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Degradation kinetics and metabolites in continuous biodegradation of isoprene
Bioresource Technology 206 (2016) 275–278
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Degradation kinetics and metabolites in continuous biodegradation of isoprene Navnita Srivastva a, Ram S. Singh b, Siddh N. Upadhyay b,1, Suresh K. Dubey a,⇑ a b
Department of Botany, Institute of Science, Banaras Hindu University, Varanasi 221005, India Department of Chemical Engineering and Technology, Institute of Technology, Banaras Hindu University, Varanasi 221005, India
h i g h l i g h t s Isoprene degradation kinetics in the bioreactor has been evaluated. Biofilter unit has shown better degradation efficiency than the bioscrubber unit. Major portion of inlet isoprene (62–75%) gets converted to carbon dioxide. Metabolites analysis has shown the oxidative cleavage of double bond of isoprene. The isoprene degradation pathway in Pseudomonas sp. has been elucidated.
a r t i c l e
i n f o
Article history: Received 4 December 2015 Received in revised form 14 January 2016 Accepted 17 January 2016 Available online 3 February 2016 Keywords: Isoprene Bioscrubber Biofilter Kinetics GC–MS
a b s t r a c t The kinetic parameters of isoprene biodegradation were studied in a bioreactor, comprising of bioscrubber and polyurethane foam packed biofilter in series and inoculated with Pseudomonas sp., using a Michaelis–Menten type model. The maximum elimination capacity, EC max ; substrate constant, K s and EC max /K s values for bioscrubber were found to be 666.7 g m3 h1, 9.86 g m3 and 67.56 h1, respectively while those for biofilter were 3333 g m3 h1, 13.96 g m3 and 238.7 h1, respectively. The biofilter section exhibited better degradation efficiency compared to the bioscrubber unit. Around 62–75% of the feed isoprene got converted to carbon dioxide, indicating the efficient capability of bacteria to mineralize isoprene. The FTIR and GC–MS analyses of degradation products indicated oxidative cleavage of unsaturated bond of isoprene. These results were used for proposing a plausible degradation pathway for isoprene. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Isoprene (2-methyl-1,3-butadiene) is the most abundant nonmethane volatile organic compound (NMVOC) emitted in the atmosphere. Various biogenic and anthropogenic sources lead to its global emission of 450–700 TgC yr1 (Ashworth et al., 2010). It readily photo-oxidizes to generate tropospheric ozone, secondary organic aerosols and carbon monoxide. Its exposure affects skin and respiratory system of humans and it is also reported to possess carcinogenic properties (IARC, 1994). Hence, its removal from the contaminated environment is imperative. The pioneer work on isoprene biodegradation was carried out by Hou et al. (1981) using Methanotrophs and Xanthobacter. This was followed by identification of other isoprene degrading bacteria (Alvarez ⇑ Corresponding author. Tel.: +91 0542 2307147. 1
E-mail address: [email protected] (S.K. Dubey). DAE-Raja Ramanna Emeritus Fellow.
http://dx.doi.org/10.1016/j.biortech.2016.01.070 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.
et al., 2009). The information on biochemical pathway for isoprene degradation is rather in infancy. Vlieg et al. (1999) reported isoprene degradation through epoxidation by Rhodococcus AD45. In another study, dioxygenase was shown to be responsible for degradation of isoprene by Pseudomonas sp. (Boyd et al., 2000). Studies on biofiltration of isoprene are scanty (Yoon et al., 2002; Srivastva et al., 2015) and lack information on the biodegradation kinetics. In our previous study, efficacy of Pseudomonas sp. (NCBI accession number: KM226326) for isoprene biodegradation in shake flasks and a bioscrubber-cum-biofilter unit packed with polyurethane foam were evaluated (Srivastva et al., 2015). In this follow up study, the biodegradation kinetics in the bioreactor system is studied and the relevant kinetic parameters are evaluated using a Michaelis–Menten type model. The various metabolites produced during biodegradation are analyzed and correlated with earlier reports to propose a more complete and plausible degradation pathway for isoprene.
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respectively. Mass spectra of the peaks were compared with compounds present in NIST database (NIST08.LIB). Compounds with >90% similarity were selected.
2. Methods 2.1. Biodegradation of isoprene The bioscrubber-cum-biofilter units mounted vertically and connected in series were inoculated with Pseudomonas sp., for investigating biodegradation in continuous mode. The lower part of the bioreactor was operated as the bioscrubber and the upper part packed with polyurethane foam as the conventional biofilter. Details of these units are given elsewhere (Srivastva et al., 2015). Isoprene loaded air (isoprene concentration: 0.03–17.4 g m3) was fed to the bottom of the bioscrubber at four different flow rates. The outlet from this served as feed to the biofilter. The biodegradation was carried out for a total period of 130 days and performance of the bioreactor system was assessed during operation by measuring the concentration of isoprene at the inlet of bioscubber and the oulets of bioscubber and filter sections.
