Eciency and Kinetics of Assam Crude Oil Degradation by Pseudomonas Aeruginosa AKS1 and Bacillus Sp. AKS2

We report kinetics of Assam crude oil degradation by Pseudomonas aeruginosa AKS1 and Bacillus sp. AKS2, both isolated from Assam renery sediments. The isolates exhibited appreciable degrees of hydrophobicity, emulsication index and biosurfactant production. Crude oil degradation eciency of isolates was assessed in (1) liquid medium amended with 1% v/v crude oil and (2) microcosm sediments (125 mg crude oil/ 10 g sand). In liquid culture, the biodegradation rate (k) and half-life (t 1/2 ) values were found to be 0.0383 day -1 and 18.09 days for P. aeruginosa AKS1, and 0.0204 day -1 and 33.97 days in case of Bacillus sp. AKS2. In microcosm sand sediments, the estimated biodegradation rate (k) and half-life (t 1/2 ) values were 0.0138 day -1 and 50 days for P. aeruginosa AKS1, and 0.0113 day -1 and 61.34 days in case of Bacillus sp. AKS2. The level of nutrient treatment in microcosm sand sediment was 125 µg N & 62.5 µg P/g sediment in case of P. aeruginosa AKS1 and 375 µg N & 37.5 µg P/g sediment in case of Bacillus sp. AKS2. In microcosms without inorganic nutrients, biodegradation rate (k) and half-life (t 1/2 ) values were found to be 0.0069 day -1 and 100 days for P. aeruginosa AKS1 and for Bacillus sp. AKS2, the respective values were found to be 0.0046 day -1 and 150.68 days. Our data provides important information for predictive hydrocarbon degradation in liquid medium and contaminated sediments.


Introduction
Hydrocarbon pollution due to natural seeps and anthropogenic activities is a major problem worldwide posing serious threat to local ora and fauna. Developing economy like India generate a large amount of petroleum sludge from crude oil re ning activities carried out to meet the mass production of hydrocarbons for use in pharmaceuticals, solvents, fertilizers, pesticides and plastics, besides fuel oil and gasoline. A part of the low molecular weight hydrocarbon fractions from contaminated sediments is removed by evaporation leaving behind relatively recalcitrant high molecular weight fractions.
Reclamation of such contaminated sediments is very di cult task and requires intervention of physical, chemical and biological methods. The hydrocarbons are ultimately biodegraded by microbial populations. Bioremediation of hydrocarbon polluted sites using native bacterial population is considered as the most e cient, sustainable, environmentally friendly, and cost-effective approach. Therefore there is a need to identify and test the hydrocarbon degradation potential of native bacteria for bioremediation.
The success of bioremediation is largely governed by the survival, adaptability and catabolic activities of the introduced microorganisms. Some of the bacterial strains successfully used for this purpose include Pseudomonas aeruginosa (san ai) ( (Belhaj et al. 2002) including recalcitrant and toxic polycyclic aromatic hydrocarbons (PAHs) (Bugg et al. 2000;Tian et al. 2002). Several Pseudomonads are also known to produce glycolipid type biosurfactants (Rahman et al. 2003 In a previous study we have characterized the kinetic parameters of hydrocarbon degradation of Pseudomonas aeruginosa AKS1 and Bacillus sp. AKS2 isolated from hydrocarbon contaminated sediments collected from Guwahati re nery, India (Chettri et al. 2016). In present study, both isolates have been tested for their ability to produce biosurfactant and characterized for crude oil degradation activity using rst-order kinetics.

Bacterial growth
Growth of both isolates was assessed in liquid Bushnell-Haas (BH) medium supplemented with 1% (v/v) n-hexadecane. The experiments were carried out in 100 ml Erlenmeyer asks containing 50 ml BH medium inoculated with exponentially growing cells at a nal concentration of OD 600 equal to 0.035. The culture asks were then incubated at 25 ± 0.5°C in a rotary shaker at 100 rpm under darkness. Growth was assessed as increase in cell density and total protein content (Lowry et al. 1951). Growth in BH medium without n-hexadecane served as control.

