INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 3, No 1, Copyright by the authors - Licensee IPA- Under Creative Commons license 3.

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1 INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 3, No 1, 2012 Copyright by the authors - Licensee IPA- Under Creative Commons license 3.0 Research article ISSN The study of growth kinetics of Bacillus subtilis BMT4i (MTCC 9447) using diesel as the sole carbon and, Bahuguna Ashutosh, Bhatt Kamlesh Kumar, Dangwal Koushalya Department of Biotechnology, Modern Institute of Technology (MIT), Dhalwala, Rishikesh , Uttarakhand, India. kdangwal1@yahoo.co.in doi: /ijes ABSTRACT The present study aimed at studying the growth kinetics of an efficient high molecular weight polycyclic aromatic hydrocarbon (HMW-PAH) degrader Bacillus subtilis BMT4i (MTCC 9447) using diesel oil as the sole carbon and. The study was carried out in order to understand and reveal the morphological and growth variations in benzo-a-pyrene (BaP) degrading BMT4i imposed by the stress environment of diesel oil. The morphological variations were determined by growing Bacillus subtilis BMT4i in basal salt medium (BSM) with diesel oil (2%: BSMD) as sole carbon and at various time durations using Gram staining and spore staining (Schaeffer-Fulton method) followed by viability and growth assessment using colony forming units (CFU)/ml method. The result demonstrated that the diesel oil has visible effect on the shape and size of BMT4i cells. Majority of the BMT4i cells undergone endospore formation within 24 h of diesel oil exposure and some of them were released as exospores (free spores) after 24 h. The growth kinetics demonstrated exponential increase in the cell number of BMT4i in BSMD with increase in incubation duration up to 3 days reaching maxima of 4.16 x showing approx. 2x fold increase in cell number and there after it declined. About 2x10 11 fold enhancements in the CFU number is directly correlated with the ability of Bacillus subtilis BMT4i to utilize diesel oil as the sole source of carbon and energy which leads to high increase in cell number within just 3 days. Therefore, it could be concluded that Bacillus subtilis BMT4i (MTCC 9447) is very efficient in using diesel oil as the sole source of carbon and energy and it could be employed for bioremediation of diesel oil and PAH contaminated sites. Keywords: Bacillus subtilis BMT4i (MTCC 9447), Benzo-a-pyrene (BaP), Bioremediation, Degradation, Diesel oil. 1. Introduction Hydrocarbons such as diesel fuel, crude oil and petroleum distillates are some of the world s most widely used primary energy and fuel resources, due to the energy they produce when combusted (Watanabe, 2001). Huge quantities of fuel are required to power industry, automobiles and heat homes and with the number of times each gallon of petroleum is stored, transported, or transferred, accidents and leakages are inevitable (Surridge, 2007), making these hydrocarbons the most common global environmental pollutants. Their contamination is hazardous to the health of plants and these are carcinogenic, mutagenic and potent immuno-toxicants, posing a serious threat to human and animal health (Altas, 1981; Zhou and Crawford, 1995; Liebeg and Cutright, 1999; Ting and HuTan, 1999; Vasudevan and Rajaram, 2001). After critical removal of large amounts of the oil by various physical and chemical methods, microbial degradation process aids the elimination of spilled oil from the Received on May 2012 Published on July