3.1. Biodegradation kinetics The experimentally achieved elimination capacity (EC) remained almost constant with the increasing inlet loading rate during Phases II and III of the bioscrubber. While in the biofilter section, the experimentally calculated EC increased with increase in the inlet loading rate during those phases (Srivastva et al., 2015). This indicated the absence of inhibition in the bioreactor system during the operation. Hence, the Michaelis–Menten type model:
V 1 Ks 1 1 þ ¼ ¼ QðC in C out Þ EC EC max C ln EC max
2.2. Analysis of degradation products 2.2.1. Carbon balance For carbon balance the concentration of carbon in the inlet was taken as 100% and the carbon in other fractions were estimated in reference to this value. The inlet and outlet concentrations of isoprene were determined by gas chromatography (GC) as described earlier (Srivastva et al., 2015). Carbon dioxide in the outlet was also determined chromatographically using GC (Agilent Technologies, 7820A GC system, California, USA) as per manufacturer’s method. The analysis of total organic carbon content of the leachate was performed using a TOC analyzer (Analytik Jena Multi N/C 2100, UK) as per manufacturer’s protocol. Biomass carbon was calculated by subtracting the sum of outlet isoprene carbon, leachate carbon and CO2-C from the inlet isoprene carbon using equation proposed by Lu et al. (2002):
C inlet ¼ C outlet þ C L þ C CO2 þ C bio
3. Results and discussion
ð1Þ
Here, C inlet is the percentage of carbon in inlet gas stream, C outlet is percentage of carbon in outlet gas stream, C L is total organic carbon percentage in leachate, C CO2 is percentage of carbon in carbon dioxide and C bio is the carbon percentage in biomass. 2.2.2. Fourier transform infrared spectroscopy (FTIR) The leachate sample was centrifuged at 10000 rpm for 10 min to remove the bacterial cells and the supernatant (1 mL) was used for FTIR analysis (ALPHA, Bruker Optics, Billerica, MA) in the mid IR region of 500–4000 cm1. Same volume of pure isoprene (Merck Company, Germany) was used as control. The peaks interpretation was carried out using IR Pal 1.0 software. 2.2.3. Gas chromatography–mass spectrometry (GC–MS) The metabolites were extracted from the leachate (10 mL) in npentane as described earlier (Srivastva et al., 2015). One lL of the extracted sample was injected into Shimadzu GCMS-QP2010 Ultra (Serial No. O205249, USA) equipped with RxiÒ 5 ms capillary column (30 m 0.25 mm 0.25 lm), using a Hamilton gas-tight syringe (Model: 701 N). Same volume of pure isoprene (Merck Company, Germany) was used as control. Helium (99.99% purity) was used as the carrier gas at 100 kPa pressure and 11.9 mL min1 flow rate. The injector temperature was set at 250 °C and the oven temperature at 40 °C for 5 min and then ramped to 90 °C at the rate of 5 °C min1 for 2 min followed by ramping to 130 °C at the rate of 10 °C min1 for 2 min and finally to 260 °C at the rate of 10 °C min1 for 2 min. Split ratio was chosen as 4. The MS was operated in full scan mode with m/z ranging from 35 to 200. Ion source and interface temperatures were set at 250 and 300 °C,
ð2Þ
modified for continuous system (Mathur et al., 2006) was used for evaluating the kinetic parameters from the steady state experimental data of Phases I to III. Here, V is the working volume (m3), Q is the volumetric flow rate (m3 h1), C in is the inlet concentration (g m3), C out is the outlet concentration (g m3), EC is the elimination capacity (g m3 h1), EC max is the maximum elimination capacity (g m3 h1), K s is the saturation constant of substrate (isoprene) out (g m3) and C ln f¼ Cin C C in g is the logarithmic average of inlet and
lnC
out
outlet concentrations of isoprene. 1 The equations corresponding to the best fit straight line for EC vs 1 plots for bioscrubber and biofilter were generated using the C ln
least-squares method (Fig. 1). The EC max and K s for bioscrubber were found to be 666.7 g m3 h1 and 9.86 g m3, respectively (Fig. 1A) while those for biofilter were 3333 g m3 h1 and 13.96 g m3, respectively (Fig. 1B). The value of EC max /K s obtained for bioscrubber and biofilter units were 67.56 h1 and 238.7 h1, respectively. It has been established that the biofilter operates in the plugflow regime while the bioscrubber exhibits the well-mixed behavior (Yadav et al., 2014). The EC max for the bioscrubber obtained from the analysis of the data is comparable to the EC (567 g m3 h1) obtained experimentally (Srivastva et al., 2015) which indicates that the bioscrubber is operating at the near optimum condition. Further, the lower K s value compared to that for batch mode (Srivastva et al., 2015) indicates that this unit is more efficient than the batch operation. This can be attributed to the absence of oxygen limitation and better mixing due to bubbling of isoprene loaded air. Further, the bioscrubber is also advantageous in two ways – it acts as humidifier and prevents organic overloading of biofilter. The degradation capability of the biofilter, as evident from the kinetic parameter – EC max /K s , however, is better than the bioscrubber. This ratio reflects the efficiency of substrate degradation by bacterial community and is considered as an useful index for the enzymatic reaction. This kinetic study is also consistent with the previous report (Srivastva et al., 2015) where removal efficiencies of biofilter (89% in Phase II and 66% in Phase III) were found to be better than those of bioscubber (31% in Phase II and 17% in Phase III). Schlegelmilch et al. (2005) also reported 29% removal in the bioscrubber and 97–99% for the bioscrubber/biofilter combination. The EC max value for the biofilter unit obtained through analysis of data is around 2.6 times greater than the highest EC value (1256 g m3 h1) observed experimentally (Srivastva et al., 2015). This shows that the biofilter could be operated at even higher inlet loading rates.
