A comparative study on chemical characterization and properties of surface active compounds from Gram-positive Bacillus and Gram-negative Ochrobactrum strains utilizing pure hydrocarbons and waste mineral lubricating oils

Mineral lubricating oils are widely used in various industrial sectors for their applications in maintenance and functioning of machineries. However, indiscriminate dumping of these used oils have resulted in polluting the natural reservoirs which subsequently destroys ecological balance. Bacteria can emulsify or lower surface tension between phases of immiscible substrates and can acquire them as their carbon and energy sources. Such a phenomenon is mediated by production of extracellular polymers which can function as eminent surface active compounds based on their surfactant or emulsifying nature. The comparison between bacterial strains (Gram-positive Bacillus stratosphericus A15 and Gram-negative Ochrobactrum pseudintermedium C1) on utilization of pure straight chain hydrocarbons, waste mineral lubricating oils as sole carbon source and chemical characterization of the synthesized surface active compounds were studied. Characterization analysis by Ultraviolet Visible spectrophotometry, Fourier transform infrared spectroscopy, Nuclear Magnetic Resonance spectroscopy, Carbon–Hydrogen–Nitrogen analysis has given detailed structural elucidation of surface active compounds. The contrasting nature of bacterial strains in utilization of different hydrocarbons of waste mineral lubricating oils was observed in Gas Chromatography-Mass Spectroscopy analysis. The variation between both strains in utilization of hydrocarbons can be manifested in chemical structural differences and properties of the produced surface active compounds. Scanning Electron Microscopy has given detailed insight into the microstructural difference of the compounds. The utilization of lubricating oils can address waste disposal problem and offer an economical feasible approach for bacterial production of surface active compounds. Our results suggest that these surface active compounds can maneuver applications in environmental bioremediation and agriculture, pharmaceuticals and food as functional biomaterials.


Introduction
The detrimental effect on environment due to hazardous waste disposal has been a matter of concern since many decades. Mineral lubricating oils are often required in automobiles, textiles, metallurgical industrial sectors for maintenance of machineries. However, during servicing and repair works, such oils are generally dumped onto the natural reservoirs which can cause a serious threat to living organisms (US EPA 2004;US EPA 2018;Meiners 2020;Guerin 2008). Mainly consisting of straight chain and branched hydrocarbons, PAH (Poly Aromatic Hydrocarbons), PCB (Poly Chloro Biphenyls) and heavy metals, these oils contribute up to 76% of world's waste oil disposal. Irrational disposal of such waste oils in natural reservoirs can cause bioaccumulation of non-biodegradable, carcinogenic chemical components in animals and plants which ultimately cause serious implications in human health (Bhattacharya et al. 2015;Kajdas 2014). Therefore, researchers are seeking newer environment friendly remediation methods involving hydrocarbon utilizing bacterial species in minimizing pollution caused by these hazardous pollutants present in waste mineral lubricating oils. Extensive studies involving degradation of these components by bacterial metabolic genes and enzymes have been a topic of great interest since 1980s (Panday and Arora 2020; Bonilla et al. 2005;Vasconcellos et al. 2011;Martínez-Checa et al. 2002;Zhou et al. 2016).The hydrocarbons present in the waste mineral oils provides necessary carbon source for production of bacterial extracellular polymers (Misra and Pandey 2005;US EPA 2004;Das and Chandran 2011). The hydrophilic and lipophilic functional groups of extracellular polymers mediate easier uptake of such hydrocarbons by acting as surface active compound (SAC) (Vasconcellos et al. 2011). Surface active compounds like synthetic emulsifiers or surfactants can emulsify or reduce surface tension or interfacial tension between two immiscible liquids (Uzoigwe et al. 2015). These extracellularly produced surface active compounds by using cost effective carbon sources have gained immense importance due to their biodegradable and less toxic nature. Therefore, these bacterial surface active compounds can be a better alternative with potential applications in environmental bioremediation and agriculture, as additives in daily household products and pharmaceuticals and as preservatives in food while simultaneously reducing pollution caused by chemically synthesized surfactants and emulsifiers Banat et al. 2014).
Different myriad species of bacteria like Acinetobacter, Bacillus, Variovorax, Azotobacter, Pseudomonas, Ochrobactrum, Geobacillus, Paenibacillus, Exiguobacterium, Halomonas, Alcaligenes, Aeribacillus are known to be producer organisms of various surface active compounds Sana et al. 2017a;Cai et al. 2017;Zhou et al. 2016;Nerurkar et al. 2009;Toledo et al. 2008). Few studies involving gram-negative, aerobic, rod shaped bacilli Ochrobactrum sp. of family Brucellaceae has been investigated for production of extracellular polymers having surfactant and emulsifier properties (Bezza et al. 2015;Zarinviarsagh et al. 2017;Calvo et al. 2008). Ochrobactrum pseudintermedium has shown ability to degrade pure hydrocarbons and waste oils with simultaneous production of surface active compounds (Bhattacharya et al. , 2015. Similarly, Bacillus sp. in presence of varied short and long chain hydrocarbons has produced extracellular polymeric emulsifiers and surfactants in many studies (Gurjar et al. 1995;Toledo et al. 2008;Jemil et al. 2016;Dadrasnia and Ismail 2015;Ayed et al. 2014). But studies involving production of extracellular polymeric substances from gram-positive, rod shaped Bacillus stratosphericus belonging to the family Bacillaceae are few (Hentati et al. 2016). Our earlier work has reported about production of SACs by utilization of fish fat as the only substrate (Sana et al. 2017b). Here in this work, Bacillus stratosphericus A15 has been explored for production of SACs from different petroleum hydrocarbon substrates and compared alongside with that of O. pseudintermedium C1.
Our work focuses on utilization of pure straight chain hydrocarbons and industrial waste mineral lubricating oils for production of extracellular surface active compounds by gram-negative Ochrobactrum pseudintermedium C1 and gram-positive Bacillus stratosphericus A15. The study further compares their chemical composition, surface activity, morphological, microstructural and thermal stability properties. Waste mineral lubricating oils provide an economical carbon source for production of biodegradable SACs and additionally provide a better alternative in minimizing pollution caused by oil disposal.

