Exploring the crude oil biodegradation and biosurfactant production abilities of isolated Agrobacterium fabrum SLAJ731
3.2. Materials and methods
3.2.1. Microbial strain and chemicals used
In this study, a gram-negative bacterium Agrobacterium fabrum SLAJ731 was used, which was earlier isolated by our group from a core sample of the Assam oil field, India. Crude oil (API gravity 34.97°) used in this study was obtained from IOCL, Noonmati, Assam India., in sterile sampling bottles and stored at 4°C till further use. Bushnell Hass (BH) media (Himedia M350-500G) was used for all optimization studies. Nutrient Broth (M002-500G), Luria Bertani (LB) media (M1245-500G), Nicotinamide Adenine Dinucleotide Hydride (NADH, RM393), n-hexadecane (RM2238-100ML), Yeast extract (RM027-500G) Glucose (MB037-500G), 3-[(3-Cholamidopropyl) dimethyl ammonio]- 1-propanesulfonate (CHAPS, MB084), Trifluoroacetic acid (RM874), Acetonitrile (AS029), n-hexane (AS097), Methanol (AS059) and Ethyl acetate (AS051), were purchased from Himedia, India.
Deuterated chloroform (570699), and Sinapinic acid (85429) were procured from Sigma, India. MiliQ (18 MΩ, Millipore system) water was used throughout the experiments.
3.2.2. Screening for biosurfactant production
Pre-culture of A. fabrum SLAJ731 was used as inoculum (1 %, v/v) in sterile BH media supplemented with 1 % (v/v) hexadecane as a model C source, at 30 °C and 150 rpm for 24 h. After incubation, the culture was centrifuged at 8000 rpm for 10 min at 4 °C. The supernatant was collected and explored for
screening of biosurfactant production ability of the bacteria using drop collapse test, oil displacement test and emulsification activity (E24) (Datta et al., 2018).
3.2.2.1. Drop collapse test
Drop collapse test examines the destabilizing ability of oil droplets by the action of biosurfactant. In this assay, 2 µL of oil was added to the cleaned glass slide and allowed to equilibrate at 37 °C for 1 h.
Next, 5 µL of cell-free supernatant was added to the oil droplet and left for 1 min. The collapsing of oil drop indicated the reduction in the interfacial tension between the polar and non-polar solvents interface, indicating positive surface activity of the biosurfactant (Persson and Molin, 2004).
3.2.2.2. Oil displacement test
The oil displacement test examines the ability of biosurfactants to displace the oil layer by lowering its surface tension. For this, 100 µL of oil was poured on 20 mL of distilled water in a Petri plate, and a uniform oil film was allowed to form. 10 µL of cell-free supernatant was added to this oil film, and immediately, the diameter of the clear zone formed was measured. The diameter of the clear zone is directly proportional to the surface activity of the biosurfactant (Morikawa et al., 1993).
3.2.2.3. Emulsification activity
E24 technique is used to determine the emulsion forming ability of biosurfactants in the non-polar and polar solvent system (Datta et al., 2018). Estimation of E24 was performed by mixing an equal volume of n-hexane (non-polar solvent) with the obtained cell-free supernatant. The mixture was vigorously homogenized for 2 min and left at room temperature for 24 h. Later, the height of the emulsion layer formed in the mixture was measured, and the E24 was calculated using equation 3.1,
𝑬𝟐𝟒= 𝐇𝐞𝐢𝐠𝐡𝐭 𝐨𝐟 𝐞𝐦𝐮𝐥𝐬𝐢𝐟𝐢𝐞𝐝 𝐥𝐚𝐲𝐞𝐫 (𝐦𝐦)
𝐓𝐨𝐭𝐚𝐥 𝐡𝐞𝐢𝐠𝐡𝐭 𝐨𝐟 𝐥𝐢𝐪𝐮𝐢𝐝 𝐜𝐨𝐥𝐮𝐦𝐧 (𝐦𝐦)× 𝟏𝟎𝟎 (3.1)
3.2.3. Optimization of biosurfactant production
Upon confirming the ability of the present strain for biosurfactant production, its culture conditions were optimized using the one-factor-at-a-time approach (OFAT) as discussed in Chapter 2, section 2.5.2.1 (Devaraj et al., 2019; Jimoh and Lin, 2019; Parthipan et al., 2017). In each case, the 24 h grown culture of A. fabrum SLAJ731 was used as inoculum (5 %, v/v) at 1.4× 107 CFU/mL and incubated in BH broth at listed pH, temperature and 150 rpm for 24 h (Table 3.1). After incubation under each listed condition, the cells were pelleted down by centrifugation at 8000 rpm for 10 min at 4 °C and weighed for the dry biomass estimation. The obtained supernatant was tested for E24.