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typically found in isoprene polymer (Gualtieri et al., 2005). The leachate sample also showed some additional peaks that were not observed in the control (Supplementary data, Fig. S1B). Peaks at 1000 and 1075 cm1 represent the C–O stretching of alcohol while peaks at 1475 cm1 indicate the bending of C–H in alkane. The peak at 1500 cm1 exhibits the N–H bending of amine group. The stretching of hydroxyl group of alcohol has resulted in peaks at 2300, 3550 and 3650 cm1. The appearance of peaks corresponding to carbonyl, hydroxyl and amine group in the leachate indicate oxidative cleavage of isoprene. The identification of compounds with such functional groups was done through GC–MS analysis.
Fig. 1. Lineweaver–Burk plot of Michaelis–Menten equation for (A) bioscrubber unit and (B) biofilter unit.
3.2. Biodegradation products analysis 3.2.1. Carbon balance The major portion of inlet carbon (62–77%) was converted into CO2, indicating efficient biodegradation potential of Pseudomonas sp. A small portion of carbon (4–20%) as undegraded isoprene was detected in the outlet. The loss of carbon between inlet and outlets can be assumed to be retained as biomass. The higher carbon content in the biomass (32% Cbio) during acclimation phase could be due to low isoprene concentration and fast initial growth of bacteria. As the flow rate increased during different phases, the growth slowed down and hence the biomass carbon also declined to 16% and then stabilized at 15%. The distribution of carbon in biomass and CO2 is in accordance with the results of earlier studies on biofiltraion of hydrocarbons (Marchal et al., 2003). Carbon observed in the leachate (1–2%) could be attributed either to the undegraded dissolved isoprene or the biodegradation products of isoprene; hence, the leachate components were further analyzed through FTIR.
3.2.2. FTIR analysis The FTIR spectra for control and leachate samples are depicted in the Supplementary data (Fig. S1). Peaks for the control sample at 750, 800 and 900 cm1 represent the C–H bond stretching of alkene (Supplementary data, Fig. S1A). The absence of these peaks in the leachate indicates biodegradation of isoprene. However, some common peaks i.e., 1425, 1500, 2850 and 2920 cm1 were found in both, control and leachate sample (Supplementary data, Fig. S1). Peaks at 1425 and 1500 cm1 indicate C– H stretching of aromatic compound. The presence of aromatic compounds could be due to polymerization of isoprene monomers (Mazza et al., 2000). The peaks observed at 2850 and 2920 cm1 represent the CH2 stretching (symmetric and asymmetric)
3.2.3. GC–MS analysis For control samples the gas chromatogram generated 5 peaks at different retention times (Supplementary data, Fig. S2) while for the extracted leachate samples 9 peaks were obtained (Supplementary data, Fig. S3) which are listed in Table 1. The MS spectrum for each peak was analyzed (Supplementary data, Figs. S4 and S5). In case of the control samples the most prominent peak was that of isoprene. However, the polymerized compounds of isoprene monomer such as limonene, D-limonene and 3,7-dimethyl-1,3,6octatriene are also observed. The dimerization products of isoprene during normal operating conditions have been reported by other workers also (Estevez et al., 2014). The polymerization of isoprene generating cyclic compounds is also well documented (Mazza et al., 2000). In the case of leachate samples, beside solvent (n-pentane) and dissolved CO2, the MS spectrum shows the presence of some compounds common with the control (Table 1). This could be due to the presence of undegraded isoprene in the leachate which further polymerized under the operating condition of GC–MS. The presence of estragole, a sesquiterpene, could be due to the degradation of isoprene polymers (Mazza et al., 2000). Further, some alipahtic compounds such as 3-methyl-2-butanone; 2,7-dimethyloctane; 3-methylbutanal and 2-aminopropanol were also observed. The presence of these compounds is in accordance to the functional groups identified through FTIR spectra. These compounds represent the isoprene biodegradation products. Earlier studies also showed alcohol and ketone as the metabolites produced during isoprene biodegradation by Pseudomonas sp. (Boyd et al., 2000). Unlike earlier reports, unsaturated aliphatic alkenes are not detected rather saturated alkane and aldehyde are observed. 3.3. Degradation pathway It is interesting to note that the compounds 3-methylbutanal, 2,7-dimethyloctane and 2-aminopropanol have been detected for the first time as biodegradation products of isoprene. Using these results and available published information on biodegradation of isoprene a plausible pathway has been proposed (Supplementary data, Fig. S6). The key enzymes that could play role in oxidation of isoprene in Pseudomonas sp. are dioxygenase, dehydrogenase, transaminase and some hydrogen adding enzymes. The biodegradation of isoprene is initiated with oxidative cleavage of unsaturated bond by dioxygenase generating 1,2-dihydroxy, 3-methyl, 3-butene or 1,2-dihydroxy, 2-methyl, 3-butene. Through a series of reactions mediated by dehydrogenase and hydrogen adding enzyme, 1,2-dihydroxy, 3-methyl, 3-butene gets converted to 3-methyl, 1,2-butanediol (Boyd et al., 2000). This compound could further undergo oxidation with the aid of dehydrogenase, particularly alcohol dehydrogenase, to produce 3-methyl-2-butanone and 3-methylbutanal. The presence of NAD-dependent alcohol dehydrogenase in Pseudomonas sp. has been shown to catalyze oxidation of primary and secondary alcohols to the corresponding aldehydes and ketones using NAD as coenzyme (Levin et al., 2004). Simultaneously, dehydrogenase could act on 3-methyl,
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Table 1 GC–MS analysis of control and leachate samples (RT: retention time). Peak No.