Chemicals and media required for the study
Waste mineral lubricating oils like spindle oil, compressor oil, hydraulic oil were collected from an oil factory named Asianol Pvt Ltd., Kolkata, India. Pure alkane hydrocarbons like nonane, tetradecane, hexadecane, eicosane, octacosane and chemical reagents of analytical grade were purchased from Sigma Aldrich Co. USA. Bacteriological media (Nutrient agar and Bushnell-Haas broth) were purchased from Hi-Media Laboratories, India.

Culture conditions, maintenance and production of surface active compounds (SACs)
The growth of the bacterial strains and subsequent production of SACs was done using Bushnell Haas broth (BH) [in g/L: 1.0 KH 2 PO 4 , 1.0 K 2 HPO 4 , 1.0 NH 4 NO 3 , 0.2 MgSO 4 ·7H 2 O, 0.02 CaCl 2 ·2H 2 O, 0.05 FeCl 3 ·6H 2 O]. Erlenmeyer flasks containing 50 ml BH broth were sterilized prior to the experiment. Pure hydrocarbons and waste mineral lubricating oils as sole carbon sources were added to the BH broth containing flasks. Fresh overnight grown cultures were inoculated in sterilized BH broth (OD 600 = 1.0) and aseptically added to the flasks.

Parameters maintained for synthesis of bacterial SACs in culture broth
Determination of optimal culture conditions by considering parameters like temperature, pH, incubation period, percentage of carbon source and aeration was done. The conditions maintained for growth of bacterial strains and production of SACs were 25-40 °C, pH 2-10, 1-15 days and 2-10% of waste mineral lubricating oils (v/v). The flasks were then kept in orbital shaker incubator (ORBITEK-LJE, Scigenics Biotech Pvt. Ltd, India) under aerobic conditions. Control flasks (non-inoculated flasks) were incubated in the same conditions to verify that no abiotic loss take place in aerobic conditions.

Extraction and drying of SACs
The methods described by Sengupta et al. 2019 were followed for extraction, purification and drying of surface active compounds. After centrifugation (Remi C24 Plus centrifuge, India) and discarding of bacterial cell pellet, n-hexane was added to culture supernatant to collect and separate residual oil for GC-MS analysis (Adebusoye et al. 2007). Precipitated surface active compounds were collected aseptically in microcentrifuge tubes and were kept in vacuum desiccators filled with fused anhydrous CaCl 2 as desiccant. The surface active compounds were again dried in Abderhalden's drying pistol apparatus for further characterization analysis.