2 environment (Ijah and Okang, 1993). This is possible because microorganisms have enzyme systems to degrade and utilize diesel oil as a source of carbon and energy (Ijah and Antai, 1988; Antai and Mgbomo, 1993; Ezeji et al., 2005). We have reported previously the utilization of carcinogenic BaP, a pentacyclic compound released by the incomplete combustion of petroleum; as the sole source of carbon and energy by a novel strain Bacillus subtilis BMT4i (MTCC 9447) which is able to degrade more than 80% of BaP after 28 days of growth via an inducible chromosomally encoded pathway (Lily et al., 2009; 2010). In addition, BMT4i is also able to degrade a range of other PAHs such as naphthalene, anthracene and dibenzothiophene. It is of great significance to ensure the ability of BMT4i to use fuel such as diesel oil as the sole carbon and and hence their degradation. Therefore, to reveal the morphological and growth variations in BMT4i imposed by the stress environment of diesel oil, the present study was designed to study the growth kinetics of Bacillus subtilis BMT4i (MTCC 9447) using diesel oil as the sole carbon and. 2. Materials and methods Chemicals and reagents: Tryptone, peptone, beef extracts, bacto-agar, yeast extract, dextrose and staining reagents used in the present study were purchased from HiMedia Laboratories (Mumbai, India). General chemicals, including constituents of basal salt mineral medium (BSM) of analytical grade were purchased from Glaxo (Mumbai, India) and Merck (Mumbai, India). 2.1 Growth kinetics of BMT4i in diesel and glucose media In order to determine the potential of Bacillus subtilis BMT4i to use diesel oil as the sole source of carbon and energy, the growth kinetics of BMT4i in BSMD (2% diesel oil) was studied. Gram staining and spore staining (Schaeffer-Fulton method) of BMT4i cultures at various time durations were performed in order to study the morphological variations. In addition, the viability of BMT4i was also checked by CFU method as mentioned previously (Lily et al., 2009; 2011). All the experiments were set up in triplicates. To study the growth kinetics of BMT4i, a single colony of BMT4i was inoculated in 250 ml of nutrient broth, grown at 37 o C with constant stirring at rpm till the A 600 reaches to 1.0 (approx. 1x10 8 cells/ml). Afterwards, the Gram Staining and spore staining of the culture was done to ensure the purity and the absence of spores respectively. Thereafter, the culture was centrifuged at 8000 rpm for 10 minutes and washed thrice with BSM to remove trace of nutrient broth. The pellet of BMT4i was suspended in 25 ml of BSM and cell number was adjusted to 10 8 cells /ml in BSM. For the time course studies, the test and control flasks were set up. For the test, 2.0 ml of BSM suspension culture of BMT4i (2x10 6 cells /ml) was inoculated in 250 ml flask containing 100 ml BSMD. For the comparison of growth of BMT4i in the diesel oil, 2.0 ml culture was separately inoculated in another 250 ml flask containing 100 ml of BSM with 2.0% glucose (BSMG), followed by incubation in shaker incubator at 37 C at 150 rpm for various time periods along with their respective controls devoid of BMT 4i. At various time points (0, 1, 2, 3, 7, 14, 21, 28, 35, 42 days), 1.0 ml culture of BMT4i was withdrawn from all the test flasks i.e. BSMD and BSMG cultures. First of all, the morphology of BMT4i cells and presence of spores in the culture were checked by Gram staining and spore staining respectively. Afterwards, the CFU/ml of BMT4i was checked to ascertain the viability and growth of BMT4i in the above mentioned media. For CFU/ml measurement, 100 µl cultures from all the test and control flasks were diluted up to 10-14, plated over BSMG agar (1.5%) 21

3 plates and further incubated at 37 o C. After 24 h, the numbers of colonies were counted and the CFU/ml was calculated. The growth curve was prepared by plotting log 10 CFU/ml against incubation time. 3. Results and discussion 3.1 Growth kinetics of Bacillus subtilis BMT 4i (MTCC 9447) in BSMD The present study was aimed to study the growth kinetics of a novel bacterial strain Bacillus subtilis BMT4i (MTCC 9447) using diesel oil as the sole source of carbon and energy to understand and reveal the morphological and growth variations in BMT4i imposed by the stress environment of diesel oil. For that, the BMT4i was grown in diesel oil media for various time points and the morphological features of BMT4i were studied by Gram staining and spore staining followed by viability assessment of BMT4i by CFU method. The Gram staining of BMT4i grown in BSMD demonstrated that the diesel oil has visible effect on the shape and size of BMT4i cells. Just after 24 h, the thickness and the size of rods of BMT4i decreased, although the shape remained the same up to 7 days. The chain arrangement of the BMT4i cells that was visible at 0 day was lost within 24 h. Thereafter, the BMT4i were replaced by rounded pleomorphic cells which remained abundant up to 28 days (Figure 1). The spore staining of BMT4i grown in diesel oil media revealed that majority of the BMT4i cells started undergoing endospore formation within 24 h of diesel oil exposure and some of them were released as exospores (free spores) after 24 h. The spores became enormous and their number increased up to 21 days, thereafter remained stationary (Figure 1). The present study is the first report demonstrating the morphological transformation of any bacterial species induced by diesel oil in the growth medium. The change in the thickness and size of the rods in addition to induction of sporulation in the diesel media is quite justifiable since diesel oil is not the regular carbon source and BMT4i has to induce diesel oil degradation pathway in order to adapt to the stress condition imposed by diesel oil. It has been previously reported that microorganisms enter into a resting state remaining inactive for an extended period of time under nutritional limitation (Errington, 2003). Specialized differentiated cell types are used by a wide range of bacteria as a method of dealing with starvation and the survival of harsh conditions. Some of the specialized cells are called spores. For example, Bacillus subtilis attains a robust resting state, termed as an endospore that can remain dormant for years. It is known that the bacterial endospore formation is an energy intensive and time consuming process. The growth kinetics experiment revealed that the BMT4i possess ability to utilize diesel oil for its growth. As shown in the plot of log 10 of CFU/ml (cell number) against incubation time, starting with an initial log 10 CFU/ml of BMT4i (2.12 x 10 8 ), the cell number of BMT4i increased exponentially with increase in incubation duration up to 3 days, declines slightly and remained stationary up to 21 days (Figure 2). Thereafter, the cell number declined drastically. At the beginning, log 10 CFU/ml of BMT4i (2.12 x 10 8 ) of BMT4i was added to the BSMD. The CFU/ml started increasing linearly after 24 h ( log 10 CFU/ml) attaining maximum log 10 CFU/ml (4.16 x ) on 3 rd day showing approx. 2x fold increase in the growth. The CFU/ml declined slowly after 3 rd day reaching log 10 CFU/ml (8.06 x ) after 7 th day remaining almost stationary up to 21 day, thereafter the viability declined sharply. About 2x10 11 fold enhancements in the CFU number is directly correlated with the ability of Bacillus subtilis BMT4i to utilize diesel oil as the sole source of carbon and energy which leads to high increase in cell number within just 3 days. 22