RT (min)
Area%
Height%
Compounds
Control 1 2
1.33 10.68
93.88 0.36
81.02 1.06
D-Limonene
3 4 5
10.79 13.27 13.37
0.81 3.86 1.09
2.28 11.95 3.69
Limonene 3,7-Dimethyl-1,3,6-octatriene 1,5-Dimethyl-1,5-cyclooctadiene
Leachate 1 2 3 4 5 6 7 8 9
1.28 11.86 13.27 13.38 25.58 26.57 28.20 29.61 29.77
94.62 0.36 1.78 0.53 0.38 0.16 1.56 0.44 0.17
92.48 0.42 2.52 0.79 0.46 0.28 1.76 0.94 0.35
n-Pentane Carbon dioxide 3,7-Dimethyl-1,3,6-octatriene 1,5-Dimethyl-1,5-cyclooctadiene Estragole 3-Methyl-2-butanone 2,7-Dimethyloctane 3-Methylbutanal 2-Aminopropanol
2-Methyl-1,3-butadiene
1,2-butanediol to remove hydroxyl group and generating saturated alkane which could further dimerize to produce 2,7dimethyloctane. The synthesis of extracellular hydrocarbons has been reported in Pseudomonas sp. They are assumed to play a role in cell aggregation (Nikolaev et al., 2001). The sequential action of dehydrogenase and transaminase could produce 2-aminopropanol from 1,2-dihydroxy, 2-methyl, 3-butene. The 2-aminopropanol, also known as L-alaninol, has been reported to further undergo oxidation to generate an amino acid, alanine, in Pseudomonas sp. (Wasch et al., 2002).
4. Conclusions The kinetic parameters show the superior efficiency of biofilter for isoprene degradation compared to bioscrubber. Study also indicates that the system could be operated at higher inlet loading rates without inhibition. The conversion of major portion of inlet isoprene into carbon dioxide shows the efficacy of Pseudomonas sp. for isoprene abatement. The FTIR and GC–MS results have been successfully used for proposing a more realistic pathway for isoprene biodegradation through enzymes: dioxygenase, dehydrogenase, transaminase and some hydrogenases.
Acknowledgments One of the authors (NS) is thankful to CSIR, New Delhi, India, for the financial support in the form of JRF and SRF. Authors also thank Jindal Steel and Power Ltd., Raigarh, for GC–MS analysis of leachate.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2016.01. 070. References Alvarez, L.A., Exton, D.A., Timmis, K.N., Suggett, D.J., McGenity, T.J., 2009. Characterization of marine isoprene-degrading communities. Environ. Microbiol. 11, 3280–3291. Ashworth, K., Wild, O., Hewitt, C.N., 2010. Sensitivity of isoprene emission estimated using MEGAN to the time resolution of input climate data. Atmos. Chem. Phys. 10, 1193–1201. Boyd, D.R., Clarke, D., Cleij, M.C., Hamilton, J.T.G., Sheldrake, G.N., 2000. Bacterial biotransformation of isoprene and related dienes. Monatsh. Chem. 131, 673– 685. Estevez, Y., Gardrat, C., Berthelot, K., Grau, E., De Jeso, B., Ouardad, S., Peruch, F., 2014. Unexpected dimerization of isoprene in a gas chromatography inlet. A study by gas chromatography/mass spectrometry coupling. J. Chromatogr. A 1331, 133–138. Gualtieri, M., Andrioletti, M., Mantecca, P., Vismara, C., Camatini, M., 2005. Impact of tire debris on in vitro and in vivo systems. Part. Fibre Toxicol. 2, 1–14. Hou, C.T., Patel, R.N., Laskin, N., Barnabe, N., Barist, I., 1981. Epoxidation and hydroxylation of C4- and C5-branched-chain alkenes and alkanes by methanotrophs. Ind. Microbiol. 23, 477–482. IARC (International Agency for Research on Cancer), 1994. Isoprene. In some industrial chemicals. IARC monographs on the evaluation of carcinogenic risk of chemicals to humans. Lyon France 60, 215–232. Levin, I., Meiri, G., Peretz, M., Burstein, Y., Frolow, F., 2004. The ternary complex of Pseudomonas aeruginosa alcohol dehydrogenase with NADH and ethylene glycol. Protein Sci. 13, 1547–1556. Lu, C., Lin, M.R., Chu, C., 2002. Effects of pH, moisture, and flow pattern on tricklebed air biofilter performance for BTEX removal. Adv. Environ. Res. 6, 99–106. Mathur, A.K., Sundaramurthy, J., Balomajumder, C., 2006. Kinetics of the removal of mono-chlorobenzene vapour from waste gases using a trickle bed air biofilter. J. Hazard. Mater. 137, 1560–1568. Marchal, R., Penet, S., Solano-Serena, F., Vandecasteele, J.P., 2003. Gasoline and diesel oil biodegradation. Oil Gas Sci. Technol. 58, 441–448. Mazza, A., Sironi, S., Fioretti, L., Andreoni, V., Botta, D., 2000. Limonene biodegradation: a tool for investigating the odors’ formation in the composting plants. In: Tangorra, F.M., Restelli, G.L. (Eds.), 9th Workshop of the Network, General Theme: Technology Transfer, FAO. Nikolaev, Y.A., Panikov, N.S., Lukin, S.M., Osipov, G.A., 2001. Saturated C21–C33 hydrocarbons are involved in the self-regulation of Pseudomonas fluorescens adhesion to a glass surface. Microbiology 70, 74–81. Schlegelmilch, M., Streese, J., Biedermann, W., Herold, T., Stegmann, R., 2005. Odour control at biowaste composting facilities. Waste Manage. 