Influence of SACs on cell surface hydrophobicity
The adhesion of bacterial cells to hydrocarbons was estimated by modifying the technique of Rosenberg et al (1980). Waste oils were added to test-tubes containing 5 ml of bacterial cell suspension (OD 600 = 1.0) and vortexed for 2 min. After keeping them undisturbed for 1 h, the separated aqueous phase was carefully collected by micropipette and optical density was measured at 600 nm. Bacterial adhesion to hydrocarbons (BATH) is expressed as-

Assessment of surfactant and emulsifying property of SACs
The surfactant property of surface active compounds was estimated at room temperature by following du Nuoy ring method (Lunkenheimer and Wantke 1981) using a tensiometer (Data physics DCAT 11, Germany). Emulsification Index (EI) was estimated by following method of Cooper and Goldenberg (1987) against hydrophobic substrates tetradecane, hexadecane, motor oil, diesel, kerosene, vacuum gas oil, engine oil and crude oil. The emulsification index was measured after 24 h according to the following equation (Cooper and Goldenberg 1987).
where, h emulsion is the height of emulsion layer formed in between immiscible phases by emulsifying property of SAC and h total is the total height of the liquid mixture.
Structural elucidation of the SACs produced by bacterial strains utilizing waste mineral lubricating oil as substrates.

Chemical analysis of the purified SACs
The carbohydrate content was estimated by following phenol-sulphuric acid method for total carbohydrates according to Dubois et al. (1956) and the protein content was estimated according to Lowry's method for proteins

Fourier transform infrared (FTIR) spectroscopy
FTIR spectra were obtained in the range 4000-400 cm −1 after preparation of pellets by mixing finely grounded dried potassium bromide and surface active compounds (Perkin Elmer, USA, Spectrum version 10.5.1).

H 1 Nuclear Magnetic Resonance (H 1 -NMR) spectroscopy
SACs were dissolved in CDCL 3 (for surface active compounds obtained from bacterial utilization of pure hydrocarbons as sole carbon source) and DMSO-d6 (for surface active compounds obtained from bacterial utilization of waste spindle oil as sole carbon source) and vortexed thoroughly. The samples were then subjected to 400 MHz NMR (Bruker Avance 400 Spectrometer, Germany) and 500 MHz NMR (Jeol ECZ 500 spectrometer, Japan) for H 1 -NMR analysis up to spectral width of 15 ppm. The chemical shifts were recorded in ppm relative to the resonance of tetramethylsilane as the internal standard.

Elemental (CHN) analysis
4-5 mg of the Purified SACs were taken for estimation of Carbon, Hydrogen, Nitrogen content (Thermofinnigan Flash 1112 Elemental Analyzer, Italy).

Microstructural analysis by Scanning Electron Microscopy (SEM)
Dried SACs were observed after glutaraldehyde fixation, sequential dehydration and sputtering with platinum followed by recording images at 15 kV (Zeiss Evo 18 Special Edition, Germany).

Thermal stability studies
Thermal stabilities of 12 mg dried, purified SACs were determined by using Thermogravimetric (TG) and Differential thermal (DT) analysis (Perkin Elmer Diamond, USA). Initial and final temperature were set at 30 °C and 800 °C respectively. The heating rate was maintained at 10 °C/min by gradually increasing the temperature.

Gas Chromatography-Mass Spectrometry (GC-MS) analysis of waste mineral lubricating oil before and after bacterial inoculation
The n-hexane extracted residual oils before and after bacterial incubation were evaporated for removal of the solvent (EYELA Rikakikai Rotary Evaporator, Japan). The oil samples were then diluted 10 times with HPLC-grade n-hexane and volume of 1 µl were injected into a gas chromatograph equipped with DB5MS column (Thermo Scientific Trace 1300 series, USA). Helium was used as the carrier gas with flowrate 1.5 ml/min. Chromatograms were analyzed by Chromeleon 7.0 program and a library (NIST 2007) search was performed for the identification of peaks.

Selection of carbon source
Gram-positive Bacillus stratosphericus A15 and Gram-negative Ochrobactrum pseudintermedium C1 showing growth turbidity and better yield of surface active compounds in hydrocarbon substrates were sub-cultured and preserved in sterilized nutrient agar slants at 4 °C. BH broth was enriched with two types of hydrocarbons i.e., pure straight chain alkanes and waste mineral lubricating oils as carbon sources. Carbon sources influencing maximum bacterial growth and SAC production were selected. Ochrobactrum pseudintermedium C1 and Bacillus stratosphericus A15 showed optimal growth when hexadecane and nonane were carbon sources, respectively. Among mineral lubricating oils, waste spindle oil (specific gravity 0.8488) produced maximum growth and yield of SACs by both bacterial strains.

pH of the culture medium
The study was performed under varying pH conditions (pH 2-10). However, yield of surface active compounds increased at alkaline and neutral to slightly alkaline culture medium for Bacillus stratosphericus A15 and Ochrobactrum pseudintermedium C1, respectively.