Detection of catabolic genes by PCR
Genomic DNA was isolated from BH-crude oil grown cultures using Genomic DNA extraction Kit (Bioline).
The conserved fragments of catabolic alkane hydroxylase gene was ampli ed using the primer pairs and at annealing temperature listed in Supplementary Table 1. A 25 µl PCR reaction mixture contained 50 ng template DNA, 2.5 µl 10X reaction buffer, 1.25 µl of 25 mmol/L MgCl 2 , 0.5 µl of 10 mmol/L dNTPs, 0.5 µl of 10 pmol/µl each primer and 1 U Taq DNA polymerase. Final volume was adjusted by adding sterile double distilled water. PCR conditions were as follows: initial denaturation at 94°C for 4 min, followed by 30 cycles of denaturation at 94°C for 30 s, primer annealing for 30 s, elongation at 72°C for 30 s, and a nal extension step at 72°C for 10 min using an ABI 2720 thermal cycler. The PCR amplicons were then separated on 1.2% agarose gel in 1x TAE buffer and visualized after staining with ethidium bromide (5 µg/ml) and photographed using a Bio-Rad Gel Doc™ XR + imaging system.

Cell surface hydrophobicity test
Cell hydrophobicity was measured according to Rosenberg (Rosenberg et al. 1980

Emulsi cation index
The emulsi cation activity was determined as described (Berg et al. 1990). To culture supernatant (0.5 ml) same volume of TM buffer (20 mM Tris[tris(hydroxymethyl)-aminomethane]/HCl buffer, pH7.0 and 10 mM MgSO 4 ) and 0.1 ml of kerosene was added. The tube was vortexed for 1 min, held stationery for 3 min, and then visually examined for turbidity of a stable emulsion. Emulsi cation index was calculated using the formula E 24 = (Height of emulsion formed/ Total height of solution)*100

Isolation of biosurfactant
Supernatant of bacterial culture grown for 24 and 48 hours in 0.5% (v/v) n-hexadecane amended nutrient broth were collected by centrifugation at 10,000 rpm for 15 min at 4°C. The pH of cell free supernatant was then adjusted to 2.0 using concentrated HCl and kept at 4°C overnight to allow formation of precipitate. The precipitate was collected by centrifugation at 17,000 rpm for 15 min at 4°C, dissolved in water and lyophilized (Heto LyoLab 3000 Lyophilizer, Germany). The recovery of lyophilized biosurfactant sample was expressed as g biosurfactant.g − 1 dry cell wt.

Characterization of biosurfactant
The partially puri ed biosurfactant was characterized based on simple colorimetric assays.
2.6.1. Carbohydrate: The carbohydrate content was measured using Phenol-sulphuric acid method with glucose as standard. In brief, 5 mg of the biosurfactant sample was dissolved in 1 ml of water. One hundred µl of this suspension was mixed with 200 µl of phenol reagent (5% v/v water) followed by addition of 1 ml concentrated H 2 SO 4 and incubation at room temperature for 10 min. The mixture was vortexed vigorously and allowed to stand for 30 min. Finally absorbance was read at 490 nm against a reagent blank.
2.6.2. Protein: The extracellular protein in partially puri ed biosurfactant was measured at 750 nm using bovine serum albumin as standard according to Lowry (Lowry et al. 1951).

Crude oil degradation in liquid BH medium
Degradation of crude oil by individual bacterial isolate was carried out in biometric asks containing 100 ml BH-medium (Himedia, India) supplemented with 1% Assam crude oil (v/v) as the sole source of carbon and energy. Bacterial cells grown in BH-medium supplemented with glucose (200 mg L − 1 ) were harvested, washed at 10000 rpm for 10 min in BH-medium and then re-suspended in the same. The cell suspension was inoculated at a nal OD 600 equal to 0.035. Non-inoculated asks were used as abiotic controls. The culture asks were incubated as described in Sect. 2.1. Crude oil degradation activity of bacteria was assessed in terms of CO 2 evolution (Zibilski 1994). For estimation of total/residual petroleum hydrocarbons (TPH), each sample was extracted in an equal volume of dichloromethane (DCM) at regular interval and separated from aqueous phase. The DCM phase was then dehydrated with anhydrous sodium sulphate and remaining TPH was extracted using a Soxhlet apparatus. Weight of the residual TPH after rota-evaporation was determined gravimetrically.