4 The ability of BMT4i to utilize diesel as the carbon and is not surprising since BMT4i has been isolated from PAH contaminated soil and it possess the inherent capability to utilize hydrocarbons for its growth 9. However, it is unexpected that despite of huge morphological transformation and early induction of sporulation BMT4i cells are continue to divide and attain their peak growth. The reason for which is uncertain. The sustenance of high growth in the form of stationary phase could be explained by the fact that bacterial growth and degradation of PAH as a sole source of carbon and energy in the liquid culture depends upon the availability of aqueous soluble fraction of the PAH which further depends upon the aqueous dissolution rate of PAH 7. When the PAH consumption by the increasing population exceeds the PAH aqueous dissolution rate, the dissolved PAH concentration drops and exponential growth ceases, even though the culture media contains sufficient amount of PAH which is not available due to low aqueous dissolution rate (Johnsen et al., 2005). In the present study, it is possible that the metabolic demand of exceedingly high cell number of BMT4i in peak growth could not be fulfilled by the aqueous soluble fraction of diesel oil and hence the cell number drops. Figure 1: Micrograph showing Gram Staining and spore staining of Bacillus subtilis BMT4i grown for 0, 3, 7, 14, 21, 35 and 42 days in BSMD (BSM with 2% diesel oil) medium. (Magnification 1000 X) 23

5 25 log10cfu/ml BMT4i in BSMD B Incubation Time (in days) Figure 2: Growth (log 10 CFU/ml) of Bacillus subtilis BMT4i (MTCC 9447) in BSMD (BSM with 2% diesel oil) for various incubation duration (days). Each point represents the average value obtained with three independent experiments. 3.2 Growth kinetics of Bacillus subtilis BMT 4i (MTCC 9447) in BSMG To compare the growth kinetics of BMT4i in BSMD to that in BSMG in which glucose is the usual carbon source, the BMT4i was grown in BSMG for different time durations followed by their morphological examination by Gram staining and spore staining in addition to the viability measurement by CFU/ml method. The Gram staining revealed negligible effect on the shape and size of BMT4i cells up to 10 days. However, after 10 day, the chain arrangement of the BMT4i cells was lost. Thereafter, the BMT4i were replaced by small sized cells after 21 days (Figure 3). Later on the number of live cells declined and most of the cells morphology became distorted by 42 days. The spore staining of BMT4i grown in BSMG demonstrated the absence of spore formation by BMT4i cells up to 7 days. On 10 th day, few exospores were observed on spore staining of BMT4i cell. Afterwards, the number of spores enhanced and became abundant by 42 days. The observations clearly demonstrated regular morphological pattern as thick rods showing chain arrangement and no spore formation up to 7 days in the BSMG. It is not unexpected, since glucose is a readily utilizable carbon source so cells remained healthy in its presence for more duration. After 7 days of good growth, due to exhaustion of glucose as carbon source, the stress response was generated by BMT4i cells and they undergone spore formation which became evident by release of few spores by 10 days. The growth kinetics experiment in BSMG demonstrated that the cell number of BMT4i increased exponentially with increase in incubation duration up to 3 day and there after it became almost static up to 10 days and later on it started declining slowly (Figure 4). The initial 1.2 x 10 8 BMT4i cells ( log 10 of CFU/ml) inoculated on 0 day started increasing linearly after 24 h ( log 10 CFU/ml) attaining maximum log 10 CFU/ml (1.05 x ) on 3 rd day of incubation which is almost equivalent to 1x fold enhancement in cell number. Thereafter, the cell number remained almost static up to 10 th day ( log 10 of CFU/ml) followed by decline in viability. 24

