Bacteria-mediated aerobic degradation of hexacosane in vitro conditions

Bioresource Technology 170 (2014) 62–68

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Bacteria-mediated aerobic degradation of hexacosane in vitro conditions Nitanshi Jauhari, Shweta Mishra, Babita Kumari, S.N. Singh ⇑ Plant Ecology & Environmental Science Division, CSIR-National Botanical Research Institute, Lucknow, Uttar Pradesh, India

h i g h l i g h t s  Hexacosane degradation by two bacteria in isolation and combination.  No report till date is available on bacterial degradation of hexacosane.  Involvement of alkane hydroxylase & alcohol dehydrogenase in hexacosane degradation.  Impact of cell surface hydrophobicity, surface tension and emulsification index.  A drop in medium pH indicates formation of acidic intermediates.

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Article history: Received 16 May 2014 Received in revised form 21 July 2014 Accepted 23 July 2014 Available online 1 August 2014 Keywords: Hexacosane Bacteria Degradative enzymes Surface tension Cell surface hydrophobicity

a b s t r a c t In vitro degradation of hexacosane (C26H54), a HMW n-alkane, was studied in MSM by two bacterial strains i.e., Pseudomonas sp. BP10 and Stenotrophomonas nitritireducens E9, isolated from petroleum sludge, in isolation and combination. The results revealed that both the strains were able to metabolize hexacosane by 82% in isolation and 98% in their consortium after 7 days. An enhancement of 16% in hexacosane degradation by the consortium indicated an additive action of bacterial strains. However, in control, a degradation of 21% was attributed to abiotic factors. During incubation with hexacosane, both the bacteria continued to multiply in isolation and consortium, which reflected that hexacosane was utilized by bacteria as a carbon and energy source. Activities of alkane hydroxylase and alcohol dehydrogenase were differentially expressed in isolation and combination, indicating their involvement in hexacosane degradation. Enhanced cell surface hydrophobicity and emulsification index and reduced surface tension also supported the degradation process. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Alkanes are found highly abundant in the environment due to extensive use of petroleum fuels and their products. These are generally classified into linear (n-alkanes), cyclic (cyclo-alkanes) or branched (iso-alkanes). Low molecular weight alkanes are usually volatile in nature and easily degradable, while high molecular weight alkanes are highly persistent in the environment. As alkanes are aliphatic compounds and chemically very inert (Labinger and Bercaw, 2002), their metabolism by microbes faces challenges of water solubility and accumulation in the cell membranes and the energy to activate the molecule. A number of microorganisms, including bacteria, filamentous fungi and yeasts, have been reported to metabolize alkanes ⇑ Corresponding author. Address: Plant Ecology & Environmental Science Division, CSIR-National Botanical Research Institute, Lucknow 226001, India. Tel.: +91 522 2297823; fax: +91 522 2205836. E-mail address: [email protected] (S.N. Singh). http://dx.doi.org/10.1016/j.biortech.2014.07.091 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

through different degradative pathways (van Beilen and Funhoff, 2005; Wentzel et al., 2007) However, some recently characterized bacterial species, called hydrocarbonoclastic bacteria (HCB), are highly specialized for hydrocarbon degradation and play an important role in the removal of hydrocarbons from polluted and nonpolluted environments (Harayama et al., 2004; Head et al., 2006; Yakimov et al., 2007; Wang et al., 2010). Rojo (2009) observed that direct uptake of alkane molecules from the water phase was only possible for low molecular weight alkanes which are soluble and easily transported into cells. For medium-and long-chain n-alkanes, microbes find access to these compounds as facilitated by the hydrophobic cell surface or biosurfactant produced. Biosurfactants have been reported to increase the uptake and assimilation of hexadecane in the liquid medium (Beal and Betts, 2000; Noordman and Janssen, 2002). A key process for alkane degradation is oxygenation of terminal methyl group (Rehm and Reiff, 1981). Since alkane-degrading bacteria possess multiple genes for alkane hydroxylases, they are highly capable of degrading a wide range of alkanes (van Beilen

