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Biodesulfurization of benzonaphthothiophene by an isolated Gordonia sp. IITR100
International Biodeterioration & Biodegradation 104 (2015) 105e111
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Biodesulfurization of benzonaphthothiophene by an isolated Gordonia sp. IITR100 Ashok Kumar Chauhan a, Abrar Ahmad a, Surya Pratap Singh b, Ashwani Kumar a, * a Environmental Biotechnology Division, Indian Institute of Toxicology Research (Council of Scientific & Industrial Research), M.G. Marg, Lucknow 226001, India b Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 January 2015 Received in revised form 24 May 2015 Accepted 29 May 2015 Available online xxx
Studies on the desulfurization of three or more ringed-compounds, which are considered to inhibit biodesulfurization of crude oil, are rare. In this paper, desulfurization of a three-ringed compound benzo [b]naphtho[2,1-d]thiophene (BNT) by an isolated strain Gordonia sp. IITR100 is described. The bacterium mediates desulfurization of BNT and utilizes the released sulfur for its growth. The reaction is accompanied with the formation of metabolites BNT-sulfone and BNT-sulfinate, in addition to the reported BNT-hydroxide. Recombinant E. coli cells, harboring DszC or DszA, were also able to mediate the metabolism of BNT to BNT-sulfone, or of BNT-sulfone to BNT-sulfinate, respectively. Desulfurization of BNT, both by IITR100 and E. coli-DszC cells was strongly inhibited in the presence of dibenzothiophene. The results are discussed in the context of the biodesulfurization of petroleum fractions where several organosulfur compounds are present together. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Biodesulfurization Gordonia sp. IITR100 Benzonapthothiophene Dibenzothiophene Crude oil
1. Introduction Numerous organosulfur compounds are present in the petroleum products (Liu et al., 2010), and pose various serious problems e.g. (i) sulfurous emissions in the air upon their burning, causing health problems and acid rain (ii) souring of refinery equipment, and (iii) increase in viscosity of a large fraction of crude oil, rendering it non-amenable to refinery process (Korte and Boedefeld, 1978; Lewtas, 2007). Biodesulurization is an attractive option for reduction of the sulfur content of crude-oil and its fractions (Kilbane, 2006). Studies with the model compound dibenzothiophene (DBT) and its derivatives have revealed that the desulfurization occurs by a ‘4S’ pathway that includes serial activity of the enzymes DszC, DszA and DszB, resulting in the formation of hydroxy-biphenyl as end product (Gallagher et al., 1993; Oldfield et al., 1997; Santos et al., 2007; Mohebali and Ball, 2008). Genes for these proteins i.e. dszC, dszA and dszB have been characterized from several organisms, where these are present together as dszABC in an operon and are co-ordinately regulated (Denome et al., 1994;
* Corresponding author. E-mail addresses: [email protected] (A.K. Chauhan), abrar.ahmadg@ yahoo.com (A. Ahmad), [email protected] (S.P. Singh), ashwani.iitr26@gmail. com (A. Kumar). http://dx.doi.org/10.1016/j.ibiod.2015.05.024 0964-8305/© 2015 Elsevier Ltd. All rights reserved.
Mohebali and Ball, 2008). Pathway for the desulfurization of benzothiophene, at least in part, is analogous to the ‘4S’ pathway and formation of the metabolites BT-sulfoxide & BT-sulfone has been reported (Gilbert et al., 1998; Tanaka et al., 2001). Metabolism of BTsulfone is followed with the formation of benzo[c][l,2]oxathiin Soxide that undergoes transformation into either 2-(20 -hydroxypheny1) ethan-1-al (Kilbane, 2006) or o-hydroxystyrene (Tanaka et al., 2001), as seen in the strains Gordonia sp. 213E and Sinorhizobium sp. KT55, respectively. Recently, three genes designated as bdsABC, which are distinct from dszABC that were identified earlier for DBT-desulfurization, have been characterized from a Gordonia terrae strain C-6, whose products mediate the transformation of BT into o-hydroxystyrene (Wang et al., 2013). Many of the isolated organisms have also been shown to mediate the desulfurization of crude oil and its various fractions. In most cases, while the desulfurization of light-oil fractions is reasonable (~95%), the desulfurization of various crude oil is modest (~50%) (Kilbane, 2006; Mohebali and Ball, 2008; Bhatia and Sharma, 2010, 2012). It is now recognized that the desulfurization of heavy oils is inhibited progressively in the presence of increasing concentrations of the three- or more ringed-sulfur compounds (Choudhary et al., 2008). Studies on these compounds, however, are scarce. The organisms Bacillus subtilis WU-S2B (Kirimura et al., 2001) and Rhodococcus erythropolis XP (Yu et al., 2006) have been
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shown to utilize BNT as sulfur source, and the reaction in both cases was accompanied with the formation of BNT-hydroxide. But the intermediates formed during the process, or the enzymes involved in the activity were not studied. Kinetics of desulfurization of organosulfur compounds, when these are present individually, has been shown to be very different compared to when these are present along with other sulfur compounds. For example, a Rhodococcus sp. strain K1bD could mediate the desulfurization of DBT and 1,4-dithiane, when these were present individually in the growth medium. But, when the two were provided together only the desulfurization of DBT was observed (Kirkwood et al., 2005). Similarly, the desulfurization of benzothiophene or 1,4-dithiane, by Rhodococcus sp. strain JVH1, was delayed significantly in the presence of benzyl sulfide in the culture medium (Kirkwood et al., 2007). In another study, biodesulfurization of BNT, by a mixed culture, was decreased significantly in the presence of DBT (Jiang et al., 2014). In the present study, we evaluated the desulfurization of BNT by an isolated Gordonia sp. IITR100, when it was present either individually, or together with DBT, by isolated IITR100 and recombinant E. coli cells. 2. Materials and methods 2.1. Chemicals Benzo[b]naphtho[2,1-d]thiophene (BNT; CAS Number 239-350), dibenzothiophene (DBT), dibenzothiophene sulfone (DBT-sulfone), silylating mixture [ N,O-bis(trimethylsilyl) trifluoro acetamide (BSTFA) and trimethylchlorosilane (TMCS); 99:1], 2,6dichloroquinone-4-chloroimide, and acetonitrile, were purchased from SigmaeAldrich chemical co., St. Louis, USA. Crude oil was obtained from Indian Oil Corporation Limited, Faridabad, India. All the other chemicals used were of analytical grade. 