6 Figure 3: Micrograph showing Gram Staining and spore staining of Bacillus subtilis BMT4i grown for 0, 3, 7, 10, 21, and 35 days in BSMG (BSM with 2% glucose) medium. (Magnification 1000 X) log 10C FU /ml of BMT4i in BSMG Incubation Time (in days) Figure 4: Growth (log 10 CFU/ml) of Bacillus subtilis BMT4i (MTCC 9447) in BSMG (BSM with 2% Glucose) for various incubation duration (days). Each point represents the average value obtained with three independent experiments. 25

7 4. Conclusion The present study is the first report demonstrating the effect of diesel oil on the bacterial morphology and growth kinetics (Bacillus subtilis BMT4i). The study evidently revealed diesel induced morphological transformation and early sporulation in BMT4i in comparison to that of glucose. Acknowledgement This work was supported by the Modern Institute of Technology, Rishikesh, Uttarakhand, India that is gratefully acknowledged. 5. References 1. Antai, S.P., and Mgbomo, E., (1993), Pattern of Degradation of Bonny light crude oil by Bacillus Spp. and Pseudomonas Spp isolated from oil spilled site, World African Journal of Biological and Applied Chemistry, 38(1-4), pp Atlas, R.M., (1981), Microbial degradation of petroleum hydrocarbons: an environmental perspective, Microbiology Reviews, 45, pp Errington, J., (2003), Regulation of endospore formation in Bacillus subtilis, Nature Reviews Microbiology, 1, pp Ezeji, E.U., Anyanwu, B.N., Onyeze, G.O.C., Ibekwe, V.I., (2005), Studies on the utilization of petroleum hydrocarbon by microorganism isolated from oil contaminated soil, International Journal of Natural and Applied Sciences, 1(2),pp Ijah, U.J.J., Antai, S.P., (1988), Degradation and mineralization of crude oil by bacteria, Nigerian Journal of Biotechnology, 5, pp Ijah UJJ., Okang CN., (1993), Petroleum degrading capabilities of bacteria isolated from soil, World African Journal of Biological and Applied Chemistry, 38(1-4), pp Johnsen, A.R., Wick, L.Y., Harms, H., (2005), Principles of microbial PAHdegradation in soil. Environmental Pollution, 133,pp Liebeg, E.W., Cutright, T.J., (1999), The investigation of enhanced bioremediation through the addition of macro and micronutrients in a PAH contaminated soil. International Biodeterioration and Biodegradation, 44, pp Lily, M.K., Bahuguna, A., Dangwal, K., Garg, V., (2009), Degradation of benzo[a]pyrene by a novel strain Bacillus subtilis BMT4i (MTCC 9447), Brazilan Journal of Microbiology, 40 (4), pp Lily, M.K., Bahuguna, A., Dangwal, K., Garg, V., (2010), Optimization of an inducible chromosomally encoded benzo[a]pyrene (BaP) degradation pathway in Bacillus subtilis BMT4i (MTCC 9447), Annals of Microbiology, 60(1), pp

8 11. Lily, M.K., Garg, V., Dangwal, K., (2011), Biodegradation of benzo-a-pyrene (BaP) by Bacillus subtilis BMT4i (MTCC 9447): Isolation, identification of BaP degrading bacteria and characterization of BaP degradation activity, LAP LAMBERT Academic Publishing Gmbh and Co. KG. ISBN Surridge, A.K.J., (2007), Denaturing gradient gel electrophoresis characterisation of microbial communities in polycyclic aromatic hydrocarbon and polychlorinated biphenyl contaminated soil, PhD Thesis, University of Pretoria, South Africa, pp Ting, Y.P., HuTan, H.M., (1999), Bioremediation of petroleum hydrocarbons in soil microcosms, Resources Environment and Biotechnology, 2, pp Vasudevan, N., Rajaram, P., (2001), Bioremediation of oil sludge contaminated soil, Environment International, 26, pp Watanabe, ME., (2001), Can bioremediation bounce back? Natural Biotechnology, 19, pp Zhou, E., Crawford, R., (1995), Effects of oxygen, nitrogen, and temperature on gasoline biodegradation in soil, Biodegradation, 6, pp