Diesel and Kerosene Degradation by Pseudomonas desmolyticum. 2112) and Nocardia hydrocarbonoxydans NCIM 2386 (Nh

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1 Curr Microbiol (8) 56: DOI 1.17/s Diesel and Kerosene Degradation by Pseudomonas desmolyticum NCIM 2112 and Nocardia hydrocarbonoxydans NCIM 2386 Satish Kalme Æ Ganesh Parshetti Æ Sushma Gomare Æ Sanjay Govindwar Received: 4 April 7 / Accepted: June 7 / Published online: 11 March 8 Ó Springer Science+Business Media, LLC 8 Abstract Pseudomonas desmolyticum NCIM 2112 (Pd 2112) and Nocardia hydrocarbonoxydans NCIM 2386 (Nh 2386) demonstrated an ability to degrade diesel and kerosene. Triton X-1 had enhanced the diesel degradation process by reducing the time required for the maximum utilization of total petroleum hydrocarbon. Fourier transform infrared spectroscopy spectrum of degraded diesel indicates the presence of aliphatic and aromatic aldehydes, C=C aromatic nuclei, and substituted benzenes. Surface tension reduction and stable emulsification was increased using consortium when compared to individual strains. Triton X-1 showed increase in microbial attachment to hydrocarbon among the various chemical surfactants tested. For generating a rapid assay to screen microorganisms capable of degrading kerosene, the acetaldehyde produced in the degradation process could be used as an indicator of degradation. These results indicate diesel and kerosene degradation ability of both of the strains. Introduction Industrialization and accidental spillage have increased the pollution of hydrocarbon compounds in the soil as well as water. The major constituents of kerosene are alkanes and cycloalkanes (68.6%); benzene and substituted benzene (13.7%); and naphthalene and substituted naphthalene. Kerosene possesses moderate to high acute toxicity to biota S. Kalme G. Parshetti S. Gomare S. Govindwar (&) Department of Biochemistry, Shivaji University, Kolhapur 4164, India spg_biochem@unishivaji.ac.in with product-specific toxicity related to the type and concentration of aromatic compounds [13]. Even though alkanes are the most abundant compounds in crude oil, it is necessary to search for microorganisms that are also able to oxidize alkylated polycyclic aromatic hydrocarbons (PAHs). This was observed in Pseudomonas sp. F21, isolated in a mineral medium with Arabian crude oil. Strain F21, which degrades all the n-alkanes and branched alkanes of low molecular weight, also causes selective depletion of methylated naphthalenes, phenanthrenes, chrysenes, and pyrenes [3]. Pseudomonas and Nocardia sp. are often isolated from hydrocarbon-contaminated sites and hydrocarbon degrading cultures [2, 14]. There is a need to prepare the consortium of microorganisms that can degrade alkanes as well as PAH. Keeping these points in mind, our objective for this study was to access the potential of both of the strains for diesel degradation in the presence of individual culture and consortium. A simple, rapid assay procedure for kerosene biodegradation has been developed by modifying the method of Jacobs et al. [5]. This can help to screen microorganisms having potential to degrade kerosene by transforming kerosene to alcohol. Materials and Methods Bacterial Strain and Growth Conditions Pseudomonas desmolyticum NCIM 2112 (Pd 2112) and Nocardia hydrocarbonoxydans NCIM 2386 (Nh 2386) were obtained from National Center for Industrial Microorganisms, India. Pure culture was maintained on nutrient agar slants at 4 C. Both of the strains were pregrown in nutrient broth medium at 3 C for 24 h and used for diesel

2 582 S. Kalme et al.: Diesel and Kerosene Degradation by Microbes degradation studies. Bushnell Hass medium containing (g/l) K 2 HPO 4, 4.74, KH 2 PO 4,.56 (Hi-Media, Mumbai), and mineral medium containing (g/l) K 2 HPO 4, 1.71, KH 2 PO 4, 1.32, NH 4 Cl, 1.26, MgCl 2,.11, CaCl 2,.2 were used for degradation studies cells/ml of Pd 2112 or Nh 2386 were inoculated in the media for the studies unless stated. Chemicals The nonionic chemical surfactants such as Triton X-1, Tween, and Igepol CO-89 were purchased from Sisco Research Laboratories, Sd fine, and Sigma-Aldrich, respectively. Diesel and kerosene were purchased from the gasoline filling station and local market, respectively. Diesel Oil Degradation One hundred milliliters of Bushnell Hass medium or mineral medium, in a 5-mL Erlenmeyer flask, containing 1% of diesel (v/v,.814 g/ml of density) were inoculated with Pd 2112 or Nh 2386 cells. Efficiency of consortium for diesel degradation in presence