Temperature of the culture medium
The study was done under varying temperature conditions (30-40 °C). Production of SACs by Ochrobactrum pseudintermedium C1 were highest at temperatures between 35 and 37 °C, while Bacillus stratosphericus A15 has produced SACs between 32 and 33.5 °C.

Percentage of carbon source, incubation time on bacterial growth and yield of SAC
It was observed that 4% of the pure and waste hydrocarbons as sole carbon sources provided optimum growth of bacterial strains and subsequent production of SACs. In this study, bacterial strains were grown in pure straight chain hydrocarbons i.e., starting from hexane (C-6) to octacosane (C-28). It was observed that bacterium Ochrobactrum pseudintermedium C1 can degrade long chain hydrocarbons (C-14 to C-18) and produce SACs whereas, on the contrary, Bacillus stratosphericus A15 can degrade short chain hydrocarbons (C-8 to C-12) and produce SACs. Identical degradation pattern of alkanes was reported in a study by Zheng et al. 2011. The maximum growth was observed between 10th and 12th day of incubation. The maximum optical densities were 0.21 and 0.5 for Bacillus stratosphericus A15 and Ochrobactrum pseudintermedium C1 respectively (Fig. 1a). The yield of SACs were highest between 12th and 13th day for Bacillus stratosphericus A15 and between 14th and 15th day for Ochrobactrum pseudintermedium C1. The SAC 3 produced by strain A15 utilizing spindle oil was ethanol Fig. 1 a Growth of Bacillus stratosphericus A15 and Ochrobactrum pseudintermedium C1 in Bushnell-Haas broth with hydrocarbons as carbon source with variation in days b Isolation of SACs (i) Before precipitation (ii) After precipitation and drying precipitated and the total yield was around 150 mg/L, while strain C1 has produced SAC 4 with total yield of 300 mg/L following the same process. Ethanol precipitated SACs after isolation and extraction have shown an amorphous nature. These compounds were then subjected to various characterization and chemical analysis to further evaluate their surface activity and to elucidate their chemical structure and composition (Fig. 1bi, bii). A comparative table on various parameters for bacterial production of SACs in culture medium is given in Table 1A.

Assessing the emulsifying, surfactant and cell surface hydrophobicity of SACs
Gram-positive Bacillus stratosphericus A15 and Gramnegative Ochrobactrum pseudintermedium C1 have shown 38% and 56% adherence to waste spindle oil with decrease in absorbances of the aqueous phase to 0.62 and 0.57 at 600 nm respectively as estimated by BATH assay. Such phenomenon is further explained by the tendency of the strain Bacillus stratosphericus A15 to produce SAC 1 and SAC 3 after utilization of nonane and waste spindle oil with the capacity of lowering the surface tension from 71 mN m −1 to 53 mN m −1 and 51.7 mN m −1 respectively (Fig. 2ai).
On the contrary, SACs produced by Ochrobactrum pseudintermedium C1 have shown no significant surface tension reduction. SAC 2 and SAC 4 produced from strain Ochrobactrum pseudintermedium C1 could reduce surface tension to 63.9 mNm −1 and 61 mNm −1 when hexadecane and waste spindle oil, respectively, were present as carbon sources. Unlike SACs produced by Bacillus stratosphericus A15, Ochrobactrum pseudintermedium C1 has produced surface active compounds with emulsifying properties and exhibited better results when diesel, engine oil and mustard oil were used as hydrophobic substrates in comparison to SACs produced by strain A15. SAC 4 produced by Ochrobactrum pseudintermedium C1 has shown emulsification index of diesel (2 %), vacuum gas oil (2.5 %), hexadecane (10 %), tetradecane (12.5 %), mustard oil (37.5 %), motor oil (35 %), crude oil (50 %) and engine oil (88 %) after 24 h. The emulsions remained stable for up to 48 h ( Fig. 2aii) ( Table 1B). The findings are in correlation with earlier reported studies where petroleum hydrocarbons were present in the growth medium (Toledo et al. 2008;Gudiña et al. 2015).

Chemical analysis, Ultraviolet Visible Spectrophotometry and CHN analysis
The surface active compounds were found to be composed of carbohydrates, protein and lipid in varying percentages.
The obtained results were further corroborated by CHN analysis ( Table 2). The results obtained can be further interpreted by FTIR and H 1 -NMR analysis. In previous studies by Zheng et al. (2011) and Kourmentza et al. (2019), bacterial surface active compounds with similar varied constituents was reported.