2.2. Bacterium A bacterial strain Gordonia sp. IITR100, obtained earlier from a contaminated soil from around a refinery in Gujarat, India by selective enrichment on 4,6-dimethyl-DBT as sulfur source (Ahmad et al., 2014), was used. It harbors dszABC genes whose nucleotide sequence (KC693733.1) is >99% identical to those described from Gordonia alkanivorans strains IB (AY678116.1) and RIPI90A (EU364831.1). 2.3. Desulfurization activity The bacterial strain IITR100, grown in 20 ml medium1(Na2HPO4, 2.0 g; KH2PO4, 1.0 g; MgCl2.6H2O, 0.4 g; NH4Cl, 0.4 g; SnCl2.2H2O, Al(OH)3, 0.1 g; 0.5 g; KI, 0.05 g; LiCl, 0.01 g; MnCl2.4H2O, 0.8 g; ZnCl2, 0.1 g; H3BO3, 0.05 g; CoCl2.6H2O, 0.1 g; NiCl2.6H2O, 0.1 g; BaCl2, 0.05 g; (NH4)6Mo7O24.4H2O, 0.05 g, l1) that contained 50 mM sucrose and 0.3 mM DBT as carbon and sulfur source, respectively, at 30 C with shaking at 220 rpm for five days, when the OD600 reached around 3.0 (~108 cfu ml1), was used as inoculum for all the experiments. To study the desulfurization of BNT, 18 flasks, each containing 20 ml medium-1, 50 mM sucrose and 0.3 mM BNT were inoculated with 0.1 ml inoculum. Identical un-inoculated sets were run in parallel as controls. After incubation as above, three flasks were removed after specified time intervals, and 3.0 ml medium from each was removed for the estimation of growth (OD600) and evaluation of the formed hydroxy-metabolites by Gibbs's assay (described below). The remaining culture medium was acidified to pH < 2.0, and extracted three times with equal volume of ethyl
acetate. The pooled extracts were dried by evaporation of the solvent at room temperature, and the residue was dissolved in a suitable solvent for further analysis of the formed metabolites. Desulfurization of DBT was also studied likewise. In the reactions, where influence of DBT on the desulfurization of BNT was to be studied, 0.3 mM of both BNT and DBT were added to the medium. For desulfurization of crude oil, same set up was used except that 10 ml oil was mixed with 50 ml medium-1, instead of BNT or DBT. After incubation for 15 days, the top crude oil layer was removed by aspiration, and 2.0 ml of it was extracted twice with 5 ml 150 mM NaOH, each time. The pooled extract was acidified to pH < 2.0 and re-extracted with ethyl acetate, as described above, for further analysis of the formed metabolites. Only trace amounts of the metabolite(s) were seen after the same extraction done with the aqueous layer of the culture medium. 2.4. Desulfurization by recombinant E. coli cells Recombinant E. coli-DszA and E. coli-DszC cell that were expressing enzymes DszA and DszC, respectively, were prepared as described earlier (Macwan et al., 2012). Briefly, the amplified dszA or dszC (KC693733.1) were ligated with pET-28a (þ) (Novagen, Darmstadt, Germany) vector, and cloned in E. coli BL21 cells. After their growth in 1.0 L Luria broth (LB) that contained kanamycin (50 mg/ml), and induction with 0.2 mM IPTG, the induced cells were washed with medium-1 and re-suspended in 100 ml of the same medium containing 50 mM sucrose for desulfurization studies. To evaluate the metabolism of BNT by E. coli-DszC, a set of twelve flasks, each containing 5.0 ml suspension of E. coli-DszC and 0.3 mM BNT, was incubated at 30 C for 0, 0.5, 2, 4, 8 and 12 h. Two flasks were harvested for each time point and the reaction was stopped by acidification of the content to pH < 2.0 with 0.2N HCl. Residual BNT and the formed metabolites were analyzed by TLC and HPLC, as described below. For experiments where the metabolism of DBT, or of BNT in the presence of DBT, was to be evaluated, the reactions were set up in the same manner, except that 0.3 mM DBT or a mixture of BNT and DBT (0.3 nmM each) was used as substrate. Metabolism of the metabolite M, formed by the activity of DszC on BNT, was studied by E. coli-DszA, as described above for E. coli-DszC. Control reactions were run in parallel by using recombinant E. coli that carried the vector (pET-28a) only. 2.5. Analytical methods Presence of hydroxy-metabolites was evaluated by Gibb's assay, as described before (Kayser et al., 1993). Briefly, to 2.5 ml microbial culture, whose pH was adjusted earlier to 8.0 with 10% (w/v) sodium carbonate, 25 ml Gibb's reagent (1% 2,6-Dichloroquinone-4chloroimide in ethanol) was mixed and incubated at 30 C for 30 min. After incubation, the cells were removed by centrifugation and the formed color in the supernatant was measured at 610 nm. For thin layer chromatography (TLC), the extracted samples were dissolved in 100 ml acetone:heptane (1:1), and suitable aliquots were analyzed on silica gel 60 F254 plates (Merck, Germany) that were developed in chloroform and toluene (1:3). The separated chemicals were visualized under UV254 and also after spraying with Gibb's reagent. For HPLC analysis, extracted samples were dissolved in acetonitrile, and suitable aliquots were analyzed by using a Waters instrument (Milford, MA, USA) that was equipped with a PDA 996 detector and LiChrospher® 100 RP-18 column (5 mm, 4.6 250 mm). The run conditions were: temperature; 25 C, mobile phase; 100% Acetonitrile, and the flow rate 0.75 ml min1. For FT-IR spectroscopy of the formed metabolites, their potassium
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Fig. 1. Desulfurization of benzonaphthothiophene (BNT) by IITR100. (a) Growth (dry cell weight) of IITR100 in the presence (closed circle) and absence (open circle) of BNT. Reduction in residual BNT, in the presence (closed triangle) and absence (open triangle) of IITR100 is also shown. (b) TLC of BNT and the formed metabolites (T1, T2 & T3), after incubation with IITR100 cells. (c) HPLC depicting desulfurization of BNT with IITR100 and formation of Metabolites H1 & H2, after 0, 4, 6, and 8 days of incubation.
bromide pellets were analyzed on a Spectrum Two™ IR spectrometer (Perkin Elmer, Waltham, MA, USA). For Gas chromatography and mass-spectroscopy, metabolites were purified by TLC and a portion of the sample was derivatized
by adding 40 ml of pyridine and 60 ml of silylating mixture, followed by incubation at 70 C for 30 min. Analysis was performed on Trace GC Ultra coupled with TSQ Quantum XLS Mass spectrometer, equipped with Triplus Auto Sampler (Thermo Scientific,
Fig. 2. (a) Metabolism of BNT by E. coli-DszC, seen by TLC (lanes: 1; BNT, 2; BNTþ E. coli-pET28a, 3; BNTþ E. coli-DszC) and by HPL. (b) Transformation of metabolite M by E. coliDszA (lanes: 1; M, 2; Mþ E. coli-DszA, 3; T1 formed by IITR100). TLC in panel b was run twice for better separation.