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et al., 2002). Alkane degradation is initiated by alkane hydroxylase to transform alkane to alkanols. Usually, there are three types of alkane hydroxylases which degrade short, medium and long chain alkanes (van Beilen and Funhoff, 2007). Methane monooxygenase usually hydroxylates gaseous short-chain alkanes (C1–C4), while medium chain alkanes (C5–C16) are oxidized by membrane-bound non-heme alkane monooxygenase (Alk-B) (van Beilen et al., 1994) and cytochrome P450 monooxygenase. As compared to other bacteria, Stenotrophomonas maltophilia strain M2, S. maltophilia strain Q2 and Tsukamurella tyrosinosolvens strain Q3 could degrade hexadecane two times faster due to high emulsification activity and presence of Alk-B gene (Tebyanian et al., 2013). Besides, other bacteria belonging to the genera Acinetobacter, Rhodococcus and Pseudomonas, have been reported for the direct uptake of aliphatic hydrocarbons, as facilitated by changing the structure of their outer membrane and by enhancing the cell surface hydrophobicity (Van Hamme and Ward, 2001). Hexacosane (C26H54, Mol Wt. 366.71), a long chain n-alkane which is invariably found in the crude oil, is very persistent to microbial degradation. However, a few reports of hexacosane degradation by bacteria are available. Mohanty and Mukherji (2008) found two naturally occurring bacterial cultures, Exiguobacterium aurantiacum and Burkholderia cepacia, highly capable of degrading a wide range of n-alkanes including hexacosane in non-aqueous phase liquid (NAPL). da Cruz et al. (2011) observed degradation of hexacosane by 99%, 64% and 44% by aerobic (Bacillus, Brevibacterium, Mesorhizobium and Achromobacter), anaerobic facultative strains (Bacillus and Acinetobacter) and mixed consortium (Stenotrophomonas, Brevibacterium, Bacillus, Rhizobium, Achromobacter and uncultured bacteria), respectively, in petroleum oil deep sea reservoirs. Very recently, biodegradation of hexacosane was also observed by Elango et al. (2014) in oil in sand aggregates by Mycobacterium sp. in coastal environment. In our study, an in vitro degradation of hexacosane was investigated by two strains of the bacteria separately and in combination over a period of 7 days incubation. Besides, the role of two enzymes involved in degradation process was also ascertained. The bacterial degradation of hexacosane in MSM was directly linked to cell surface hydrophobicity and emulsification index. 2. Methods 2.1. Materials

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for7 days in an orbital shaker set at 37 °C and 180 rpm. After 7 days of incubation, residual amount of hexacosane was extracted with hexane (20 ml) thrice and concentrated to 2 ml on rota evaporator. Samples were analyzed using a Gas chromatograph (Agilent 7890A) with FID detector and a capillary BP5 column (5% phenyl methyl polysiloxane column, 30 m  0.32 mm  0.25 lm). Both injection and detector temperatures were maintained at 280 °C. Initial oven temperature was maintained at 80 °C for 2 min and then elevated to 300 °C gradually with 10 °C increase per minute. 2.3. Experimental set up 2.3.1. Preparation of inoculum Two selected bacterial strains and their consortium were first grown in nutrient broth (composition: 5 g of peptic digest of animal tissue, 5 g of sodium chloride, 1.5 g of beef extract and 1.5 g of yeast extract in 1 L of media) separately, for 48 h in an orbital shaker set at 37 °C and 150 rpm. Bacterial cells were harvested by centrifugation at 5000 rpm at 4 °C for 10 min. Pellet of bacterial cells was washed with MSM twice and again re-suspended in 20 ml MSM. The absorbance (OD600) of bacterial cells was measured with an UV–Vis spectrophotometer and adjusted to OD 1. 2.3.2. Bacterial degradation of hexacosane For hexacosane degradation study, 20 ml of MSM with 50 ppm hexacosane as a substrate were prepared in 100 ml Erlenmeyer flasks and then sterilized in an autoclave set at 121 °C and15 lb/ square inch. Then after, bacterial strains (BP10 and E9) and their consortium (by mixing half of each bacterial inoculum) were inoculated separately in flasks with three replicates. The control flasks were also prepared in the same manner, but without bacterial culture. All flasks were incubated in an orbital shaker set at 37 °C and 150 rpm for 7 days. After every 24 h intervals, hexacosane depletion in MSM was measured and specific degradation rate was calculated by the formula as below:

dx=x0  dt where, dx = change in concentration of substrate; x0 = substrate concentration; dt = time interval. 2.3.3. Degradation kinetics As degradation of hexacosane fits to first order reaction kinetics, it can be expressed by a formula as below:

InCt ¼ InC0  Kt Minimal salt medium (MSM), nutrient agar (NA) and nutrient broth (NB) were procured from Hi-media, while hexacosane, folin–phenol reagent, NADH, NAD+, bovine serum albumin (BSA) were purchased from Sigma–Aldrich. However, hexane (HPLC grade) was obtained from Qualigen.

where, Ct is the residual hydrocarbon concentration at any time, C0 is the initial hydrocarbon concentration; K is the speed constant which reflects the degradation rate and t is time (day). The half-life period of hexacosane was also calculated by the formula as below:

t1=2 ¼ ln2=k 2.2. Isolation and screening of different bacterial strains

where, k is the biodegradation rate constant (day1).

Ten bacterial strains were isolated from petroleum hydrocarbon-contaminated soil through enrichment method and grown on MSM agar plate (composition: 7 g dipotassium phosphate, 2 g monopotassium phosphate, 0.5 g sodium citrate, 1 g ammonium sulfate, 0.1 g magnesium sulfate in 1 L medium (pH 7.0 ± 0.2) supplemented with 25 and 50 ppm hexacosane. Petri plates were incubated at 37 °C for 5 days in an incubator. Among these strains, six bacterial strains, designated as BP8, E9, PSM10, BP10, E3 and PSA5, were selected based on their better growth performance. These strains were further screened based on their degradation ability of hexacosane in MSM broth. For this purpose, these strains were incubated in 20 ml of MSM broth contained in 100 ml Erlenmeyer flask and supplemented with 50 ppm of hexacosane

2.3.4. Extraction of hexacosane and analysis Residual amount of hexacosane after degradation by isolated bacteria and their consortium, was extracted from MSM by liquid–liquid extraction (1:1 v/v MSM:Hexane) method. Samples were extracted three times with hexane and pooled together. The extracts were concentrated by evaporation and remaining hexacosane was measured with a Gas chromatograph (Agilent 7890A). 2.4. Growth of bacterial isolates in MSM with hexacosane For determination of bacterial growth, enzyme assay, protein and other properties of bacteria, 100 ml Erlenmeyer flasks were prepared in a similar way as for the hexacosane degradation study

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(containing MSM supplemented with 50 ppm hexacosane and bacterial inoculums). However, control flasks contained MSM and bacterial inoculums, but without any carbon source i.e., hexacosane. The flasks were sterilized, inoculated with bacteria and incubated in an orbital shaker set at 37 °C and 150 rpm for 7 days. At every 24 h intervals, flasks were taken out and growth of each strain (BP10 and E9) and their consortium was measured at OD600 with an UV–Vis spectrophotometer (Perkin Elmer lambda 35). Specific growth rate of bacteria and yield coefficient were calculated by the formulae as below:

Specific growth rateðlÞ ¼ dx=x0  dt where, dx = mass of cells produced; x0 = original mass of the cell; dt = time interval.

2.9. Surface tension of the medium Two bacterial strains (BP10 and E9) and their consortium were tested for biosurfactant production by measuring a reduction in surface tension of the medium during degradation of hexacosane (50 ppm). The surface tension was measured by stalagmometer using the following formula:

c1 =c2 ¼ d1 n2 =d2 n1  n2 =n1 where, c1 = surface tension of culture supernatant, c2 = surface tension of water, n1 = no. of drops formed from same amount of culture supernatant, n2 = no. of drops formed from same amount of water, d1 = density of culture supernatant, d2 = density of water.

Yield coefficientðYÞ ¼ dx=ds

2.10. Cell surface hydrophobicity (CSH)

where, dx = mass of new cells; ds = mass of substrate consumed.