Characterization of SACs by FTIR studies
The surface active compounds produced by bacterial strain A15 (SAC 1) and C1 (SAC 2) utilizing pure hydrocarbons nonane and hexadecane respectively were primarily characterized by FTIR followed by detailed structural elucidation by H 1 -NMR. It is worth mentioning that when compared to spectra of pure nonane and hexadecane, significant differences were observed in bacterial produced surface active compounds. The bands between 3300 and 3450 cm −1 and 2000-2500 cm −1 can be attributed to the hydroxyl -OH and alkyne stretch respectively. Further bands at 1650 cm −1 , 1385 cm −1 and 1050 cm −1 corresponds to alkene C=C stretch or carbonyl stretching of amides, CH 3 bends and C-O stretch of ether respectively in both the structures (Beltrani et al. 2015;Amaral et al. 2006). Presence of small bands in the fingerprint region (858-934 cm −1 ) indicated characteristic presence of carbohydrates (Beltrani et al. 2015) (Table 3A) (Fig. 2bi).
The FTIR spectra of surface active compounds produced by strain A15 (SAC 3) and strain C1 (SAC 4) utilizing waste spindle oil have shown some differences (Table 3B) (Fig. 2bii). Hydroxyl -OH, alkyne stretch and C=C stretch of alkenes or carbonyl stretching of amides (C=O) were present in both SAC 3 and SAC 4 (Amaral et al. 2006). The C=O stretch of amide group was further supported by bands at 1461 cm −1 and 1570 cm -1 which corresponds to C-N stretch and N-H bends respectively in SAC 4 (Amaral et al. 2006;Beltrani et al. 2015). Sharp bands (2925, 2855 cm −1 ) in SAC 4 were due to CH stretch of CH 2 and CH 3 groups respectively (Beltrani et al. 2015;Gudina et al. 2015). Additional bands in fingerprint region denoted presence of ether and anomeric carbon of carbohydrates in both the spectra Sengupta et al. 2019;Beltrani et al. 2015).

Characterization of SACs by 1 H-NMR studies
H 1 -NMR analysis of SAC 1 and SAC 2 produced by bacterial strains A15 and C1 utilizing pure hydrocarbons nonane and hexadecane as sole carbon sources respectively have shown distinct differences in structures when compared to proton NMR spectra of pure hydrocarbons. Aliphatic saturated hydrocarbon chain of lipids, unsaturated hydrocarbons and ether functional groups were present in both the structures. A strong signal at 2.984 ppm in SAC 2 can be assigned to the protons attached to carbonyl group. Presence of isopropyl groups in SAC 1 is attributed by signals between 3.690 and 3.755 ppm (Table 3A) (Fig. 3ai, aii). Structural differences by H 1 -NMR analysis of surfaceactive compounds produced from utilizing spindle oil were obtained (Table 3b) (Fig. 3bi, bii). Multiple signals between 0.7 and 1.4 ppm denote protons in aliphatic saturated chain of lipid moiety i.e., alkyl CH 3 , CH 2 and CH or cholesterol in the structures (Watts 2013). This is followed by unsaturated or carbonyl protons at 1.8-2.4 ppm and protons of ether functional group at 3.6-3.8 ppm. Further downfield, signals between 4.2 and 5 ppm can confirm the presence of anomeric sugars (Duus et al. 2000). Presence of isopropyl groups in SAC 4 was evident by characteristic signals between 1.409 and 1.475 ppm and 4.1-4.2 ppm. However, in SAC 3 isopropyl group was observed in the region between 1.4 and 1.5 ppm. The presence of isopropyl group suggest the branched nature of the structures. Fig. 2 a Fourier transform infrared spectra of (i) SAC 1 and SAC 2 produced by Bacillus stratosphericus A15 and Ochrobactrum pseudintermedium C1 utilizing pure hydrocarbons nonane and hexadecane as sole carbon sources respectively (ii) SAC 3 and SAC 4 produced by Bacillus stratosphericus A15 and Ochrobactrum pseudintermedium C1 utilizing waste spindle oil as sole carbon source respectively. bi Surface tension lowering property of surface active compounds produced by bacterial strain A15 utilizing pure straight chain hydrocarbons (nonane, tetradecane and hexadecane) and waste spindle oil bii Emulsification index of surface active compounds produced by bacterial strain C1 utilizing waste spindle oil against hydrophobic substrates: A hexadecane B tetradecane C mustard oil D vacuum gas oil E motor oil F crude oil observed after 24 h

Microstructural studies of SACs by scanning electron microscopy
The SACs produced by both strains have shown some contrasting differences. Strain A15 produced surface active compound with few elongated rod structures in low magnifications whereas in higher magnifications presence of spherical structures were more prominent (Fig. 4ai, aii) (Table 1B). Surface active compound produced from strain C1 has shown irregular spherical structures in lower magnifications whereas in higher magnifications few elongated rod structures and globular spherical structures adjacent to web like porous matrix was observed (Fig. 4bi, bii) (Table 1B).