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Fig. 3. Mass spectrum of the metabolites M, M1 T2and the silylated product of T2 (T2-Si). Based on mass spectrum, metabolites M, M1 and T2 were tentatively identified as BNTsulfone, BNT-sulfinate and BNT-hydroxide, respectively.
USA) and a TG-5MS capillary column (30 m 0.25 mm I.D. 0.25 mm film thickness). The GC conditions were, oven temperature was maintained at 100 C (2.0 min), followed with increment to 150 C at the rate of 15 C min1 (0 min), and further increased to 200 C at the rate of 3 C min1 (0 min) and finally increased to 260 C at the rate of 30 C min1 (6.0 min). The other conditions were: injector temperature; 250 C, CT split value; 20, mass range; 50e600 amu, source temperature; 220 C, and transfer line temperature; 290 C. Helium was used as carrier gas at a flow rate of 1.0 ml min1.
3. Results 3.1. Desulfurization of BNT by IITR100 Addition of IITR100 to the liquid medium that was containing 0.3 mM BNT led to its growth, which was maximal after six days of incubation and leveled off thereafter (Fig. 1a). This growth was accompanied with >99% decrease in the residual BNT during the incubation period. The maximal specific growth rate for the strain was 0.021 h1 during the reaction, and the specific desulfurization
Fig. 4. Desulfurization of BNT and DBT by IITR100 (a), and by E. coli-DszC (b), when present separately or together. The starting concentration of substrate in all the reactions was 0.3 mM (100%).
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Fig. 5. (a) TLC of the extracted metabolites formed from crude oil, without (UI) and with treatment (I) with IITR100, and viewed after spraying Gibb's reagent. (b) Mass spectrum of the metabolites hydroxy-biphenyl (I), 1-methyl hydroxy-biphenyl (II) and BNT-hydroxide (III), obtained from the treated sample, extracted from the region of TLC marked by the double headed arrow.
rate was 1.2 mmol BNTg1 (DCW) h1. Some growth of IITR100 was also observed in the absence of BNT, possibly due to the presence of residual sulfur that might have been present in the ingredients used for the preparation of the mineral salt medium. Growth of IITR100 in the presence of BNT was accompanied with the formation of hydroxylated metabolites T1, T2 and T3, which formed color after reaction with the Gibb's reagent (Fig. 1b). Exposure of T2 to UV light led to the formation of T3 (data not shown), suggesting that T3 might be formed due to the abiotic transformation of T2 during the incubation. HPLC analysis (Fig. 1c) also revealed a progressive decrease in the levels of BNT (Rt 6.6 min), which was accompanied with transient accumulation of a metabolite H1 (Rt 4.4 min) after four days of incubation, and formation of another metabolite H2 (Rt 3.9 min) that continued to increase till eight days of incubation.
136 as major ions (Fig. 3), which identified it as BNT-sulfone. Metabolite T1, seen in TLC after incubation of BNT with IITR100, and the metabolite M1, formed after the incubation of M with E. coli-DszA, exhibited same retention time as the metabolite H1 on HPLC. Its mass spectrum consisted of (m/z) 428, 413, 261, 218, 210 & 189, and identified it as BNT-sulfinate. Similarly, metabolite T2 that was seen in TLC after incubation of BNT with IITR100 had the same retention time as H2 on HPLC. Its mass spectrum, consisting of (m/z) 220, 191, 189, 165 & 94 as major ions identified it as BNT-hydroxide. This spectrum was comparable to that reported earlier for BNThydroxide (Yu et al., 2006). Further, an increase in its mass by 72 units after silylation reaction, from 220 to 292 (Fig. 3), confirmed it to be a mono-hydroxy product. Identity of metabolite T3 could not be ascertained, but its migration in GC and its GCeMS spectrum was comparable to that of BNT-hydroxide, which could be formed by thermal disintegration of T3 during the analysis.