or absence of Triton X-1 (.24 mm) was studied only in Bushnell Hass medium. Flasks were incubated at 3 C at 1 rpm on an orbital shaker. A control for each was incubated without microorganism to measure abiotic loss. Percent removal of total petroleum hydrocarbon (TPH) was studied up to 7 weeks at 1-week intervals. Measuring dry weight of the cells monitored bacterial growth. TPH was extracted from culture broth supernatant by using dichloromethane (1:1, v/ v) [16]. The degraded diesel was analyzed using the Fourier transform infrared spectroscopy (FTIR) (Perkin-Elmer) in the region of 4 4/cm. Surface Tension Culture broth of Bushnell Hass medium used for diesel oil degradation was centrifuged at 8 g for 3 min after incubation of 48 and 168 h. The biosurfactant production was estimated by reporting change in surface tension measured with a Du-Nouy tensiometer by the method of Grishchenkov et al. [4]. Surface tension of culture liquid extracted immediately after addition of microorganism was used as a control ( h). Microbial Attachment of Hydrocarbons Cells of Pd 2112 and Nh 2386 were grown at 3 C in Bushnell Hass medium containing 1% (v/v) hexadecane for 48 h, keeping the flask on orbital shaker at 1 rpm. Cells were harvested by centrifugation and resuspended in 5 mm phosphate buffer (ph 7.4) giving optical density of.72 for Pd 2112 and consortium and.4 for Nh 2386 at 4 nm. The attachment of microorganisms to the hydrocarbon was calculated as the percent decrease in the optical density [16]. The effect of chemical surfactants on microbial attachment was studied by using nonionic surfactants such as Triton X-1 (CMC.24 mm), Tween (CMC.8 mm), and Igepol CO-89 (CMC.9 mm) at half of their critical micelle concentration. For this study,.2 ml surfactant buffer was used in reaction mixture and attachment studied without surfactant was kept as control. Emulsification Assay Pd 2112 and Nh 2386 were grown in 1% (v/v) diesel in Bushnell Hass medium for 48 h at 3 C on orbital shaker at 1 rpm. Cells were recovered by centrifugation at 8 g for 3 min and resuspended in 5-mM phosphate buffer ph 7.. The method of Nadarajah was followed to study the deemulsification of hydrocarbon water emulsion [7]. In this study, emulsification by individual strains was compared to values generated by consortium. Enzyme Activities Preparation of Cell Free Extract Pd 2112 and Nh 2386 cells were grown in nutrient broth at 3 C for 24 h and 48 h, respectively, centrifuged at 6 g for min. These cells were suspended in 5-mM potassium phosphate buffer (ph 7.4) for sonication (Sonics- Vibracell ultrasonic processor), keeping sonifier output at 4 amp and maintaining temperature below 4 C and giving eight strokes of 5 s each with 2-min interval. This extract was used without centrifugation as enzyme source. Enzyme Assays The test of Jacobs et al. [5] was conducted to detect the biodegradation of kerosene, by a monooxygenase biodegradation pathway, as the following reactions: Ethanol þ nicotinamide adenine dinucleotide phosphate NADP þ #" Alcohol dehydrogenase Acetaldehyde þ NADP þ þ H þ Acetaldehyde þ Nash # 58 C/ min Diacetyldihydrocollidine yellow colored compound, k max 388 nm Assay for kerosene biodegradation contained.5-ml 5-mM HEPES buffer (ph 7.8),.2-mL NADPH-generating

3 S. Kalme et al.: Diesel and Kerosene Degradation by Microbes 583 system (NADP, 2.6 mm: glucose 6-phosphate, 12.5 mm; glucose 6-phosphate dehydrogenase, 4 units),.5-ml cellfree extract, and.4 ml of kerosene in reaction tube. The reaction mixture was incubated at 37 C for 3 min with constant orbital shaking at 1 rpm. The reaction was terminated by adding 1 ml % ice-cold trichloroacetic acid solution. The amount of acetaldehyde liberated was determined at 388 nm by using Nash reagent [8]. Induction of Kerosene Degradation Activity Cells grown in nutrient broth were inoculated in the mineral medium containing one organic compound at a time, as an inducer at concentration indicated in Table 2. The cells were harvested after incubation of 24 h at 3 C with constant orbital shaking at 1 rpm and cell-free extract was prepared as described above. This cell-free extract was used as enzyme source to study induction in kerosene degradation activity. Cells harvested immediately after the addition of inducer were treated as control. Statistical Data Analyses Data were analyzed by one-way analysis of variance (ANOVA) with Tukey-Kramer multiple comparisons test. Removal of TPH (%) Time (days) Dry weight (mg) Fig. 1 Biodegradation of diesel in mineral medium by Pd 