Thermal stability studies of SACs
Thermogravimetric analysis (TGA) of SACs produced by bacterial strains A15 and C1 by utilizing waste spindle oil have shown initial weight reduction in between 80 and 150 °C. This phenomenon can be attributed due to loss of moisture and solvents in both cases. Thermogram of samples displayed similar gradual downward curve with increase in temperature thus implying decomposition of organic matter. During the second phase i.e., from 150 to 350 °C rapid degradation in both the samples was observed. Maximum degradation has started from 350 °C and continued till 800 °C. Upon completion of the analysis, residual weights of SAC 3 and SAC 4 were found to be 58% and 66% respectively suggesting more thermal resistant nature of SAC 4 when compared to SAC 3 (Fig. 5a) (Table 1B). Similar thermal degradation of surface active extracellular polymeric substances have been reported in earlier studies (Sengupta et al. 2019). Thermal Analysis (DTA) of both samples validate with their respective TGA graphs. The endothermic peak near 100 °C is characteristic of dehydration reaction. Another significant exothermic peak was observed at 338.06 °C and 380.54 °C Fig. 3 a Nuclear magnetic resonance spectra of (i) SAC 1 (ii) SAC 2 produced by Bacillus stratosphericus A15 and Ochrobactrum pseudintermedium C1 utilizing pure hydrocarbon (nonane and hexadecane). b Nuclear magnetic resonance spectra of (i) SAC 3 (ii) SAC 4 pro-duced by Bacillus stratosphericus A15 and Ochrobactrum pseudintermedium C1 utilizing waste spindle as sole carbon source respectively for SAC 3 and SAC 4 respectively corresponding to organic oxidation of both the compounds (Fig. 5b).

GC-MS analysis of waste industrial mineral lubricating oils before and after bacterial incubation
GC-MS chromatogram analysis of waste spindle oil before and after bacterial incubation, have revealed some interesting differences (Fig. 5c). Complete disappearance of few peaks corresponding to cyclohexane, hexadecynol and heptadecyne derivatives in the chromatogram of Ochrobactrum pseudintermedium C1 utilized spindle oil fraction was observed whereas there has been decrease in relative abundance to the peaks corresponding to hexadecynal, eicosane intermediates in C1 utilized fraction of spindle oil (Fig. 5 di, diii). Comparing chromatograms before and post incubation by bacterial strains, less degradation capability of hydrocarbons by strain A15 was observed (Fig. 5 di, dii). The peak corresponding to hexadecynal was observed in bacterial strains utilized chromatograms. However, decrease in concentration of hexadecynal in strain C1 utilized fraction when compared to strain A15 utilized fraction of waste spindle oil suggested better utilization of long chain hydrocarbons by strain C1 (Fig. 5 di, dii, diii). Presence of organic acid hexadecadienoate and alcoholic intermediates for instance, dodecadienol, octadecatetraenol in the chromatogram of spindle oil utilized by strain C1 also suggests bacterial metabolism and bioconversion of complex hydrocarbon substrates into intermediate compounds.
Ochrobactrum pseudintermedium C1 has shown similar characteristics in degrading long chain hydrocarbons of waste oils (Bhattacharya and Biswas 2014; Bhattacharya et al. 2015;Sengupta et al. 2019). Although, Bacillus stratosphericus A15 has produced surface active compound by utilizing fish fat as carbon source (Sana et al. 2017b), this study reports production of surface active compounds and GC-MS analysis of residual hydrocarbon post bacterial inoculation.

