbiol. 74 (2008) 6320–6326. [38] J. Mainprize, J.S. Knapp, A.G. Callely, Fate of benzothiazole-2-sulfonic acid in biologically treated industrial effluents, J. Appl. Bacteriol. 40 (1976) 285–291. [39] H. De Wever, H. Verachtert, 2-Mercaptobenzothiazole degradation in laboratory fed batch systems, Appl. Microbiol. Biotechnol. 42 (1994) 623–630. [40] N. Haroune, B. Combourieu, P. Besse, M. Sancelme, T. Reemtsma, A. Kloepfer, A. Diab, J.S. Knapp, S. Baumberg, A.M. Delort, Benzothiazole degradation by Rhodococcus pyridinovoransstrain PA: Evidence of a catechol 1,2-dioxygenase activity, Appl. Environ. Microbiol. 68 (2002) 6114–6120. [41] A.M. Warhurst, K.F. Clarke, R.A. Hill, R.A. Holt, C.A. Fewson, Production of catechols and muconic acids from various aromatics by the styrene-degrader Rhodococcus rhodochrous NCIMB 13259, Biotechnol. Lett. 16 (1994) 513–516. [42] H. De Wever, K. Vereecken, A. Stolz, H. Verachtert, Initial transformations in the biodegradation of benzothiazoles by Rhodococcus isolates, Appl. Environ. Microbiol. 64 (1998) 3270–3274. [43] F. Junker, T. Leisinger, A.M. Cook, 3-Sulfocatechol 2 3-dioxygenase and other dioxygenases (EC 1.13.11.2 and EC 1.14.12.) in the degradative pathways of 2-aminobenzenesulphonic, benzenesulphonic and 4-toluenesulphonic acids in Alcaligenes sp. strain O-1, Microbiology 140 (1994) 1713–1722.

Please cite this article in press as: U. B., R. Rajaram, Microaerobic degradation of 2-Mercaptobenzothiazole present in industrial wastewater, J. Hazard. Mater. (2016), http://dx.doi.org/10.1016/j.jhazmat.2016.09.061