25, 917–927. Srivastva, N., Shukla, A.K., Singh, R.S., Upadhyay, S.N., Dubey, S.K., 2015. Characterization of bacterial isolates from rubber dump site and their use in biodegradation of isoprene in batch and continuous bioreactors. Bioresour. Technol. 188, 84–91. Vlieg, H.J.E.T., Kingma, J., Kruizinga, W., Janssen, D.B., 1999. Purification of a glutathione S-transferase and a glutathione conjugate-specific dehydrogenase involved in isoprene metabolism in Rhodococcus sp. strain AD45. J. Bacteriol. 181, 2094–2101. Wasch, S.I.de A., van der Ploeg, J.R., Maire, T., Lebreton, A., Kiener, A., Leisinger, T., 2002. Transformation of Isopropylamine to L-Alaninol by Pseudomonas sp. strain KIE171 involves N-Glutamylated intermediates. Appl. Environ. Microbiol. 68, 2368–2375. Yadav, M., Srivastva, N., Singh, R.S., Upadhyay, S.N., Dubey, S.K., 2014. Biodegradation of chlorpyrifos by Pseudomonas sp. in a continuous packed bed bioreactor. Bioresour. Technol. 165, 265–269. Yoon, I.N., Kim, C.N., Park, C.H., 2002. Optimum operating conditions for the removal of volatile organic compounds in a compost-packed biofilter. Korean J. Chem. Eng. 19, 954–959.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Degradation kinetics and metabolites in continuous biodegradation of isoprene Navnita Srivastva a, Ram S. Singh b, Siddh N. Upadhyay b,1, Suresh K. Dubey a,⇑ a b
Department of Botany, Institute of Science, Banaras Hindu University, Varanasi 221005, India Department of Chemical Engineering and Technology, Institute of Technology, Banaras Hindu University, Varanasi 221005, India
h i g h l i g h t s Isoprene degradation kinetics in the bioreactor has been evaluated. Biofilter unit has shown better degradation efficiency than the bioscrubber unit. Major portion of inlet isoprene (62–75%) gets converted to carbon dioxide. Metabolites analysis has shown the oxidative cleavage of double bond of isoprene. The isoprene degradation pathway in Pseudomonas sp. has been elucidated.
a r t i c l e
i n f o
Article history: Received 4 December 2015 Received in revised form 14 January 2016 Accepted 17 January 2016 Available online 3 February 2016 Keywords: Isoprene Bioscrubber Biofilter Kinetics GC–MS
a b s t r a c t The kinetic parameters of isoprene biodegradation were studied in a bioreactor, comprising of bioscrubber and polyurethane foam packed biofilter in series and inoculated with Pseudomonas sp., using a Michaelis–Menten type model. The maximum elimination capacity, EC max ; substrate constant, K s and EC max /K s values for bioscrubber were found to be 666.7 g m3 h1, 9.86 g m3 and 67.56 h1, respectively while those for biofilter were 3333 g m3 h1, 13.96 g m3 and 238.7 h1, respectively. The biofilter section exhibited better degradation efficiency compared to the bioscrubber unit. Around 62–75% of the feed isoprene got converted to carbon dioxide, indicating the efficient capability of bacteria to mineralize isoprene. The FTIR and GC–MS analyses of degradation products indicated oxidative cleavage of unsaturated bond of isoprene. These results were used for proposing a plausible degradation pathway for isoprene. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Isoprene (2-methyl-1,3-butadiene) is the most abundant nonmethane volatile organic compound (NMVOC) emitted in the atmosphere. Various biogenic and anthropogenic sources lead to its global emission of 450–700 TgC yr1 (Ashworth et al., 2010). It readily photo-oxidizes to generate tropospheric ozone, secondary organic aerosols and carbon monoxide. Its exposure affects skin and respiratory system of humans and it is also reported to possess carcinogenic properties (IARC, 1994). Hence, its removal from the contaminated environment is imperative. The pioneer work on isoprene biodegradation was carried out by Hou et al. (1981) using Methanotrophs and Xanthobacter. This was followed by identification of other isoprene degrading bacteria (Alvarez ⇑ Corresponding author. Tel.: +91 0542 2307147. 1
E-mail address: [email protected] (S.K. Dubey). DAE-Raja Ramanna Emeritus Fellow.