The BATH assay was used to measure the cell surface hydrophobicity of selected strains to n-hexadecane as a substrate. It represents the percent adherence of bacterial cell to hydrophobic substrates. At mid-growth phase, cells were grown in nutrient broth, harvested, and washed with PUM buffer (7.26 g KH2PO4, 22.1 g K2HPO4, 1.8 g urea and 0.2 g MgSO4 in 1 L DDW, pH 7.1) and again suspended in the same buffer. Then after, absorbance of bacterial cultures was taken at 600 nm with an UV–Vis spectrophotometer, when initial absorbance was adjusted between 1.0 and 1.2. In a test tube, 1.2 ml of culture solution was taken separately and then added 0.2 ml sterile n-hexadecane. It was then incubated in an orbital shaker set at 30 °C and 150 rpm for 10 min. The test tubes were vortexed for 2 min and kept for 30 min for the separation of aqueous and organic phases. The lower aqueous phase was removed carefully and its turbidity was measured at 600 nm. The percentage of cells adhering to hydrocarbons was calculated by the following equation:

2.5. Protein estimation For determination of bacterial cell protein, selected bacterial strains, grown in MSM with 50 ppm hexacosane, were harvested by centrifugation at 8000 rpm at 4 °C for 10 min and then washed in potassium phosphate buffer (pH = 7) twice. The cells pellet was again suspended in the same buffer, sonicated for 5 min at 0 °C and then centrifuged at 20,000 rpm for 25 min at 4 °C. The supernatant was preserved at 2 °C and used for both protein determination and enzyme assay. The protein content in bacterial strains was measured with UV–Vis spectrophotometer at 660 nm, following the method of Lowry et al. (1951), using BSA (bovine serum albumin) as a standard. 2.6. Alkane hydroxylase activity The harvested bacterial cells were washed twice and then re-suspended in 2 ml of 20 mM Tris–HCl buffer (pH = 7.4). Cells were disrupted by probe sonicator (Fisher model 300) and centrifuged at 8000 rpm at 4 °C for 10 min. The cell free supernatant was used for determination of alkane hydroxylase activity by a decrease in absorbance at 340 nm as measured with an UV–Vis spectrophotometer. 1 ml of reaction mixture contained 20 mM Tris–HCl and 0.15% CHAPS buffer (pH 7.4), 0.1 mM NADH, 10 ll of hexadecane solution (1% hexadecane in 80% DMSO) and 50 ll crude extract in 1 ml volume. The reaction was initiated by addition of 10 ll of hexadecane solution. Specific activity of alkane hydroxylase was expressed as 1 mmol of NADH oxidized per minute.

%Adherence ¼ 100  ½1  ðOD600 of the aqueous phase= OD600 of the cell suspensionÞ

2.11. Emulsification index and stability The emulsification index of the biosurfactant produced by cells of two selected bacteria was determined by following the method of Hassanshahian et al. (2012). For this purpose, the cell suspension and hexadecane were taken in a tube (1:1 v/v), vortexed for 2 min and left for 24 h. After 24 h, the emulsified layer was measured and emulsification index was calculated by the formula as below:

Emulsification index ð%Þ ¼ Height of emulsified layer=Total height 2.7. Alcohol dehydrogenase activity The harvested bacterial cells were washed twice with 10 mM potassium phosphate buffer (pH = 7.0) and re-suspended in the same buffer. After this, cells were sonicated for 5 min and then centrifuged at 4 °C and 8000 rpm for 10 min. Cell free supernatant was used for assay of alcohol dehydrogenase activity and measured with an UV–Vis spectrophotometer at 340 nm. 1 ml of reaction mixture contained 1 M Tris–HCl buffer (pH 8.8), 4 mM NAD+, 100 ll ethanol (99% pure) and 50 ll of crude extract. Specific activity of alcohol dehydrogenase was expressed as 1 mM NADH formed per minute. 2.8. pH of the medium A reduction in pH of the medium containing bacterial cells and their consortium during degradation (50 ppm) of hexacosane was measured with a pH meter (Orion EA940).

 100: Emulsification stability was measured for 7 days of incubation during which the degradation of hexacosane was studied in vitro conditions. 3. Results and discussion 3.1. Screening of bacterial strains for hexacosane degradation Out of six bacterial strains (BP8, E9, PSM10, BP10, E3 and PSA5), two bacterial strains i.e., E9 and BP10 were found to be highest degraders of hexacosane (50 ppm) after 7 days of incubation in MSM broth supplemented with 50 ppm hexacosane. Hence, these two strains were selected for the further study of in vitro degradation of hexacosane (50 ppm) in MSM, separately and in combination.