3.2. Metabolism of BNT by recombinant E. coli Incubation of BNT with recombinant E. coli-DszC led to its desulfurization at the rate of 2.6 mmol BNTg1 (DCW) h1 and >99% decrease in the concentration of BNT after eight hours of incubation (Fig. 2a). This faster rate, compared to IITR 100, could be due to the presence of higher amount of enzyme in the recombinant cells. TLC and HPLC analysis revealed the formation of a metabolite M during this reaction. Incubation of the formed metabolite M (purified from TLC after the reaction of BNT with E. coli-DszC) with the recombinant E. coli-DszA resulted in the formation of another metabolite M1. Its mobility on TLC and ability to react Gibb's reagent was same as the metabolite T1, formed after the reaction of BNT with IITR100 (Fig. 2b). 3.3. Characterization of the formed metabolites Mass spectrum of the metabolite M, formed after the incubation of BNT with E. coli-DszC, consisted of (m/z) 266, 237, 221, 218, 189 &
3.4. Desulfurization of BNT and DBT, when present individually or together Addition of IITR100 to the reaction medium that was containing 0.3 mM of either BNT or DBT as sulfur source led to desulfurization and almost all of BNT or DBT disappeared after 6e8 days of incubation (Fig. 4). The reactions, as expected, were accompanied with the formation of BNT-hydroxide or hydroxy-biphenyl, respectively. However, in the reaction, where 0.3 mM DBT and 0.3 mM BNT were presented together as sulfur source, the desulfurization of DBT occurred at a rate comparable to when it was presented alone, but the desulfurization of BNT was completely inhibited for up to four days. The desulfurization of BNT was observed between fourth and the sixth day of incubation, when the level of DBT dropped to <0.01 mM. The reaction stopped again thereafter (Fig. 4). Similar to IITR100, the desulfurization rates of BNT and DBT with E. coli-DszC were also different, when these were present either separately or together. Thus, when present separately, while >95%
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occurs via the formation of metabolites BNT-sulfone, BNT-sulfinate, and BNT-hydroxide (Fig. 6). While formation of BNT-hydroxide is in agreement with earlier reports (Kirimura et al., 2001, Yu et al., 2006), identification of the metabolites BNT-sulfone, BNT-sulfinate is demonstrated for the first time. Results also revealed that the metabolism of BNT to BNT-sulfone and of BNT-sulfone to BNTsulfinate occurs by the enzymes DszA and DszC, respectively. Further metabolism of the formed BNT-sulfinate could not be evaluated in this study, because our efforts towards the expression of DszB in E. coli in soluble fraction were not successful. However, formation of BNT-hydroxide, which is similar to hydroxy-biphenyl, formed by the desulfurization of DBT, suggests that it was possibly formed by the activity of DszB. Thus the metabolism of BNT by IITR100 is analogous to DBT, and is mediated by the same enzyme system. The E. coli-DszC cells mediated almost complete metabolism of BNT but only 60% of DBT could be metabolized under the same conditions. Although not evaluated in the present study, but this could be due to feed back inhibition by the formed DBTsulfone. Results also revealed that the desulfurization rates of various sulfur compounds are different, when these are present separately than when these are present together as mixtures. Thus, when BNT and DBT were presented together to the IITR100 cells, the desulfurization of BNT was almost completely inhibited, and was relieved only when the level of DBT fell below 0.1 mM in the culture medium. Similar inhibition was also observed during the metabolism of BNT by E. coli-DszC into BNT-sulfone, suggesting that the competition between two substrates sets in the first step itself. The results are consistent with an earlier study (Jiang et al., 2014) where biodesulfurization of BNT by a mixed culture decreased significantly in the presence of >0.1 mM DBT. The results suggest that caution should be exercised in extrapolation of the data, obtained on the desulfurization of individual sulfur compounds in liquid culture, to complex mixtures like petroleum fractions where several organosulfur compounds are present together. 5. Conclusion Fig. 6. Proposed scheme for the desulfurization of benzo[b]naphtho-[2,1-d] thiophene (BNT) by IITR100. Metabolite shown in bracket was not observed in the experiment, but is expected to be formed, based on the similarity with DBT-desulfurization.
BNT was metabolized after eight hours of incubation (Fig. 4), the metabolism of DBT was slower and its ~40% was remaining after the same incubation period. In reactions, where the two chemicals were present together, the desulfurization of DBT occurred at the rate comparable to when it was present alone, but desulfurization of BNT was almost completely inhibited (Fig. 4). 3.5. Desulfurization of BNT in a crude oil Incubation of IITR100 with a crude oil also led to the formation of hydroxylated products, as seen by their reaction on TLC with Gibb's reagent (Fig. 5). GCeMS analysis of the formed metabolites, extracted from the TLC, identified the presence of hydroxybiphenyl, 1-methyl hydroxy-biphenyl and BNT-hydroxide, which possibly were formed from DBT, 1-methyl DBT and BNT, respectively. 4. Discussion The study describes desulfurization of a three-ringed compound BNT by an isolated organism Gordonia sp. IITR100. The reaction
The isolated strain Gordonia sp. IITR100 mediates desulfurization of benzo[b]naphtho[2,1-d]thiophene (BNT) and utilizes the released sulfur for its growth. Similar to the desulfurization of model substrate dibenzothiophene, the reaction is accompanied with the formation of metabolites BNT-sulfone, and BNT-sulfinate, and BNT-hydroxide. Kinetic analysis revealed that the rates of desulfurization of sulfur compounds differ, when these are present individually or in the presence of other sulfur compounds. Conflict of interest The authors do not have any conflict of interest. Acknowledgments Authors AKC and AA sincerely thank Indian Council of Medical Research (ICMR) and Council of Scientific and Industrial Research (CSIR), respectively, for fellowship support. Financial assistance by a grant SIP-08 from CSIR, India, is gratefully acknowledged. The funding sources had no role in study design; in the collection, analysis and interpretation of data. We are also thankful to Dr. Vineeta Singh, Department of Microbiology, Central Drug Research Institute, Lucknow, and our colleagues in analytical facility of Indian Institute of Toxicology Research, Lucknow, for their help in Kinetic, IR, HPLC and GCeMS analysis.