http://dx.doi.org/10.1016/j.biortech.2016.01.070 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.
et al., 2009). The information on biochemical pathway for isoprene degradation is rather in infancy. Vlieg et al. (1999) reported isoprene degradation through epoxidation by Rhodococcus AD45. In another study, dioxygenase was shown to be responsible for degradation of isoprene by Pseudomonas sp. (Boyd et al., 2000). Studies on biofiltration of isoprene are scanty (Yoon et al., 2002; Srivastva et al., 2015) and lack information on the biodegradation kinetics. In our previous study, efficacy of Pseudomonas sp. (NCBI accession number: KM226326) for isoprene biodegradation in shake flasks and a bioscrubber-cum-biofilter unit packed with polyurethane foam were evaluated (Srivastva et al., 2015). In this follow up study, the biodegradation kinetics in the bioreactor system is studied and the relevant kinetic parameters are evaluated using a Michaelis–Menten type model. The various metabolites produced during biodegradation are analyzed and correlated with earlier reports to propose a more complete and plausible degradation pathway for isoprene.
276
N. Srivastva et al. / Bioresource Technology 206 (2016) 275–278
respectively. Mass spectra of the peaks were compared with compounds present in NIST database (NIST08.LIB). Compounds with >90% similarity were selected.
2. Methods 2.1. Biodegradation of isoprene The bioscrubber-cum-biofilter units mounted vertically and connected in series were inoculated with Pseudomonas sp., for investigating biodegradation in continuous mode. The lower part of the bioreactor was operated as the bioscrubber and the upper part packed with polyurethane foam as the conventional biofilter. Details of these units are given elsewhere (Srivastva et al., 2015). Isoprene loaded air (isoprene concentration: 0.03–17.4 g m3) was fed to the bottom of the bioscrubber at four different flow rates. The outlet from this served as feed to the biofilter. The biodegradation was carried out for a total period of 130 days and performance of the bioreactor system was assessed during operation by measuring the concentration of isoprene at the inlet of bioscubber and the oulets of bioscubber and filter sections.
3.1. Biodegradation kinetics The experimentally achieved elimination capacity (EC) remained almost constant with the increasing inlet loading rate during Phases II and III of the bioscrubber. While in the biofilter section, the experimentally calculated EC increased with increase in the inlet loading rate during those phases (Srivastva et al., 2015). This indicated the absence of inhibition in the bioreactor system during the operation. Hence, the Michaelis–Menten type model:
V 1 Ks 1 1 þ ¼ ¼ QðC in C out Þ EC EC max C ln EC max
2.2. Analysis of degradation products 2.2.1. Carbon balance For carbon balance the concentration of carbon in the inlet was taken as 100% and the carbon in other fractions were estimated in reference to this value. The inlet and outlet concentrations of isoprene were determined by gas chromatography (GC) as described earlier (Srivastva et al., 2015). Carbon dioxide in the outlet was also determined chromatographically using GC (Agilent Technologies, 7820A GC system, California, USA) as per manufacturer’s method. The analysis of total organic carbon content of the leachate was performed using a TOC analyzer (Analytik Jena Multi N/C 2100, UK) as per manufacturer’s protocol. Biomass carbon was calculated by subtracting the sum of outlet isoprene carbon, leachate carbon and CO2-C from the inlet isoprene carbon using equation proposed by Lu et al. (2002):
C inlet ¼ C outlet þ C L þ C CO2 þ C bio
3. Results and discussion
ð1Þ
Here, C inlet is the percentage of carbon in inlet gas stream, C outlet is percentage of carbon in outlet gas stream, C L is total organic carbon percentage in leachate, C CO2 is percentage of carbon in carbon dioxide and C bio is the carbon percentage in biomass. 2.2.2. Fourier transform infrared spectroscopy (FTIR) The leachate sample was centrifuged at 10000 rpm for 10 min to remove the bacterial cells and the supernatant (1 mL) was used for FTIR analysis (ALPHA, Bruker Optics, Billerica, MA) in the mid IR region of 500–4000 cm1. Same volume of pure isoprene (Merck Company, Germany) was used as control. The peaks interpretation was carried out using IR Pal 1.0 software. 2.2.3. Gas chromatography–mass spectrometry (GC–MS) The metabolites were extracted from the leachate (10 mL) in npentane as described earlier (Srivastva et al., 2015). One lL of the extracted sample was injected into Shimadzu GCMS-QP2010 Ultra (Serial No. O205249, USA) equipped with RxiÒ 5 ms capillary column (30 m 0.25 mm 0.25 lm), using a Hamilton gas-tight syringe (Model: 701 N). Same volume of pure isoprene (Merck Company, Germany) was used as control. Helium (99.99% purity) was used as the carrier gas at 100 kPa pressure and 11.9 mL min1 flow rate. The injector temperature was set at 250 °C and the oven temperature at 40 °C for 5 min and then ramped to 90 °C at the rate of 5 °C min1 for 2 min followed by ramping to 130 °C at the rate of 10 °C min1 for 2 min and finally to 260 °C at the rate of 10 °C min1 for 2 min. Split ratio was chosen as 4. The MS was operated in full scan mode with m/z ranging from 35 to 200. Ion source and interface temperatures were set at 250 and 300 °C,