3.2.4. Biosurfactant extraction and characterization
The bacterial culture was grown in sterile BH media with optimized C and N (as per the C: N ratio obtained) and maintained at optimized conditions for 144 h. At the end of the incubation period, the cells were harvested using centrifugation at 8000 rpm for 10 min at 4°C. Next, the solvent extraction technique using an equal volume of ethyl acetate: methanol (4:1) and cell-free supernatant was exploited for biosurfactant extraction, as also reported by our group (Gudiña et al., 2015; Sharma et al., 2019). In this regard, the obtained supernatant was initially acid precipitated (pH 2) and incubated overnight at 4°C using 6M HCl. Later, it was solvent extracted, and the organic layer was vacuum evaporated using rotary vacuum evaporation, leaving behind a crude biosurfactant. Thus, obtained biosurfactant was weighed gravimetrically and stored at room temperature for further characterizations using FTIR spectroscopy (Perkin–Elmer, Spectrum two), 1H NMR spectroscopy (MERCURY PLUS, VARIAN), and LC-MS (Waters, Q-Tof Premier) and Tensiometer (Data physics, DCAT 11EC).
3.2.4.1. Functional group analyses
The functional group analyses of the crude biosurfactant were performed using FTIR and NMR spectroscopy. FTIR spectroscopy works based on identifying the chemical groups and functional groups present in the molecule of interest. FTIR spectroscopy uses infra-red radiation to vibrate and rotate molecules within the compound at a specific frequency recorded by the detector as a peak intensity.
The nature of the peak (stretching/bending) and its intensity describe the chemical groups present in the
chemical structure of the compound of interest. FTIR has been a widely accepted identification and characterization technique employed to study biosurfactants. Briefly, 10 µL of crude biosurfactant was mixed with 100 mg of anhydrous KBr and then compressed into a thin film/ pellet, which was analyzed using an FTIR spectrophotometer (400-4000 cm-1). The spectra resolution was set to 4 cm-1, and the scan rate was maintained to 32 scans (Elazzazy et al., 2015).
The NMR technique is an analytical technique used to determine the structure of any compound based on its magnetic spin orientation in an external magnetic field applied. Crude biosurfactant sample was prepared in deuterated chloroform and analyzed using in 400 MHz 1H NMR Spectrometer equipped with an Oxford superconducting magnet of frequency 400 MHz (9.4 Tesla) with a probe capacity of 5 mm. The obtained chemical shifts (δ) of NMR absorptions were recorded in ppm scale relative to tetramethylsilane (Zou et al., 2014).
3.2.4.2. Surface tension measurement
The cell-free supernatant was also studied for the determination of critical micelle concentration (CMC) of the crude biosurfactant based on reduction of surface tension of water upon sequentially adding varying concentrations of crude biosurfactant till inflection point was reached using a tensiometer (Data physics, DCAT 11EC) at 25 °C using a Du Nouy ring method (Pandey and Pattanayek, 2013).
3.2.5. Biodegradation analysis of crude oil
The ability of A. fabrum SLAJ731 to biodegrade crude oil was performed by inoculating at 1.4×107 CFU/mL inoculum (5 %, v/v) in sterilized BH media (100 mL) under optimized experimental parameters. The experimental setups were prepared as described in Table 3.1. In order to study the effect of optimization on the oil biodegradation ability, a comparative experimental setup was kept with no glucose, imitating sub-optimal condition. The cells were harvested using centrifugation upon incubation, and the supernatant with residual crude oil was recovered and extracted in organic solvent (n-hexane) thrice. Later, the organic layer, comprising residual oil, was pooled and subjected to vacuum evaporation to recover residual oil. Thus, obtained residual oil was quantified gravimetrically. The overall oil biodegradation was calculated using equation 3.2;
𝑶𝒊𝒍 𝒅𝒆𝒈𝒓𝒂𝒅𝒂𝒕𝒊𝒐𝒏 (% ) =(𝑨𝒎𝒐𝒖𝒏𝒕𝒊𝒏𝒊𝒕𝒊𝒂𝒍 𝒐𝒊𝒍−𝑨𝒎𝒐𝒖𝒏𝒕𝒓𝒆𝒔𝒊𝒅𝒖𝒂𝒍 𝒐𝒊𝒍) ×𝟏𝟎𝟎
𝑨𝒎𝒐𝒖𝒏𝒕𝒊𝒏𝒊𝒕𝒊𝒂𝒍 𝒐𝒊𝒍 (3.2)
Furthermore, the extracted residual crude oil was analyzed using FTIR spectroscopy and Gas chromatography-mass spectrometry (GC-MS) analyses to elaborate the change in its chemical composition. For GC-MS study (model 7890B, Agilent Technologies), DB5 column (30 m × 0.25 mm
× 0.25 μm) used. The injector temperature was maintained at 280 °C with a split ratio of 1:10. The initial oven temperature was maintained at 80 °C for 2 min and increased at ramping of 5 °C/min to 280
°C, followed by a hold for 30 min. The detector was maintained at 300 °C, and the Helium (carrier gas) flow rate was maintained at 1 mL/min. On the other hand, the aqueous layer was studied for biosurfactant production, as discussed in section 3.2.4. In addition, the chemical oxygen demand (COD) was also estimated to determine the dissolved organic content (Clesceri et al., 1998; Sajna et al., 2015).