Discussion
Bacterial species generally degrade long chain alkanes more efficiently followed by branched alkane, small aromatics and cyclic alkanes. In a study, several Gram-negative and Gram-positive species of bacteria have shown different degradation rates when exposed to crude oil sludge (Obi et al. 2016;Lăzăroaie 2010). Bacteria do so by adhering to hydrocarbon droplets or by surfactant and emulsifier production which facilitates easier consumption of such immiscible carbon sources (Rojo 2009). Many bacterial species have produced such amphiphilic molecules possessing emulsifying and surfactant properties by degradation of hydrocarbons. However, chemical architecture, nature and properties of such molecules vary and are mainly dictated by the growth parameters and nutrient sources (Rosenberg and Ron 1999;Calvo et al. 2009). It is necessary to mention that by certain evolutionary strategies, some bacteria can show affinity towards oil or hydrocarbons and consume them as their necessary energy and carbon source. They are called "hydrocarbonoclastic" bacteria for their role in bioremediation of hydrophobic pollutants like petroleum hydrocarbons and discharged oils (Xu et al. 2018;Yakimov et al. 2007). In context of environmental bioremediation, such eco-friendly practices can be done by subjecting bacteria alone or by utilizing bacterial surface active compounds for increasing availability of immiscible carbon sources Sengupta et al. 2018). Detailed structural characterization and chemical composition by various spectroscopy and spectrophotometry methods can elucidate chemical structures and hence respective functional attributes of surface active compounds ).

Bacteria synthesized SACs from pure and waste hydrocarbons as carbon sources
In this study, Gram-negative Ochrobactrum pseudintermedium C1 and Gram-positive Bacillus stratosphericus A15 strains capable of metabolizing hydrocarbon and lipid based substrates were chosen for production of surface active compounds Sengupta et al. 2019; Fig. 5 a Thermogravimetric analysis and b Differential Thermal analysis curves of SAC 3 and SAC 4 produced by Bacillus stratosphericus A15 and Ochrobactrum pseudintermedium C1. c Gas Chromatography-Mass Spectroscopy chromatograms of (i) before incubation and after incubation by bacterial strains (ii) A15 and (iii) C1 respectively d Gas Chromatography-Mass Spectroscopy chromatograms (i) before and after incubation by bacterial strains (ii) A15 and (iii) C1 Sana et al. 2017b). Both bacterial strains have shown an initial lag phase during their growth on hydrocarbon carbon sources. The final OD 600 of strain C1 was found to be greater than strain A15 suggesting its better growth and yield of SACs while utilizing long chain hydrocarbons. Interestingly, production of SACs was observed in another study by Cai et al. 2017 when hexadecane and diesel were used as carbon sources in culture medium. However, bacterial strain A15 has shown better growth and yield of SACs in presence of short chain hydrocarbons. The best yield was observed in presence of nonane and hexadecane for Bacillus stratosphericus A15 and Ochrobactrum pseudintermedium C1 with positive growth turbidity at 600 nm during incubation. Among other mineral lubricating oils, waste spindle oil with increased adherence to the cell surface of bacterial strains and yield of SACs was chosen as carbon source. The yield of SACs by both bacterial strains was found to be maximum during early logarithmic and stationary phase of growth therefore, promoting better uptake of these immiscible hydrocarbon substrates as carbon sources. Utilization of petroleum hydrocarbons like crude oil followed by production of surface active compounds from bacteria or bacterial consortium has been studied in previous works (Cooper and Goldenberg 1987;Bhattacharya et al. 2019).
Other additional parameters like pH, temperature, aeration are necessary for increasing the production of surface active compounds. Extreme conditions of pH, temperature can inhibit bacterial growth and result in inactivation of enzymes required for biosynthesis of these extracellular surface active compounds (Jin and Kirk 2018;Das and Chandran 2011).