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growth conditions. J. Appl. Microbiol. 99, 1444e1454. http://dx.doi.org/ 10.1111/j.1365-2672.2005.02723.x. Kirkwood, K.M., Foght, J.M., Gray, M.R., 2007. Selectivity among organic sulfur compounds in one- and two-liquid-phase cultures of Rhodococcus sp. strain JVH1. Biodegradation 18, 473e480. http://dx.doi.org/10.1007/s10532-0069080-8. Korte, F., Boedefeld, E., 1978. Ecotoxicological review of global impact of petroleum industry and its products. Ecotoxicol. Environ. Saf. 2, 55e103. http://dx.doi.org/ 10.1016/S0147-6513(78)80028-1. Lewtas, J., 2007. Air pollution combustion emissions: characterization of causative agents and mechanisms associated with cancer, reproductive, and cardiovascular effects. Mutat. Res. Rev. Mutat. 636, 95e133. http://dx.doi.org/10.1016/ j.mrrev.2007.08.003. Liu, P., Shi, Q., Chung, K.H., Zhang, Y., Pan, N., Zhao, S., 2010. Molecular characterization of sulfur compounds in venezuela crude oil and its SARA fractions by electrospray ionization Fourier transform ion cyclotron resonance. Energy Fuels 24, 5089e5096. http://dx.doi.org/10.1021/ef100904k. Macwan, A.S., Kukshal, V., Srivastava, N., Javed, S., Kumar, A., Ramachandran, R., 2012. Crystal structure of the hexachlorocyclohexane dehydrochlorinase (LinAType2): mutational analysis, thermostability and enantioselectivity. PloS One 7, e50373. http://dx.doi.org/10.1371/journal.pone.0050373. Mohebali, G., Ball, A.S., 2008. Biocatalytic desulfurization (BDS) of petrodiesel fuels. Microbiology 154, 2169e2183. http://dx.doi.org/10.1099/mic.0.2008/017608-0. Oldfield, C., Pogrebinsky, O., Simmonds, J., Olson, E.S., Kulpa, C.F., 1997. Elucidation of the metabolic pathway for dibenzothiophene desulphurization by Rhodococcus sp. strain IGTS8 (ATCC 53968). Microbiology 143, 2961e2973. http:// dx.doi.org/10.1099/00221287-143-9-2961. Santos, S.C., Alviano, D.S., Alviano, C.S., Goulart, F.R., de Padula, M., Leitao, A.C., Martins, O.B., Ribeiro, C.M., Sassaki, M.Y., Matta, C.P., Bevilaqua, J., Sebastian, G.V., Seldin, L., 2007. Comparative studies of phenotypic and genetic characteristics between two desulfurizing isolates of Rhodococcus erythropolis and the well-characterized R. erythropolis strain IGTS8. J. Ind. Microbiol. Biotechnol. 34, 423e431. http://dx.doi.org/10.1007/s10295-007-0214-8. Tanaka, Y., Onaka, T., Matsui, T., Maruhashi, K., Kurane, R., 2001. Desulfurization of benzothiophene by the gram-negative bacterium, Sinorhizobium sp. KT55. Curr. Microbiol. 43, 187e191. http://dx.doi.org/10.1007/s002840010285. Wang, W., Ma, T., Lian, K., Zhang, Y., Tian, H., Ji, K., Li, G., 2013. Genetic analysis of benzothiophene biodesulfurization pathway of Gordonia terrae strain C-6. PloS One 8, e84386. http://dx.doi.org/10.1371/journal.pone.0084386. Yu, B., Xu, P., Shi, Q., Ma, C., 2006. Deep desulfurization of diesel oil and crude oils by a newly isolated Rhodococcus erythropolis strain. Appl. Environ. Microbiol. 72, 54e58. http://dx.doi.org/10.1128/AEM.72.1.54-58.2006.
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International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Biodesulfurization of benzonaphthothiophene by an isolated Gordonia sp. IITR100 Ashok Kumar Chauhan a, Abrar Ahmad a, Surya Pratap Singh b, Ashwani Kumar a, * a Environmental Biotechnology Division, Indian Institute of Toxicology Research (Council of Scientific & Industrial Research), M.G. Marg, Lucknow 226001, India b Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 January 2015 Received in revised form 24 May 2015 Accepted 29 May 2015 Available online xxx
Studies on the desulfurization of three or more ringed-compounds, which are considered to inhibit biodesulfurization of crude oil, are rare. In this paper, desulfurization of a three-ringed compound benzo [b]naphtho[2,1-d]thiophene (BNT) by an isolated strain Gordonia sp. IITR100 is described. The bacterium mediates desulfurization of BNT and utilizes the released sulfur for its growth. The reaction is accompanied with the formation of metabolites BNT-sulfone and BNT-sulfinate, in addition to the reported BNT-hydroxide. Recombinant E. coli cells, harboring DszC or DszA, were also able to mediate the metabolism of BNT to BNT-sulfone, or of BNT-sulfone to BNT-sulfinate, respectively. Desulfurization of BNT, both by IITR100 and E. coli-DszC cells was strongly inhibited in the presence of dibenzothiophene. The results are discussed in the context of the biodesulfurization of petroleum fractions where several organosulfur compounds are present together. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Biodesulfurization Gordonia sp. IITR100 Benzonapthothiophene Dibenzothiophene Crude oil
1. Introduction Numerous organosulfur compounds are present in the petroleum products (Liu et al., 2010), and pose various serious problems e.g. (i) sulfurous emissions in the air upon their burning, causing health problems and acid rain (ii) souring of refinery equipment, and (iii) increase in viscosity of a large fraction of crude oil, rendering it non-amenable to refinery process (Korte and Boedefeld, 1978; Lewtas, 2007). Biodesulurization is an attractive option for reduction of the sulfur content of crude-oil and its fractions (Kilbane, 2006). Studies with the model compound dibenzothiophene (DBT) and its derivatives have revealed that the desulfurization occurs by a ‘4S’ pathway that includes serial activity of the enzymes DszC, DszA and DszB, resulting in the formation of hydroxy-biphenyl as end product (Gallagher et al., 1993; Oldfield et al., 1997; Santos et al., 2007; Mohebali and Ball, 2008). Genes for these proteins i.e. dszC, dszA and dszB have been characterized from several organisms, where these are present together as dszABC in an operon and are co-ordinately regulated (Denome et al., 1994;
* Corresponding author. E-mail addresses: [email protected] (A.K. Chauhan), abrar.ahmadg@ yahoo.com (A. Ahmad), [email protected] (S.P. Singh), ashwani.iitr26@gmail. com (A. Kumar). http://dx.doi.org/10.1016/j.ibiod.2015.05.024 0964-8305/© 2015 Elsevier Ltd. All rights reserved.