ð2Þ
modified for continuous system (Mathur et al., 2006) was used for evaluating the kinetic parameters from the steady state experimental data of Phases I to III. Here, V is the working volume (m3), Q is the volumetric flow rate (m3 h1), C in is the inlet concentration (g m3), C out is the outlet concentration (g m3), EC is the elimination capacity (g m3 h1), EC max is the maximum elimination capacity (g m3 h1), K s is the saturation constant of substrate (isoprene) out (g m3) and C ln f¼ Cin C C in g is the logarithmic average of inlet and
lnC
out
outlet concentrations of isoprene. 1 The equations corresponding to the best fit straight line for EC vs 1 plots for bioscrubber and biofilter were generated using the C ln
least-squares method (Fig. 1). The EC max and K s for bioscrubber were found to be 666.7 g m3 h1 and 9.86 g m3, respectively (Fig. 1A) while those for biofilter were 3333 g m3 h1 and 13.96 g m3, respectively (Fig. 1B). The value of EC max /K s obtained for bioscrubber and biofilter units were 67.56 h1 and 238.7 h1, respectively. It has been established that the biofilter operates in the plugflow regime while the bioscrubber exhibits the well-mixed behavior (Yadav et al., 2014). The EC max for the bioscrubber obtained from the analysis of the data is comparable to the EC (567 g m3 h1) obtained experimentally (Srivastva et al., 2015) which indicates that the bioscrubber is operating at the near optimum condition. Further, the lower K s value compared to that for batch mode (Srivastva et al., 2015) indicates that this unit is more efficient than the batch operation. This can be attributed to the absence of oxygen limitation and better mixing due to bubbling of isoprene loaded air. Further, the bioscrubber is also advantageous in two ways – it acts as humidifier and prevents organic overloading of biofilter. The degradation capability of the biofilter, as evident from the kinetic parameter – EC max /K s , however, is better than the bioscrubber. This ratio reflects the efficiency of substrate degradation by bacterial community and is considered as an useful index for the enzymatic reaction. This kinetic study is also consistent with the previous report (Srivastva et al., 2015) where removal efficiencies of biofilter (89% in Phase II and 66% in Phase III) were found to be better than those of bioscubber (31% in Phase II and 17% in Phase III). Schlegelmilch et al. (2005) also reported 29% removal in the bioscrubber and 97–99% for the bioscrubber/biofilter combination. The EC max value for the biofilter unit obtained through analysis of data is around 2.6 times greater than the highest EC value (1256 g m3 h1) observed experimentally (Srivastva et al., 2015). This shows that the biofilter could be operated at even higher inlet loading rates.
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typically found in isoprene polymer (Gualtieri et al., 2005). The leachate sample also showed some additional peaks that were not observed in the control (Supplementary data, Fig. S1B). Peaks at 1000 and 1075 cm1 represent the C–O stretching of alcohol while peaks at 1475 cm1 indicate the bending of C–H in alkane. The peak at 1500 cm1 exhibits the N–H bending of amine group. The stretching of hydroxyl group of alcohol has resulted in peaks at 2300, 3550 and 3650 cm1. The appearance of peaks corresponding to carbonyl, hydroxyl and amine group in the leachate indicate oxidative cleavage of isoprene. The identification of compounds with such functional groups was done through GC–MS analysis.
Fig. 1. Lineweaver–Burk plot of Michaelis–Menten equation for (A) bioscrubber unit and (B) biofilter unit.
3.2. Biodegradation products analysis 3.2.1. Carbon balance The major portion of inlet carbon (62–77%) was converted into CO2, indicating efficient biodegradation potential of Pseudomonas sp. A small portion of carbon (4–20%) as undegraded isoprene was detected in the outlet. The loss of carbon between inlet and outlets can be assumed to be retained as biomass. The higher carbon content in the biomass (32% Cbio) during acclimation phase could be due to low isoprene concentration and fast initial growth of bacteria. As the flow rate increased during different phases, the growth slowed down and hence the biomass carbon also declined to 16% and then stabilized at 15%. The distribution of carbon in biomass and CO2 is in accordance with the results of earlier studies on biofiltraion of hydrocarbons (Marchal et al., 2003). Carbon observed in the leachate (1–2%) could be attributed either to the undegraded dissolved isoprene or the biodegradation products of isoprene; hence, the leachate components were further analyzed through FTIR.