2112 (j) and Nh 2386 (m) reported with change in dry weight for Pd 2112 (h) and Nh 2386 (4). Removal of total petroleum hydrocarbon (TPH) was estimated as described in Materials and Methods. The values are the average of two independent experiments. The maximum standard deviation is within ± Results and Discussion Biodegradation of Diesel In mineral medium containing diesel, Pd 2112 and Nh 2386 took 7 weeks for 95% and 98% removal of TPH, respectively (Fig. 1). In Bushnell Hass medium, Pd 2112 had taken 7 weeks for 98% removal, whereas Nh 2386 showed the same percent removal of TPH within 6 weeks. As Bushnell Hass supports maximum degradation, we had tried it for the consortium, and we found that the consortium removed 97% and 98% TPH with and without Triton X-1, within 4 and 5 weeks, respectively (Fig. 2). Abiotic loss of TPH was measured at the time of last extraction from uninoculated flask, which was 22 24% within 7 weeks. In growth pattern, the time required to reach stationary growth phase was 5 weeks for Pd 2112 and 4 weeks for Nh 2386 in mineral medium (Fig. 1). In Bushnell Hass medium, both of the strains individually required 4 weeks and as a consortium with or without Triton X-1 required 3 weeks to reach stationary phase. Dry weight change showed that the Bushnell Hass medium had enhanced the growth of both of the strains as compared to mineral medium (data not shown). When a major oil spill occurs in marine and freshwater environments, the supply of carbon is dramatically increased Removal of TPH (%) Time (days) Fig. 2 Biodegradation of diesel in Bushnell Hass medium by Pd 2112 (h), Nh 2386 (j), Pd Nh 2386 (m), Pd Nh Triton X-1 (d). The values are the average of two independent experiments. The maximum standard deviation is within ±1 and the availability of nitrogen and phosphorus generally becomes the limiting factor for oil degradation [1]. Addition of nonionic surfactant such as Tween 8 has indicated enhancement of the biodegradation of hydrocarbons [9]. Bushnell Hass medium along with Triton X-1 has enhanced the uptake of hydrocarbon compounds for both of the strains and reduced the time required for the biodegradation

4 584 S. Kalme et al.: Diesel and Kerosene Degradation by Microbes Fig. 3 (A) Fourier transform infrared spectroscopy (FTIR) comparison of extracted total petroleum hydrocarbon (TPH) from control and 5-week incubated diesel with Pd (B) FTIR comparison of extracted TPH from control and 3-week incubated diesel with Nh 2386 A %T control th week extracted sample B %T control rd week extracted sample cm cm- FTIR spectrum of control sample (extracted immediately after inoculation in Bushnell Hass medium) showed characteristic peaks at 2854, 2924, and 2955/cm (C-H aliphatic stretch). CH 3 group of hydrocarbon compounds for long-chain alkanes was represented by 1378, 1459 (C-H deformation), and 194/cm. Fifth-week extracted TPH with the addition of Pd 2112 had shown appearance of two peaks at 2749, 2729/cm, and nine sharp peaks between 782 and 146/cm represent the formation of aliphatic and aromatic aldehydes. Also, a new peak at 164 (C=C aromatic nuclei) with /cm represents formation of mono, meta, para substituted benzene, which suggests biodegradation of diesel oil by Pd 2112 (Fig. 3A). FTIR of TPH extracted from Nh 2386 inoculated medium after 3 weeks of incubation had shown appearance of a new peak at 164/cm with 1464/cm (C=C aromatic nuclei) represents the skeletal vibration of benzene ring for monocyclic aromatic hydrocarbons. Sharp peaks between 7 and 9/cm showed the degradation of complex aromatics into di, tri, and tetra substituted benzene derivatives, which suggests biodegradation of diesel oil by Nh 2386 (Fig. 3B). The FTIR results suggest that both of the strains prefer C-H aliphatic stretch for degradation of long-chain alkanes. Also, they convert complex aromatic compounds into substituted benzene derivatives. Surface Tension and Emulsification Pd 2112 reduced the surface tension by 31.6% and 47.7%, Nh 2386 showed 29.7% and 46.3% reduction, and consortium of these strains showed significant reduction by 49.2% and 56.5% for 48- and 168-h incubation,