Mohebali and Ball, 2008). Pathway for the desulfurization of benzothiophene, at least in part, is analogous to the ‘4S’ pathway and formation of the metabolites BT-sulfoxide & BT-sulfone has been reported (Gilbert et al., 1998; Tanaka et al., 2001). Metabolism of BTsulfone is followed with the formation of benzo[c][l,2]oxathiin Soxide that undergoes transformation into either 2-(20 -hydroxypheny1) ethan-1-al (Kilbane, 2006) or o-hydroxystyrene (Tanaka et al., 2001), as seen in the strains Gordonia sp. 213E and Sinorhizobium sp. KT55, respectively. Recently, three genes designated as bdsABC, which are distinct from dszABC that were identified earlier for DBT-desulfurization, have been characterized from a Gordonia terrae strain C-6, whose products mediate the transformation of BT into o-hydroxystyrene (Wang et al., 2013). Many of the isolated organisms have also been shown to mediate the desulfurization of crude oil and its various fractions. In most cases, while the desulfurization of light-oil fractions is reasonable (~95%), the desulfurization of various crude oil is modest (~50%) (Kilbane, 2006; Mohebali and Ball, 2008; Bhatia and Sharma, 2010, 2012). It is now recognized that the desulfurization of heavy oils is inhibited progressively in the presence of increasing concentrations of the three- or more ringed-sulfur compounds (Choudhary et al., 2008). Studies on these compounds, however, are scarce. The organisms Bacillus subtilis WU-S2B (Kirimura et al., 2001) and Rhodococcus erythropolis XP (Yu et al., 2006) have been
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shown to utilize BNT as sulfur source, and the reaction in both cases was accompanied with the formation of BNT-hydroxide. But the intermediates formed during the process, or the enzymes involved in the activity were not studied. Kinetics of desulfurization of organosulfur compounds, when these are present individually, has been shown to be very different compared to when these are present along with other sulfur compounds. For example, a Rhodococcus sp. strain K1bD could mediate the desulfurization of DBT and 1,4-dithiane, when these were present individually in the growth medium. But, when the two were provided together only the desulfurization of DBT was observed (Kirkwood et al., 2005). Similarly, the desulfurization of benzothiophene or 1,4-dithiane, by Rhodococcus sp. strain JVH1, was delayed significantly in the presence of benzyl sulfide in the culture medium (Kirkwood et al., 2007). In another study, biodesulfurization of BNT, by a mixed culture, was decreased significantly in the presence of DBT (Jiang et al., 2014). In the present study, we evaluated the desulfurization of BNT by an isolated Gordonia sp. IITR100, when it was present either individually, or together with DBT, by isolated IITR100 and recombinant E. coli cells. 2. Materials and methods 2.1. Chemicals Benzo[b]naphtho[2,1-d]thiophene (BNT; CAS Number 239-350), dibenzothiophene (DBT), dibenzothiophene sulfone (DBT-sulfone), silylating mixture [ N,O-bis(trimethylsilyl) trifluoro acetamide (BSTFA) and trimethylchlorosilane (TMCS); 99:1], 2,6dichloroquinone-4-chloroimide, and acetonitrile, were purchased from SigmaeAldrich chemical co., St. Louis, USA. Crude oil was obtained from Indian Oil Corporation Limited, Faridabad, India. All the other chemicals used were of analytical grade. 2.2. Bacterium A bacterial strain Gordonia sp. IITR100, obtained earlier from a contaminated soil from around a refinery in Gujarat, India by selective enrichment on 4,6-dimethyl-DBT as sulfur source (Ahmad et al., 2014), was used. It harbors dszABC genes whose nucleotide sequence (KC693733.1) is >99% identical to those described from Gordonia alkanivorans strains IB (AY678116.1) and RIPI90A (EU364831.1). 2.3. Desulfurization activity The bacterial strain IITR100, grown in 20 ml medium1(Na2HPO4, 2.0 g; KH2PO4, 1.0 g; MgCl2.6H2O, 0.4 g; NH4Cl, 0.4 g; SnCl2.2H2O, Al(OH)3, 0.1 g; 0.5 g; KI, 0.05 g; LiCl, 0.01 g; MnCl2.4H2O, 0.8 g; ZnCl2, 0.1 g; H3BO3, 0.05 g; CoCl2.6H2O, 0.1 g; NiCl2.6H2O, 0.1 g; BaCl2, 0.05 g; (NH4)6Mo7O24.4H2O, 0.05 g, l1) that contained 50 mM sucrose and 0.3 mM DBT as carbon and sulfur source, respectively, at 30 C with shaking at 220 rpm for five days, when the OD600 reached around 3.0 (~108 cfu ml1), was used as inoculum for all the experiments. To study the desulfurization of BNT, 18 flasks, each containing 20 ml medium-1, 50 mM sucrose and 0.3 mM BNT were inoculated with 0.1 ml inoculum. Identical un-inoculated sets were run in parallel as controls. After incubation as above, three flasks were removed after specified time intervals, and 3.0 ml medium from each was removed for the estimation of growth (OD600) and evaluation of the formed hydroxy-metabolites by Gibbs's assay (described below). The remaining culture medium was acidified to pH < 2.0, and extracted three times with equal volume of ethyl
acetate. The pooled extracts were dried by evaporation of the solvent at room temperature, and the residue was dissolved in a suitable solvent for further analysis of the formed metabolites. Desulfurization of DBT was also studied likewise. In the reactions, where influence of DBT on the desulfurization of BNT was to be studied, 0.3 mM of both BNT and DBT were added to the medium. For desulfurization of crude oil, same set up was used except that 10 ml oil was mixed with 50 ml medium-1, instead of BNT or DBT. After incubation for 15 days, the top crude oil layer was removed by aspiration, and 2.0 ml of it was extracted twice with 5 ml 150 mM NaOH, each time. The pooled extract was acidified to pH < 2.0 and re-extracted with ethyl acetate, as described above, for further analysis of the formed metabolites. Only trace amounts of the metabolite(s) were seen after the same extraction done with the aqueous layer of the culture medium. 2.4. Desulfurization by recombinant E. coli cells Recombinant E. coli-DszA and E. coli-DszC cell that were expressing enzymes DszA and DszC, respectively, were prepared as described earlier (Macwan et al., 2012). Briefly, the amplified dszA or dszC (KC693733.1) were ligated with pET-28a (þ) (Novagen, Darmstadt, Germany) vector, and cloned in E. coli BL21 cells. After their growth in 1.0 L Luria broth (LB) that contained kanamycin (50 mg/ml), and induction with 0.2 mM IPTG, the induced cells were washed with medium-1 and re-suspended in 100 ml of the same medium containing 50 mM sucrose for desulfurization studies. To evaluate the metabolism of BNT by E. coli-DszC, a set of twelve flasks, each containing 5.0 ml suspension of E. coli-DszC and 0.3 mM BNT, was incubated at 30 C for 0, 0.5, 2, 4, 8 and 12 h. Two flasks were harvested for each time point and the reaction was stopped by acidification of the content to pH < 2.0 with 0.2N HCl. Residual BNT and the formed metabolites were analyzed by TLC and HPLC, as described below. For experiments where the metabolism of DBT, or of BNT in the presence of DBT, was to be evaluated, the reactions were set up in the same manner, except that 0.3 mM DBT or a mixture of BNT and DBT (0.3 nmM each) was used as substrate. Metabolism of the metabolite M, formed by the activity of DszC on BNT, was studied by E. coli-DszA, as described above for E. coli-DszC. Control reactions were run in parallel by using recombinant E. coli that carried the vector (pET-28a) only. 2.5. Analytical methods Presence of hydroxy-metabolites was evaluated by Gibb's assay, as described before (Kayser et al., 1993). Briefly, to 2.5 ml microbial culture, whose pH was adjusted earlier to 8.0 with 10% (w/v) sodium carbonate, 25 ml Gibb's reagent (1% 2,6-Dichloroquinone-4chloroimide in ethanol) was mixed and incubated at 30 C for 30 min. After incubation, the cells were removed by centrifugation and the formed color in the supernatant was measured at 610 nm. For thin layer chromatography (TLC), the extracted samples were dissolved in 100 ml acetone:heptane (1:1), and suitable aliquots were analyzed on silica gel 60 F254 plates (Merck, Germany) that were developed in chloroform and toluene (1:3). The separated chemicals were visualized under UV254 and also after spraying with Gibb's reagent. For HPLC analysis, extracted samples were dissolved in acetonitrile, and suitable aliquots were analyzed by using a Waters instrument (Milford, MA, USA) that was equipped with a PDA 996 detector and LiChrospher® 100 RP-18 column (5 mm, 4.6 250 mm). The run conditions were: temperature; 25 C, mobile phase; 100% Acetonitrile, and the flow rate 0.75 ml min1. For FT-IR spectroscopy of the formed metabolites, their potassium
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Fig. 1. Desulfurization of benzonaphthothiophene (BNT) by IITR100. (a) Growth (dry cell weight) of IITR100 in the presence (closed circle) and absence (open circle) of BNT. Reduction in residual BNT, in the presence (closed triangle) and absence (open triangle) of IITR100 is also shown. (b) TLC of BNT and the formed metabolites (T1, T2 & T3), after incubation with IITR100 cells. (c) HPLC depicting desulfurization of BNT with IITR100 and formation of Metabolites H1 & H2, after 0, 4, 6, and 8 days of incubation.