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3.2. In vitro degradation of hexacosane

found between E9 and BP10 during degradation of hexacosane after 7 days of incubation. Thus, it was observed that half-life time was lower with higher degradation constant in the consortium than in either of bacterial strain. Degradation of n- and branched alkanes by bacterial strains was also observed by Katsivela et al. (2003). Liu et al. (2011) reported a bacterial strain Alcanivorax dieselolei which could utilize a broad spectrum of alkanes i.e., C5–C36. Tao et al. (2012) also observed 96% and 78% degradation of hexadecane (5000 mg/ml) by Pseudomonas aeruginosa B1 and Acinetobacter RAG-I B2, respectively. In the degradation process of nC12 and nC16 alkanes through terminal and sub-terminal oxidation processes, Rhodococcus strain Q15 produced intermediates which were identified as 1-dodecanol and 2-dodecanone and 1-hexadecanol and 2-hexadecanol, respectively (Whyte et al., 1998). Matsui et al. (2014) isolated three bacteria i.e., Acinetobacter sp., Pseudomonas sp., and Gordonia sp. which showed a stable growth on n-tetracosane and assimilated a wide range of aliphatic hydrocarbons from C12 to C30, but not on alkanes shorter than C8. Of the bacterial isolates, Gordonia sp. could degrade hydrocarbons of the oil tank sludge more efficiently by dissolving the sludge in a hydrophobic solvent, while Acinetobacter sp. carried out little degradation, possibly due to the difference in the incorporation mechanism of hydrophobic substrate between proteobacteria and actinobacteria.

Bacterial degradation of hexacosane by the individual strains (BP10 or E9) continued to increase with the incubation period up to 5 days and then tended to get stabilized. However, its degradation continued to augment throughout the incubation period when a consortium of the bacteria was used. This study also revealed that both the bacterial strains i.e., BP10 and E9 were capable to degrade 82% of 50 ppm hexacosane separately after 7 days of incubation. However, 21% reduction of hexacosane was also observed in the control (Fig. 1a). If the contribution of abiotic factor is deducted, 71% of hexacosane degradation might be attributed to each bacterial strain BP10 and E9, separately. When the both bacterial strains were added in combination (with the same inoculum strength), hexacosane degradation was enhanced to 98%, showing an additive effect in the degradation process. If contribution of abiotic factors is deducted, 77% degradation of hexacosane was caused by a combination of both bacterial strains during the same period of incubation. As far as specific degradation rate of different strains and their combination was concerned, it was found to be 0.11 d1 for each bacterial strain (E9 or BP10) separately and 0.14 d1 for their consortium. However, in control without any bacteria, it was recorded to be 0.03 d1. When degradation rate constant and half- life period of bacterial strains (E9 or BP10) and its consortium were determined following the first order kinetics model equation, it was observed that rate constant was higher in consortium (k = 0.729 day1, t1/ 1 , t1/2 = 2.8 days) and 2 = 0.8 day), followed by E9 (k = 0.314 day BP10 (k = 0.309 day1, t1/2 = 2.8 days) in the decreasing order. No significant difference in rate constant and half life period was

Hexacosane degradaon (%)

a

120

Control

3.3. Growth of bacteria with hexacosane in MSM Both the bacterial strains continued to multiply during 7 days of incubation period. However, bacterial strain E9 multiplied faster than BP10 during the incubation. In the case of consortium, the

BP10

E9

Consorum

100 80 60 40 20 0 1

2

3

4

5

6

7

Incubation period (days)

Growth (600 nm)

b

E9

0.7

BP10

Consorum

0.6 0.5 0.4 0.3 0.2 0.1 0 0

1

2

3

4

5

6

7

Incubation period (days) Fig. 1. (a) Degradation of hexacosane (50 ppm) by two bacterial strains (BP10 and E9) in isolation and combination. (b) Growth pattern of two bacterial strains (BP10 and E9) in isolation and combination.

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bacterial growth was found still better than the individual strains as shown in (Fig. 1b). Bacterial growth in MSM supplemented with hexacosane (50 ppm) clearly indicates that they have utilized hexacosane as an organic carbon source and energy for cell multiplication and growth. The specific growth rate of the bacterial strains was found to be 0.56 d1 for E9, 0.48 d1 for BP10, and 0.59 d1 for consortium, while the yield coefficient was calculated to be 33.97, 44.30, 39.87, for E9, BP10 and consortium, respectively.