5 S. Kalme et al.: Diesel and Kerosene Degradation by Microbes 585 respectively, as compared to h culture broth (Table 1). These data reveal the ability of both of the strains to produce biosurfactants. Pd 2112 had an emulsification value of 16% associated with the supernatant, 5% with the cells suspended in phosphate buffer. Nh 2386 also showed increased emulsification values in supernatant compared with cells suspended in phosphate buffer, which were 22% and 11.33%, respectively. Consortium of these two strains showed emulsification of 25.33% in supernatant and 12% associated with cells (Table 1). Petroleum hydrocarbon degrading bacteria can sometimes produce biosurfactants, which improves the solubility of hydrophobic pollutants and thus, their biodegradability [6]. Van Hamme and Ward [15] had observed emulsifying abilities in Pseudomonas and Rhodococcus sp. with increased access toward the diesel. In our study we found that consortium is able to decrease surface tension and form stable emulsification more efficiently as compared to individual strain, which has helped the consortium to degrade more diesel oil. Microbial Attachment to Hydrocarbons The attachment of hexadecane grown Pd 2112 to hydrocarbon was enhanced by Triton X-1 (31.9%), Tween (31.4%), and Igepol CO-89 (28.5%) as compared to the Table 1 Properties such as change in surface tension, microbial hydrocarbon attachment, and emulsification potential of Pd 2112, Nh 2386, and consortium Experimental condition Pd 2112 Nh 2386 Pd Nh 2386 Surface tension (MN/m) h 69.5 ± ±.1 7. ±.5 48 h 47.5 a ± a ± a ± h 36.3 a ± a ± a ±.3 Microbial hydrocarbon attachment (%) Control ± ± ±.6 Igepol CO ± ± *** ±.53 Triton X * ± ** ± ±.6 Tween * ±.5. ± ±.6 Emulsification potential (%) Supernatant 16. ± ± ±.33 Cells suspended in 5 mm phosphate buffer 5. ± b ± b ±.5 Values are mean of three experiments ± SEM a Significantly different from control ( h) at P \.1 * Significantly different from control cells at P \.5. ** Significantly different from control cells, Igepol CO-89 and Tween at P \.1 and *** Significantly different from control cells, Triton X- 1 and Tween at P \.1 b Significantly different from Pd 2112 at P \.1 cells treated without external addition of chemical surfactants (26.66%). In the case of Nh 2386, Triton X-1 increased the attachment up to 28.88%, whereas Igepol CO-89 (%) and Tween (%) have not shown significant change in hydrocarbon attachment, as compared to cells treated without external addition of chemical surfactants (19.99%) (Table 1). The variation in microbial hydrocarbon attachment supported the variability in solublizing PAH by individual surfactant. Triton X-1 was found to be the best chemical surfactant to enhance the microbial attachment to hydrocarbons for both of the strains. Triton X-1 had also increased TPH removal with reduction in time required for diesel oil degradation. Prak and Pritchard have also shown that synthetic surfactant such as Triton X-1, Tween, Tween 8, and Afonic (a nonionic alkyl ethoxylate) enhances the concentration of PAHs in the aqueous phase [9]. Kerosene Degradation Activity and Its Induction A simple, rapid, and suitable method for screening microorganisms for kerosene degradation was developed with some modification of the method used by Ismail Saadoun for diesel [11] and Jacobs et al. [5]. NADH and acetaldehyde were generated by the action of alcohol dehydrogenase on ethanol in this test. We have estimated acetaldehyde by using Nash reagent [8] instead of measuring NADH as reducing equivalent for dichlorophenol indophenol (DCPIP). Pure aromatic hydrocarbons (camphor and naphthalene), medium- and long-chain alkanes (hexane and hexadecane), and methoxylated aromatic compound (1,2 dimethoxybenzene) were selected as carbon source for studying their effect as inducers of kerosene degradation activity. Pd 2112 incubated in mineral medium containing n-hexadecane, n-hexane, naphthalene, 1,2 dimethoxybenzene, and camphor for 24 h showed 487, 394, 378, 975, and 215% induction, respectively, for kerosene degradation assay with comparison of h incubated cells, whereas Nh 2386 showed 599, 233, 566, 34, and 822% induction, respectively, with comparison of h incubated cells (Table 2). All carbon sources had shown significant induction in kerosene degradation activity. These inducers might have increased the amount of soluble cytochrome P-45 in both of the strains. Kerosene degradation assay required NADPH as an essential cofactor, and this activity was inhibited by 63% (in P. desmolyticum 2112) and by 5% (in N. hydrocarbonoxydans) in presence of carbon monoxide (data not shown), indicating involvement of cytochrome P45. Similar results had been observed by Salokhe and Govindwar in Serratia marcescens [12]. Bredholt et al. had observed in Rhodococcus sp. that compounds that induce the emulsifying ability simultaneously induced the cytochrome P-45 containing alkane oxidizing system [1].