Table 3.1. Experimental designs for the study of the biodegradation of crude oil using A. fabrum SLAJ in the presence of co-substrate
Experimental setups Culture parameters
(Incubation conditions: pH 6, 30°C and 150 rpm for 144 h) With glucose
(Optimal condition)
BH media + Glucose (1 % w/v) + Crude oil (1 % v/v) + Yeast extract (1 % w/v) + Inoculum (5 % v/v)
Without glucose
(Sub-optimal conditions)
BH media + Crude oil (1 % v/v) + Yeast extract (1 % w/v) + Inoculum (5 % v/v)
Abiotic control BH media + Crude oil (1 % v/v) + Yeast extract (1 % w/v)
3.2.6. Determination of alkane hydroxylase activity
One of the major enzymes involved in crude oil degradation is alkane hydroxylase. Alkane hydroxylase is involved in the oxidation of hydrocarbons into their alcoholic derivatives (equation 3.3) (Elumalai et al., 2017)
𝑹𝑯 (𝑨𝒍𝒌𝒂𝒏𝒆) + 𝑶𝟐+ 𝑯++ 𝑵𝑨𝑫𝑯𝑨𝒍𝒌𝒂𝒏𝒆 𝒉𝒚𝒅𝒓𝒐𝒙𝒚𝒍𝒂𝒔𝒆
→ 𝑹𝑶𝑯(𝑨𝒍𝒄𝒐𝒉𝒐𝒍) + 𝑯𝟐𝑶 + 𝑵𝑨𝑫+ (3.3)
After incubation, the pelleted bacterial cells were initially washed with sterile distilled water twice and later re-dissolved in 20mM Tris-HCl (pH 7.4). Further, the cells were lysed using sonication at 4°C for 15 min to obtain crude protein extract. The lysate was centrifuged at 10000 rpm at 4°C for 30 min to recover debris-free crude protein extract. Obtained pelleted debris was dried overnight oven to calculate the dry biomass. The total protein was estimated using QuantiPro BCA assay kit (Sigma, India, Cat no.
100134331) using BSA as standard (de Andrade et al., 2016; Hasan et al., 2018; Pandey et al., 2012;
Pandey and Pattanayek, 2011; Pandey and Pattanayek, 2013).
The crude protein extract was studied for alkane hydroxylase assay. The reaction mixture comprised 0.1 mM Nicotinamide adenine dinucleotide (NADH) as a cofactor, CHAPS hydrate (0.15 % (w/v), pH 7.4), 10 μL of hexadecane as substrate (1 % hexadecane in 80 % DMSO) and 50 μL of crude enzyme extract. The alkane hydroxylase enzyme activity was analyzed based on a decrease in NADH concentration during the oxidation of hexadecane to hexadecanol by AH enzyme present in the crude extract. Such consumption of NADH per minute was estimated as measuring absorbance at 340 nm, signifying the successful oxidation of the substrate (Jauhari et al., 2014; Meng et al., 2017; Singh and Tiwary, 2017).
3.2.7. Statistical analysis
The statistical analyses were carried out using OriginPro 8.5. All the experiments were performed in triplicates, and their values were reported as average ± standard deviation. Statistically, a significant difference was considered when the significance level, p, was <0.05, using analysis of variance (ANOVA).