Microcosm design and set up
Laboratory microcosm comprising 10 g sterilized sand sediment amended with 125 mg Assam crude oil and supplemented with 125 µg N.g − 1 sediment & 62.5 µg P.g − 1 sediment for P. aeruginosa AKS1, and 375 µg N.g − 1 sediment & 37.5 µg P.g − 1 sediment for Bacillus sp. AKS2 were set up. Sodium nitrate and potassium dihydrogen phosphate were used as sources of inorganic N and P respectively. Microcosms were inoculated with exponentially growing bacterial cells and incubated at a temperature of 25 ± 0.5°C under darkness. Microbial TPH degradation activity in the sediment was assessed as a function of CO 2 evolution as described in Sect. 2.7 (Zibilski 1994), and in terms of residual TPH as described by Márquez-Rocha (Márquez-Rocha et al. 2001). In short, 10 g of microcosm sand sediment was mixed with equal amount (w/w) of anhydrous sodium sulphate and TPH was extracted in DCM using a Soxhlet apparatus. Non-inoculated or microcosms without crude oil were also incubated to account for any abiotic CO 2 production.
Biodegradability of crude oil in both liquid culture and sand microcosms were tted into rst order kinetics as described in Eq. (1) below.
C t = C 0 e − kt (1) Where, C o is the initial TPH content C t is the residual TPH content at time t k is the biodegradation rate constant (day − 1 ) Biodegradation half-life times (t 1/2 ) was calculated by Eq. (2) t 1/2 = ln2/k (2) 3. Results

General characteristics of bacterial isolates:
Growth of bacterial isolates determined in n-hexadecane supplemented BH-medium showed increase in cell density and protein content which was 20-25 folds more than zero day (Fig. 1). Detection for presence of alkane hydroxylase gene in both strains was done using newly designed primer pairs AlkB-PA-2F/AlkB-PA-2R and AlkB gene-F/AlkB gene-R. While AlkB-PA-2F & 2R produced 200 bp PCR amplicons of P. aeruginosa AKS1 only, AlkB gene-F&R generated 190 bp long amplicons of both P. aeruginosa AKS1 and Bacillus sp. AKS2 (Fig. S1). Application of primer pair as described by (Kloos et al. 2006) did not produce PCR amplicons. These results highlight the diversity of alkane hydroxylase gene in bacterial community. Since both strains showed satisfactory growth in n-hexadecane supplemented BH-medium, we determined their hydrophobicity, emulsi cation index and biosurfactant production activity. The hydrophobicity (% adherence to heptane) was 77.74% and 81.19% and the E 24 values were 39.58% and 34.06% for P. aeruginosa AKS1 and Bacillus sp. AKS2 respectively ( Fig. 2A, B). The capacity to produce biosurfactant was analysed in the supernatant of bacterial culture grown in n-hexadecane (0.5% v/v) amended nutrient broth. The partially puri ed biosurfactant was lyophilized and measured gravimetrically. Pseudomonas aeruginosa AKS1 produced 0.138 ± 0.063 g biosurfactant.g − 1 cell dry wt.  (Table 1).

Crude oil degradation kinetics
Degradation of crude oil was monitored by measuring the cumulative CO 2 production and changes in TPH concentration with time in liquid BH-medium (Fig. 3), and microcosm sand sediment (Figs. 4 and 5).
In liquid microcosm the cumulative CO 2 production was observed to be 455 ± 17.12 µmoles and 200 ± 1.11 µmoles on day 15 after inoculation with P. aeruginosa AKS1 and Bacillus sp. AKS2 respectively ( Fig. 3A, C). Their respective crude oil biodegradation rate constant (k) was observed to be 0.0383 day − 1 and 0.0204 day − 1 . The half-life (t 1/2 ) for crude oil degradation was found to be 18.09 days for P.
aeruginosa AKS1 and 33.97 days for Bacillus sp. AKS2. The resulting data tted to First-order kinetics model with R 2 value more than 0.9 (Fig. 3B, D). Our data suggest high crude oil degradation e ciency for P. aeruginosa AKS1 in liquid culture. incubation which was statistically different from N only and B only controls (one-way ANOVA; P = 9.2E-09). Thereafter no signi cant increase in CO 2 evolution was observed for the total incubation period of 28 day (one-way ANOVA; P = 0.604) (Fig. 4A). Decrease in TPH with time is shown in Fig. 4B. The initial weight of TPH added was determined by taking average of residual hydrocarbons extracted from all three categories of replicate microcosms (100.6 ± 1.20 mg /10 g sand). In N + B microcosm, the residual TPH was found to reduce by 28%, 30%, 34% and 36% after 7, 14, 21 and 28 days respectively. In B only treated microcosms, the respective residual TPH values were found to reduce by 14%, 16%, 16% and 20% of the initial average value. However, in N only control microcosm the degradation of crude oil was found to be 3% on day 7 and 9% on day 28 after incubation. First order kinetic model was used to determine the rate of crude oil degradation in sand sediment microcosms. The biodegradation rate constant (k) and half-life (t 1/2 ) of degradation for N + B microcosms was 0.0138 day − 1 and 50 days respectively. The corresponding values for B only control microcosms were 0.0069 day − 1 and 100 days respectively, and for N only control microcosm were 0.003 day − 1 and 231 days. Degradation of crude oil in N + B microcosm was signi cantly higher than in B only microcosm (one way ANOVA; P = 0.014).
The CO 2 evolution and TPH degradation activities analysed in sand sediment microcosm inoculated with Bacillus sp. AKS2 is shown in Fig. 5. In this case, total CO 2 evolved reached to 245 ± 11.5 µmoles CO 2 on 7 days and remained nearly same (267.5 ± 31 µmoles CO 2 ) on day 28 (one way ANOVA; P = 0.316) in N + B microcosm. The CO 2 evolution activities in B only microcosms was 55 ± 7.1 µmoles CO 2 and 118.8 ± 12.4 µmoles CO 2 on days 14 and 28 respectively (Fig. 5A). The degradation of crude oil by Bacillus sp.
AKS2 with time is shown in Fig. 5B. In N + B microcosm, TPH content was reduced by 15%, 21%, 24% and 29% on 7, 14, 21 and 28 days after incubation respectively. In B only microcosms, reduction in TPH content ranged from 9-14% between day 7 and day 28 while in N only microcosm the reduction was found to be 3% and 11% between day 7 and day 28. The crude oil degradation data thus obtained were tted into rst-order kinetic model to obtain the biodegradation rate constant (k) and half-life (t