6 586 S. Kalme et al.: Diesel and Kerosene Degradation by Microbes Table 2 Induction of kerosene degradation activity in Pd 2112 and Nh 2386 cells grown for 24 h with individual carbon source in mineral medium as compared to cells incubated for 2 min (control) Carbon source Conclusion The removal of TPH had shown that the consortium could degrade diesel oil more efficiently as compared to individual strains. Triton X-1 can be used for its effectiveness in increasing the availability of hydrocarbons for the degradation. Decrease in surface tension, emulsification properties, kerosene degradation activity and its induction suggests degradation of alkane and PAH by these two strains. Acknowledgments The authors thank Dr. U. V. Desai for his valuable discussion, and CFC (common facility center, Shivaji University, Kolhapur) for technical assistance. References Concentration (g/l) Kerosene degradation activity a Pd 2112 Nh 2386 Control 3.58 ± ±.31 n-hexadecane 1.93 ** ± ** ± 3.19 n-hexane ** ± * ± 1.4 Naphthalene ** ± ** ± 1.4 1,2 Dimethoxybenzene ** ± ** ±.7 Camphor * ± ** ± 1.4 a lmol of acetaldehyde liberated/min/mg protein Values are mean of three experiments ± SEM Significantly different from the control (uninduced cells) at * P \.5, ** P \.1 by one-way analysis of variance with Tukey- Kramer multiple comparisons test 1. Bredholt H, Bruheim P, Potocky M, Eimhjellen K (2) Hydrophobicity development, alkane oxidation, and crude-oil emulsification in a Rhodococcus species. Can J Microbiol 48: Chang JH, Rhee SK, Chang YK, Chang HN (1998) Desulfurization of diesel oils by a newly isolated dibenzothiophene-degrading Nocardia sp. strain CYKS2. Biotechnol Prog 14: Cho BH, Chino H, Tsuji H, Kunito T, Makishima H, Uchida H (1997) Analysis of oil components and hydrocarbon-utilizing microorganisms during laboratory-scale bioremediation of oilcontaminated soil of Kuwait. Chemosphere 35: Grishchenkov VG, Townsend RT, McDonald TJ, Autenrieth RL, Bonner JS, Boronin AM () Degradation of petroleum hydrocarbons by facultative anaerobic bacteria under aerobic and anaerobic conditions. Process Biochem 35: Jacobs CJ, Prior BA, Dekock MJ (1983) A rapid screening method to detect ethanol production by microorganisms. J Microbiol Methods 1: Kanga SA, Bonner JS, Page CA, Mills MA, Autenrieth RL (1997) Solubilization of naphthalene and methyl-substituted naphthalenes from crude oil using biosurfactants. Environ Sci Technol 31: Nadarajah N (1999) Evaluation of mixed bacterial culture for the deemulsification of water in oil petroleum emulsions. M.Sc. thesis. University of Waterloo, Waterloo, Ontario, Canada 8. Nash T (1953) The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem J 55: Prak DJL, Pritchard PH (2) Degradation of polycyclic aromatic hydrocarbons dissolved in Tween 8 surfactant solutions by Sphingomonas paucimobilis EPA 55. Can J Microbiol 48: Prince RC, Bare RE, Garrett RM, Grossman MJ, Haith CE, Keim LG (3) Bioremediation of stranded oil on an arctic shoreline. Spill Sci Technol Bullet 8: Saadoun I (2) Isolation and characterization of bacteria from crude petroleum oil contaminated soil and their potential to degrade diesel fuel. J Basic Microbiol 42: Salokhe MD, Govindwar SP (1999) Effect of carbon source on the biotransformation enzymes in Serratia marcescens. World J Microbiol Biotech 15: Song HG, Bartha R (199) Effects of jet fuel spills on the microbial community of soil. Appl Environ Microbiol 56: Ueno A, Hasanuzzaman M, Yumoto I, Okuyama H (6) Verification of degradation of n-alkanes in diesel oil by Pseudomonas aeruginosa strain WatG in soil microcosms. Curr Microbiol 52: Van Hamme JD, Ward OP (1) Physical and metabolic interaction of Pseudomonas sp. strain JA5-B45 and Rhodococcus sp. strain F9-D79 during growth on crude oil and effect of a chemical surfactant on them. Appl Environ Microbiol 67: Zhang Y, Miller RM (1994) Effect of a Pseudomonas rhamnolipid biosurfactant on cell hydrophobicity and biodegradation of octadecane. Appl Environ Microbiol 6:

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