Interpretation of characterization studies for structural elucidation, morphological, tensioactive and thermal stability properties of surface active molecules
The FTIR and H 1 -NMR characterization has revealed the presence of some chemical groups (-OH of alcohols, -OCH 3 of ethers and -CO of amides) which are not otherwise present in linear chain alkanes. Hence, it can be said that enriching of Bushnell Haas broth with these alkane hydrocarbons lead to simultaneous production of amphiphilic SACs thus increasing cellular uptake of these immiscible hydrocarbons as carbon source (Cai et al. 2017 (Gudiña et al. 2015;Zheng et al. 2011). The spectroscopy and spectrophotometry results obtained for surface active compounds from strain A15 suggest that percentage variation of protein, lipid and carbohydrate may impart their surface tension lowering properties as demonstrated in work of Dadrasnia and Ismail 2015. The difference in surface active properties can be further understood by adhesion characteristics of individual bacterial strains to waste spindle oil. Both bacterial strains on the basis of their hydrophobicity adheres to either short chain or long chain hydrocarbons. Gram-positive bacteria contains an outer cell wall mainly composed of teichoic acids and peptidoglycan layer while Gram-negative bacteria contains an additional lipopolysaccharide and phospholipid layer surrounding the periphery of the cell wall (Silhavy et al. 2010). The long chain hydrocarbon substrates therefore can adhere to the permeable lipopolysaccharide layer of strain Ochrobactrum pseudintermedium C1, followed by its uptake to the cell's interior and mediating simultaneous production of SACs with emulsifying properties (Beveridge 1999;Sengupta et al. 2019). The presence of teichoic acids and proteins can help in cellular uptake of short chain hydrocarbons by Bacillus stratosphericus A15 leading to simultaneous production of SACs with surface tension lowering properties (Silhavy et al. 2010;Sana et al. 2017b). This surface tension lowering ability can be due to adhesion of SACs to the bacterial outer surface and directing the hydrophilic part of SAC outwards thus decreasing cell surface hydrophobicity or due to alterations in cell surface functional groups during production of these compounds (Kaczorek et al. 2018). The increased and rapid adhesion of hydrocarbons by Gram-negative bacteria has been previously studied by Rosenberg et al. (1980). Chemical characterization studies (FTIR and H 1 -NMR) has depicted presence of alkene, alkyne stretch of aliphatic hydrocarbon and increased number of isopropyl groups in SAC 4 exhibiting stability at higher temperatures when compared to SAC 3. The elemental analysis has also shown high carbon, hydrogen and nitrogen content in SAC 4 that can further establish its emulsifying character and thermal stability. The scanning electron microscopy images suggest the microstructural differences in SACs produced by Bacillus stratosphericus A15 and Ochrobactrum pseudintermedium C1. The globular structures in both SACs can be attributed to the presence of chemical functional groups in proteins and carbohydrates . Detailed examination of SEM images have revealed high number of globular structures in SAC 4 when compared to SAC 3. The microstructural differences observed correlate with the structural elucidation of the structures obtained by chemical analysis, FTIR and H 1 -NMR. The characterization analysis suggest that the surface active compounds are heterogeneous mixture of lipoglycoprotein containing varied percentages of lipids, proteins and polysaccharides rendering them distinct characteristics (surfactant, emulsion forming nature and thermal stability). The structural and chemical composition of the produced extracellular surface active compounds have shown resemblance to the compounds reported in earlier published works of Peele et al. 2016 andZheng et al. 2011. Emulsan produced by Acinetobacter calcoaceticus RAG-1 has shown similar chemical compositional characteristics with polysaccharides attached to fatty acid groups distributed across the entire hydrophobic molecule. The polysaccharide groups can be replaced by proteins or can be in combination as observed in this present study (Neu 1996).

Interpretation of GC-MS studies of residual waste spindle oil before and after bacterial utilization
GC-MS studies provide a detailed insight into the differential uptake between two bacterial strains in utilization of hydrocarbons present in waste spindle oil. The abundant presence of organic acids, aldehydes and alcoholic intermediate products in the utilized fractions suggest that bacterial strains can undergo β-oxidation after consuming long chain compounds in waste spindle oil which is present in the culture medium as the sole carbon and energy source . Based on GC-MS findings, presence of hexadecynal and hexadecadienoate and complete degradation of hexadecynol in utilized fraction of waste spindle oil suggests that hexadecynol oxidizes to former two compounds which is corroborated by Adlan et al. 2020 where decreasing abundance of long chain alkanes and conversion to intermediate products proved a better cellular uptake and biodegradation. Unlike Bacillus stratosphericus A15, Ochrobactrum pseudintermedium C1 had shown better utilization of long chain hydrocarbons as evident from the abundance of hexadecynal. Further, absence of peaks corresponding to heptadecane and hexadecane intermediates establishes comparatively efficient metabolism of long chain hydrocarbon by Ochrobactrum pseudintermedium C1. This is further justified from the study by Sierra-Garcia and de Oliveira (2013) that the presence of AlkB gene, which encodes for non-haeme iron monooxygenase participate in oxidation of long or short chain hydrocarbons to respective alcoholic intermediates which might facilitate the production of SACs. Moreover, incorporation of oxygen containing chemical functional groups like hydroxyl, ether and carbonyl groups in the surface active compounds as evident from FTIR, H 1 -NMR, and chemical analysis can further confirm the involved hydrocarbon biodegradation mechanism for production of these metabolites.

Conclusion
Comparative characterization analysis of bacterial surface active compounds by utilization of pure and waste mineral lubricating oils was done. However, the compounds produced by Bacillus stratosphericus A15 and Ochrobactrum pseudintermedium C1 have exhibited difference in surface active properties and thermal stabilities. Production of such bacterial surface active compounds by using conventional nutrient growth medium can be an expensive process. Waste mineral lubricating oils represent an economic carbon source for production of bacterial surface active compounds. The presence of different chemical groups in these compounds can render them future promising applications.