BP10

Alkane hydroxylase (ηmol mg- 1 protein)

66

E9

Consort

700 600 500 400 300 200 100 0

3.4. Bacterial cell protein

0

1

2

3

4

5

6

7

Incubation period (days) The amount of the bacterial protein continued to increase with the multiplication of cells in both the bacterial strains in isolation and consortium during incubation period of 7 days. The initial concentration of cell protein varied between 0.05 and 0.1 mg ml1 and then augmented to a maximum of 0.42 mg ml1 in the consortium; followed by individual strains (BP10 or E9) which did not show significant difference in the cell protein content after 7 days of incubations (Fig. 2). Thus, cell protein was enhanced by 4 folds during the entire period of bacterial growth. 3.5. Degradative enzymes In the degradation of alkanes, two degradative enzymes are generally involved, such as alkane hydroxylase and alcohol dehydrogenase. Therefore, the induction of these two enzymes during hexacosane degradation was studied to evaluate their relative role in the degradation process.

BP10

0.5

E9

Consorum

0.4 Cell Protein (mg ml-1)

AlkB1, AlkB2 and AlkB3 were reported to be responsible for the degradation of C18–C36 alkanes (Whyte et al., 2002). This enzyme was also isolated from thermophilic bacteria i.e. Bacillus sp., Geobacillus thermodenitrificans NG80-2 which utilized a terminal oxidation pathway for the conversion of long-chain n-alkanes (C15–C36) to corresponding primary alcohols (Li et al., 2008). 3.5.2. Alcohol dehydrogenase Like alkane hydroxylase, the activity of alcohol dehydrogenase was also induced in individual strains and their consortium. In BP10, its activity continued to increase gradually till 4 days of incubation and then declined. However, in E9 and consortium, alcohol dehydrogenase continuously increased with the incubation periods. The peak activity of this enzyme was recorded as high as 2311 gmol mg1 protein in consortium, followed by E9 (1494 gmol mg1 protein) and the least (802 gmol mg1 protein) was recorded in BP10 (Fig. 4). Pirog et al. (2009) reported higher alkane hydroxylase activity than alcohol dehydrogenase in Rhodococcus erythropolis EK-1 during hexadecane degradation, but in our case, activity of alcohol dehydrogenase was found higher than alkane hydroxylase. This enzyme had been even isolated from cell-free extracts of Pseudomonas sp. strain 196Aa grown anaerobically on n-alkane (Parekh et al., 1977). Induction of this enzyme has been also reported in mesophilic, thermophilic and extreme thermophilic microorganisms by Alvarez et al. (2011). Abdel-Megeed and Muller (2009) found that alcohol dehydrogenase isolated from Pseudomonas frederiksbergensis was able to assimilate and mineralize C10–C22 n alkane as a source of carbon and energy in the temperate environment at pH 7.

0.3 0.2 0.1 0

BP10

3000

Alcohol dehydrogenase (η mol mg-1 protein)

3.5.1. Alkane hydroxylase The alkane hydroxylase enzyme was induced in both selected bacterial strains during hexacosane degradation (Fig. 3). Alkane hydroxylase activity continued to increase with incubation, attaining the peak activities in both bacterial strains after 4 days of incubation and then declined. The peak activities of this enzyme were recorded as high as 527 gmol mg1 protein in BP10 and 563 gmol mg1 protein in E9. However, in the consortium, the peak activity of alkane hydroxylase (607 gmol mg1 protein) was invariable higher than that of individual strains. After 4 days of incubation, the enzyme activity gradually decreased with the incubation period. Alkane hydroxylase initiates the aerobic degradation of alkanes by inserting oxygen atoms at the different sites of alkane terminus (Ji et al., 2013). Mishra and Singh (2012) have also observed induction of alkane hydroxylase enzyme in P. aeruginosa sp. PSA5 and Rhodococcus sp. NJ2 during n-hexadecane degradation. Enzymatic complex was genetically characterized in Pseudomonas sp., Acinetobacter sp. and Rhodococcus sp. for alkane hydroxylase systems (Wentzel et al., 2007). Rhodococcus strains (Q15 and NrrlB-16531) possessed genes encoding the synthesis of four alkane hydroxylases (AlkB1, AlkB2, AlkB3, AlkB4). Out of which,

Fig. 3. Expression of alkane hydroxylase activity in the bacterial strains E9 and BP10 in isolation and consortium during degradation of hexacosane.