3.2.2. FTIR analysis The FTIR spectra for control and leachate samples are depicted in the Supplementary data (Fig. S1). Peaks for the control sample at 750, 800 and 900 cm1 represent the C–H bond stretching of alkene (Supplementary data, Fig. S1A). The absence of these peaks in the leachate indicates biodegradation of isoprene. However, some common peaks i.e., 1425, 1500, 2850 and 2920 cm1 were found in both, control and leachate sample (Supplementary data, Fig. S1). Peaks at 1425 and 1500 cm1 indicate C– H stretching of aromatic compound. The presence of aromatic compounds could be due to polymerization of isoprene monomers (Mazza et al., 2000). The peaks observed at 2850 and 2920 cm1 represent the CH2 stretching (symmetric and asymmetric)
3.2.3. GC–MS analysis For control samples the gas chromatogram generated 5 peaks at different retention times (Supplementary data, Fig. S2) while for the extracted leachate samples 9 peaks were obtained (Supplementary data, Fig. S3) which are listed in Table 1. The MS spectrum for each peak was analyzed (Supplementary data, Figs. S4 and S5). In case of the control samples the most prominent peak was that of isoprene. However, the polymerized compounds of isoprene monomer such as limonene, D-limonene and 3,7-dimethyl-1,3,6octatriene are also observed. The dimerization products of isoprene during normal operating conditions have been reported by other workers also (Estevez et al., 2014). The polymerization of isoprene generating cyclic compounds is also well documented (Mazza et al., 2000). In the case of leachate samples, beside solvent (n-pentane) and dissolved CO2, the MS spectrum shows the presence of some compounds common with the control (Table 1). This could be due to the presence of undegraded isoprene in the leachate which further polymerized under the operating condition of GC–MS. The presence of estragole, a sesquiterpene, could be due to the degradation of isoprene polymers (Mazza et al., 2000). Further, some alipahtic compounds such as 3-methyl-2-butanone; 2,7-dimethyloctane; 3-methylbutanal and 2-aminopropanol were also observed. The presence of these compounds is in accordance to the functional groups identified through FTIR spectra. These compounds represent the isoprene biodegradation products. Earlier studies also showed alcohol and ketone as the metabolites produced during isoprene biodegradation by Pseudomonas sp. (Boyd et al., 2000). Unlike earlier reports, unsaturated aliphatic alkenes are not detected rather saturated alkane and aldehyde are observed. 3.3. Degradation pathway It is interesting to note that the compounds 3-methylbutanal, 2,7-dimethyloctane and 2-aminopropanol have been detected for the first time as biodegradation products of isoprene. Using these results and available published information on biodegradation of isoprene a plausible pathway has been proposed (Supplementary data, Fig. S6). The key enzymes that could play role in oxidation of isoprene in Pseudomonas sp. are dioxygenase, dehydrogenase, transaminase and some hydrogen adding enzymes. The biodegradation of isoprene is initiated with oxidative cleavage of unsaturated bond by dioxygenase generating 1,2-dihydroxy, 3-methyl, 3-butene or 1,2-dihydroxy, 2-methyl, 3-butene. Through a series of reactions mediated by dehydrogenase and hydrogen adding enzyme, 1,2-dihydroxy, 3-methyl, 3-butene gets converted to 3-methyl, 1,2-butanediol (Boyd et al., 2000). This compound could further undergo oxidation with the aid of dehydrogenase, particularly alcohol dehydrogenase, to produce 3-methyl-2-butanone and 3-methylbutanal. The presence of NAD-dependent alcohol dehydrogenase in Pseudomonas sp. has been shown to catalyze oxidation of primary and secondary alcohols to the corresponding aldehydes and ketones using NAD as coenzyme (Levin et al., 2004). Simultaneously, dehydrogenase could act on 3-methyl,
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Table 1 GC–MS analysis of control and leachate samples (RT: retention time). Peak No.
RT (min)
Area%
Height%
Compounds
Control 1 2
1.33 10.68
93.88 0.36
81.02 1.06
D-Limonene
3 4 5
10.79 13.27 13.37
0.81 3.86 1.09
2.28 11.95 3.69
Limonene 3,7-Dimethyl-1,3,6-octatriene 1,5-Dimethyl-1,5-cyclooctadiene
Leachate 1 2 3 4 5 6 7 8 9
1.28 11.86 13.27 13.38 25.58 26.57 28.20 29.61 29.77
94.62 0.36 1.78 0.53 0.38 0.16 1.56 0.44 0.17
92.48 0.42 2.52 0.79 0.46 0.28 1.76 0.94 0.35
n-Pentane Carbon dioxide 3,7-Dimethyl-1,3,6-octatriene 1,5-Dimethyl-1,5-cyclooctadiene Estragole 3-Methyl-2-butanone 2,7-Dimethyloctane 3-Methylbutanal 2-Aminopropanol
2-Methyl-1,3-butadiene
1,2-butanediol to remove hydroxyl group and generating saturated alkane which could further dimerize to produce 2,7dimethyloctane. The synthesis of extracellular hydrocarbons has been reported in Pseudomonas sp. They are assumed to play a role in cell aggregation (Nikolaev et al., 2001). The sequential action of dehydrogenase and transaminase could produce 2-aminopropanol from 1,2-dihydroxy, 2-methyl, 3-butene. The 2-aminopropanol, also known as L-alaninol, has been reported to further undergo oxidation to generate an amino acid, alanine, in Pseudomonas sp. (Wasch et al., 2002).
4. Conclusions The kinetic parameters show the superior efficiency of biofilter for isoprene degradation compared to bioscrubber. Study also indicates that the system could be operated at higher inlet loading rates without inhibition. The conversion of major portion of inlet isoprene into carbon dioxide shows the efficacy of Pseudomonas sp. for isoprene abatement. The FTIR and GC–MS results have been successfully used for proposing a more realistic pathway for isoprene biodegradation through enzymes: dioxygenase, dehydrogenase, transaminase and some hydrogenases.
Acknowledgments One of the authors (NS) is thankful to CSIR, New Delhi, India, for the financial support in the form of JRF and SRF. Authors also thank Jindal Steel and Power Ltd., Raigarh, for GC–MS analysis of leachate.
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