bromide pellets were analyzed on a Spectrum Two™ IR spectrometer (Perkin Elmer, Waltham, MA, USA). For Gas chromatography and mass-spectroscopy, metabolites were purified by TLC and a portion of the sample was derivatized
by adding 40 ml of pyridine and 60 ml of silylating mixture, followed by incubation at 70 C for 30 min. Analysis was performed on Trace GC Ultra coupled with TSQ Quantum XLS Mass spectrometer, equipped with Triplus Auto Sampler (Thermo Scientific,
Fig. 2. (a) Metabolism of BNT by E. coli-DszC, seen by TLC (lanes: 1; BNT, 2; BNTþ E. coli-pET28a, 3; BNTþ E. coli-DszC) and by HPL. (b) Transformation of metabolite M by E. coliDszA (lanes: 1; M, 2; Mþ E. coli-DszA, 3; T1 formed by IITR100). TLC in panel b was run twice for better separation.
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Fig. 3. Mass spectrum of the metabolites M, M1 T2and the silylated product of T2 (T2-Si). Based on mass spectrum, metabolites M, M1 and T2 were tentatively identified as BNTsulfone, BNT-sulfinate and BNT-hydroxide, respectively.
USA) and a TG-5MS capillary column (30 m 0.25 mm I.D. 0.25 mm film thickness). The GC conditions were, oven temperature was maintained at 100 C (2.0 min), followed with increment to 150 C at the rate of 15 C min1 (0 min), and further increased to 200 C at the rate of 3 C min1 (0 min) and finally increased to 260 C at the rate of 30 C min1 (6.0 min). The other conditions were: injector temperature; 250 C, CT split value; 20, mass range; 50e600 amu, source temperature; 220 C, and transfer line temperature; 290 C. Helium was used as carrier gas at a flow rate of 1.0 ml min1.
3. Results 3.1. Desulfurization of BNT by IITR100 Addition of IITR100 to the liquid medium that was containing 0.3 mM BNT led to its growth, which was maximal after six days of incubation and leveled off thereafter (Fig. 1a). This growth was accompanied with >99% decrease in the residual BNT during the incubation period. The maximal specific growth rate for the strain was 0.021 h1 during the reaction, and the specific desulfurization
Fig. 4. Desulfurization of BNT and DBT by IITR100 (a), and by E. coli-DszC (b), when present separately or together. The starting concentration of substrate in all the reactions was 0.3 mM (100%).
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Fig. 5. (a) TLC of the extracted metabolites formed from crude oil, without (UI) and with treatment (I) with IITR100, and viewed after spraying Gibb's reagent. (b) Mass spectrum of the metabolites hydroxy-biphenyl (I), 1-methyl hydroxy-biphenyl (II) and BNT-hydroxide (III), obtained from the treated sample, extracted from the region of TLC marked by the double headed arrow.
rate was 1.2 mmol BNTg1 (DCW) h1. Some growth of IITR100 was also observed in the absence of BNT, possibly due to the presence of residual sulfur that might have been present in the ingredients used for the preparation of the mineral salt medium. Growth of IITR100 in the presence of BNT was accompanied with the formation of hydroxylated metabolites T1, T2 and T3, which formed color after reaction with the Gibb's reagent (Fig. 1b). Exposure of T2 to UV light led to the formation of T3 (data not shown), suggesting that T3 might be formed due to the abiotic transformation of T2 during the incubation. HPLC analysis (Fig. 1c) also revealed a progressive decrease in the levels of BNT (Rt 6.6 min), which was accompanied with transient accumulation of a metabolite H1 (Rt 4.4 min) after four days of incubation, and formation of another metabolite H2 (Rt 3.9 min) that continued to increase till eight days of incubation.
136 as major ions (Fig. 3), which identified it as BNT-sulfone. Metabolite T1, seen in TLC after incubation of BNT with IITR100, and the metabolite M1, formed after the incubation of M with E. coli-DszA, exhibited same retention time as the metabolite H1 on HPLC. Its mass spectrum consisted of (m/z) 428, 413, 261, 218, 210 & 189, and identified it as BNT-sulfinate. Similarly, metabolite T2 that was seen in TLC after incubation of BNT with IITR100 had the same retention time as H2 on HPLC. Its mass spectrum, consisting of (m/z) 220, 191, 189, 165 & 94 as major ions identified it as BNT-hydroxide. This spectrum was comparable to that reported earlier for BNThydroxide (Yu et al., 2006). Further, an increase in its mass by 72 units after silylation reaction, from 220 to 292 (Fig. 3), confirmed it to be a mono-hydroxy product. Identity of metabolite T3 could not be ascertained, but its migration in GC and its GCeMS spectrum was comparable to that of BNT-hydroxide, which could be formed by thermal disintegration of T3 during the analysis.