E9

Consort

2500 2000 1500 1000 500 0

0

1

2

3

4

5

6

7

Incubation period (days) Fig. 2. Bacterial cell protein during the growth of both bacterial strains in isolation and combination.

0

1

2

3

4

5

6

7

Incubation periods (days) Fig. 4. Expression of alcohol dehydrogenase activity in the bacterial strains E9 and BP10 in isolation and consortium during degradation of hexacosane.

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pH

BP10 8 7 6 5 4 3 2 1 0

0

1

E9

2

Consort

3

4

Control

5

6

7

Incubation period (days)

Surface tension (mN/m)

Fig. 5. Change in surface tension of MSM with hexacosane incubated with bacterial strains in isolation and combination.

BP10

E9

1

2

Consort

Control

80 70 60 50 40 30 20 10 0 0

3

4

5

6

7

Incubation period (days) Fig. 6. Change in medium pH (MSM with hexacosane) during 7 days of incubation of bacterial strains in isolation and combination.

3.6. pH of incubation medium At the time of inoculation, the pH of the incubation medium was found to be 7.3. However, after 7 days of incubation period, the pH of the medium decreased to 6.5, 5.3 and 5.0 in BP10, E9 and consortium, respectively. However, no significant drop in pH was observed in the case of control (Fig. 5). This suggests the formation of acidic intermediates during the degradation of hexacosane by bacteria. 3.7. Surface tension (ST) The surface tension of the medium was reduced by both bacterial strains (BP10 or E9) and their consortium. Initially, the surface tension of the medium was 71 mN/m at 0 day, which was reduced to 28, 32 and 23 mN/m in BP10, E9 and consortium, respectively after 7 days of incubation (Fig. 6). However, no change in surface tension was found in the control. This indicates that both bacteria produced biosurfactants during the degradation of hexacosane. de Carvalho et al. (2009) also observed that R. erythropolis produced surface active agents which decreased the surface tension of the medium with increasing number of carbons in the carbon source from 69 mN/m (C5-grown cells) to about 23 mN/m (C14–C16). 3.8. Cell surface hydrophobicity Cell surface hydrophobicity (CSH) is major factor to determine the adhesion of hydrocarbon to cell surface. In our study, it was observed that both bacterial strains showed very high level of CSH. Cell surface hydrophobicity of the bacterial strains was recorded to be 93% in E9 and 91% in BP10 with hexadecane as a substrate. This indicated that both bacterial strains had very high

surface hydrophobicity towards alkanes. Mishra and Singh (2012) have also reported 99.86% CSH in P. aeruginosa sp. PSA5, while in Rhodococcus sp. NJ2 and Ochrobactrum sp. P2, it was found to be 96.4% in MSM enriched with hexadecane. Likewise, Tebyanian et al. (2013) reported cell surface hydrophobicity of 6%, 24% and 29% in S. maltophilia strain M2, S. maltophilia strain Q1 and T. tyrosinosolvens strain Q3, respectively with hexadecane in the medium. 3.9. Emulsification index and stability Emulsification index of cell free media was measured after incubation of E9 and BP10 in MSM with hexadecane for 7 days and found to be 42% in BP10 and 50% in E9. Tebyanian et al. (2013) reported 29%, 6.12% and 7.14% emulsification activity with hexadecane in cell free media when grown with S. maltophilia strain M2, S. maltophilia strain Q1 and T. tyrosinosolvens strain Q3, respectively. Besides, emulsification stability was recorded as 50% for E9 and 40% for BP10 throughout the degradation process of hexacosane during 7 days of incubation. 3.10. Identification of bacterial strains Both the bacterial strains BP10 and E9 were identified as Pseudomonas sp. and Stenotrophomonas nitritireducens on the basis of their homology (>99%) of DNA sequence with NCBI databases of bacteria by Chromous Biotech, Bangalore based on 16S rDNA technology. 4. Conclusion This investigation clearly indicates that both the bacterial strains BP10 and E9 are the potential degraders of a HMW alkane

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