Discussion
The bacterial isolates namely Pseudomonas aeruginosa AKS1 and Bacillus sp. AKS2 have been reported previously for hydrocarbon degrading potentials (Chettri et al. 2016). Both isolates showed good growth in liquid BH-media when amended with n-hexadecane as sole carbon source (Fig. 1A, B). They also exhibited high percentages of hydrophobicity which determines the initial adhesion of microorganisms to the interface between the NAPL (non-aqueous-phase liquid) and the aqueous phase. The direct contact between a bacterial cell and a target hydrocarbon signi cantly increase the rate of entry of hydrocarbon into the cell. The cellular metabolism of hydrocarbons subsequently promotes the bacterial growth. Both isolates were found to degrade and utilize crude oil as sole carbon source as evidenced from their CO 2 evolution activity and decrease in quantity of residual TPH. Their ability to metabolize crude oil was clearly re ected by the presence of genes for hydrocarbon degradation (Fig. S1). It is a well-established fact that biosurfactant produced by bacterial strains signi cantly reduce the surface tension by enhancing emulsi cation thereby facilitating the bioavailability and degradation of hydrocarbons in contaminated environments (Banat et (Das and Mukherjee 2007). In comparison, our data clearly re ect the high e ciency of P. aeruginosa AKS1 and Bacillus sp. AKS2 in degradation of TPH during a short term incubation period of 28 days. The degradation rate was high in liquid media and in P. aeruginosa AKS1 inoculated microcosms. The reason for reduced degradation rate in sand sediment could be due to restricted mobility of both bacteria and crude oil. Amendment of contaminated sediments with inorganic N & P signi cantly enhanced the estimated biodegradation rates and concomitant reduction of half-life time as compared to no nutrient amended controls. The biodegradation rate of petroleum hydrocarbon in soil sediment has been described using rst order kinetic equation. One such study has revealed the range of kinetic constants between 0.041 and 0.0071 day − 1 for indigenous bacterium Stenotrophomonas multophilia in ex situ bioremediation of petroleum contaminated soil (Abbassi and Shquirat 2008). Our data also tted well in the rst order kinetic equation. First order kinetics has also been used to determine the biodegradation rate of lubricating oil, mineral oil, used motor oil, crude oil and diesel oil under various nutrient supplements (Rončević et al. 2005).

Conclusions
The data presented describe the e ciency and kinetics of hydrocarbon degradation activity of P. aeruginosa AKS1 and Bacillus sp. AKS2 isolates from crude oil contaminated sediments collected from Assam re nery, India. Both isolates showed high level of biosurfactant production and emulsi cation index. The CO 2 evolution activity of P. aeruginosa AKS1 in liquid medium was more than that of Bacillus sp. AKS2. But in the sand sediment, both isolates showed almost similar rate of CO 2 evolution activity.
The biodegradation e ciency of both isolates was effectively enhanced in response to inorganic N and P nutrients treatments both in liquid medium and microcosm sand sediments. Similar analyses on this line will generate crucial inputs for developing predictive model of hydrocarbon degradation.