3.2. Metabolism of BNT by recombinant E. coli Incubation of BNT with recombinant E. coli-DszC led to its desulfurization at the rate of 2.6 mmol BNTg1 (DCW) h1 and >99% decrease in the concentration of BNT after eight hours of incubation (Fig. 2a). This faster rate, compared to IITR 100, could be due to the presence of higher amount of enzyme in the recombinant cells. TLC and HPLC analysis revealed the formation of a metabolite M during this reaction. Incubation of the formed metabolite M (purified from TLC after the reaction of BNT with E. coli-DszC) with the recombinant E. coli-DszA resulted in the formation of another metabolite M1. Its mobility on TLC and ability to react Gibb's reagent was same as the metabolite T1, formed after the reaction of BNT with IITR100 (Fig. 2b). 3.3. Characterization of the formed metabolites Mass spectrum of the metabolite M, formed after the incubation of BNT with E. coli-DszC, consisted of (m/z) 266, 237, 221, 218, 189 &
3.4. Desulfurization of BNT and DBT, when present individually or together Addition of IITR100 to the reaction medium that was containing 0.3 mM of either BNT or DBT as sulfur source led to desulfurization and almost all of BNT or DBT disappeared after 6e8 days of incubation (Fig. 4). The reactions, as expected, were accompanied with the formation of BNT-hydroxide or hydroxy-biphenyl, respectively. However, in the reaction, where 0.3 mM DBT and 0.3 mM BNT were presented together as sulfur source, the desulfurization of DBT occurred at a rate comparable to when it was presented alone, but the desulfurization of BNT was completely inhibited for up to four days. The desulfurization of BNT was observed between fourth and the sixth day of incubation, when the level of DBT dropped to <0.01 mM. The reaction stopped again thereafter (Fig. 4). Similar to IITR100, the desulfurization rates of BNT and DBT with E. coli-DszC were also different, when these were present either separately or together. Thus, when present separately, while >95%
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occurs via the formation of metabolites BNT-sulfone, BNT-sulfinate, and BNT-hydroxide (Fig. 6). While formation of BNT-hydroxide is in agreement with earlier reports (Kirimura et al., 2001, Yu et al., 2006), identification of the metabolites BNT-sulfone, BNT-sulfinate is demonstrated for the first time. Results also revealed that the metabolism of BNT to BNT-sulfone and of BNT-sulfone to BNTsulfinate occurs by the enzymes DszA and DszC, respectively. Further metabolism of the formed BNT-sulfinate could not be evaluated in this study, because our efforts towards the expression of DszB in E. coli in soluble fraction were not successful. However, formation of BNT-hydroxide, which is similar to hydroxy-biphenyl, formed by the desulfurization of DBT, suggests that it was possibly formed by the activity of DszB. Thus the metabolism of BNT by IITR100 is analogous to DBT, and is mediated by the same enzyme system. The E. coli-DszC cells mediated almost complete metabolism of BNT but only 60% of DBT could be metabolized under the same conditions. Although not evaluated in the present study, but this could be due to feed back inhibition by the formed DBTsulfone. Results also revealed that the desulfurization rates of various sulfur compounds are different, when these are present separately than when these are present together as mixtures. Thus, when BNT and DBT were presented together to the IITR100 cells, the desulfurization of BNT was almost completely inhibited, and was relieved only when the level of DBT fell below 0.1 mM in the culture medium. Similar inhibition was also observed during the metabolism of BNT by E. coli-DszC into BNT-sulfone, suggesting that the competition between two substrates sets in the first step itself. The results are consistent with an earlier study (Jiang et al., 2014) where biodesulfurization of BNT by a mixed culture decreased significantly in the presence of >0.1 mM DBT. The results suggest that caution should be exercised in extrapolation of the data, obtained on the desulfurization of individual sulfur compounds in liquid culture, to complex mixtures like petroleum fractions where several organosulfur compounds are present together. 5. Conclusion Fig. 6. Proposed scheme for the desulfurization of benzo[b]naphtho-[2,1-d] thiophene (BNT) by IITR100. Metabolite shown in bracket was not observed in the experiment, but is expected to be formed, based on the similarity with DBT-desulfurization.
BNT was metabolized after eight hours of incubation (Fig. 4), the metabolism of DBT was slower and its ~40% was remaining after the same incubation period. In reactions, where the two chemicals were present together, the desulfurization of DBT occurred at the rate comparable to when it was present alone, but desulfurization of BNT was almost completely inhibited (Fig. 4). 3.5. Desulfurization of BNT in a crude oil Incubation of IITR100 with a crude oil also led to the formation of hydroxylated products, as seen by their reaction on TLC with Gibb's reagent (Fig. 5). GCeMS analysis of the formed metabolites, extracted from the TLC, identified the presence of hydroxybiphenyl, 1-methyl hydroxy-biphenyl and BNT-hydroxide, which possibly were formed from DBT, 1-methyl DBT and BNT, respectively. 4. Discussion The study describes desulfurization of a three-ringed compound BNT by an isolated organism Gordonia sp. IITR100. The reaction
The isolated strain Gordonia sp. IITR100 mediates desulfurization of benzo[b]naphtho[2,1-d]thiophene (BNT) and utilizes the released sulfur for its growth. Similar to the desulfurization of model substrate dibenzothiophene, the reaction is accompanied with the formation of metabolites BNT-sulfone, and BNT-sulfinate, and BNT-hydroxide. Kinetic analysis revealed that the rates of desulfurization of sulfur compounds differ, when these are present individually or in the presence of other sulfur compounds. Conflict of interest The authors do not have any conflict of interest. Acknowledgments Authors AKC and AA sincerely thank Indian Council of Medical Research (ICMR) and Council of Scientific and Industrial Research (CSIR), respectively, for fellowship support. Financial assistance by a grant SIP-08 from CSIR, India, is gratefully acknowledged. The funding sources had no role in study design; in the collection, analysis and interpretation of data. We are also thankful to Dr. Vineeta Singh, Department of Microbiology, Central Drug Research Institute, Lucknow, and our colleagues in analytical facility of Indian Institute of Toxicology Research, Lucknow, for their help in Kinetic, IR, HPLC and GCeMS analysis.
A.K. Chauhan et al. / International Biodeterioration & Biodegradation 104 (2015) 105e111
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