Engine oil used in automobiles is a threat to soil and water due to recalcitrant properties of its hydrocarbons. It pollute surrounding environment which affect both flora and fauna of earth. Microbes are able to degrade hydrocarbons containing engine oil to utilize as a substrate for their growth. Our results demonstrated that Bacillus velezensis KLP2016 (Gram +ve, endospore forming; Accession number KY214239) cell-free broth recorded an emulsification index (E 24 %) from 52.3% to 65.7% against different organic solvents, such as benzene, pentane, cyclohexane, xylene, n -hexane, toluene and engine oil. The surface tension of the cell-free broth of B. velezensis grown in Luria Bertani broth at 35°C decreased from 55 to 40 mN.m -1 at critical micelle concentration 17.2 µg/mL. The active biosurfactant molecule of cell-free broth of Bacillus velezensis KLP2016 was purified by Dietheylaminoethyl-cellulose and size exclusion chromatography, followed by HPLC (RT=1.130), UV-vis spectrophotometry (210 nm) and thin layer chromatography (R f =0.90). Purified biosurfactant molecular weight was found ~1.0 kDa, on the basis of Electron Spray Ionization-MS. A concentration of 1980×10 -2 parts per million of CO 2 was trapped in a KOH solution after 15 days incubating the bacterium in Luria Bertani broth containing engine oil (1%). Results suggests that bacterium Bacillus velezensis KLP2016 may be a promising solution to the engine oil pollution problem with achieving a bioactive biosurfactant molecule for further eco-friendly application(s).
Research
Purification and characterization of a surfactin-like biosurfactant produced by Bacillus velezensis KLP2016 and its application towards engine oil degradation
https://doi.org/10.21203/rs.3.rs-94498/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 28 Jan, 2021
You are reading this latest preprint version
Environmental pollution is currently a serious problem. Engine oil used in automobiles is a very hazardous and toxic pollutant for the soil. Used engine oil that is spilled or wrongly discarded may enter storm water runoff and eventually enter into water bodies and adversely affect the environmental health of receiving water bodies and its flora and fauna [1]. Oil spills into the sea is an emerging issue, harming marine flora and fauna [2]. To save the flora and fauna of the water bodies, treatment of engine oil (main polluting agent) is very important and is the demand of the time. Different types of chemicals are used in petroleum industries for various operations, mainly in oil recovery [3]. The process releases contaminants and causes water contamination, posing health risks to living beings [4–5]. Recalcitrant hydrocarbon (present in engine oil) degrading microbes of genus Bacillus produce biosurfactants of a diverse chemical nature and molecular size, with different active role(s). These microbial biosurfactants have the capability to degrade hydrocarbons enhancing bioavailability of these hydrophobic organic compounds present in engine oil [6]. In recent years, biosurfactants have received attention due to their capacity to degrade hydrocarbons properties like biodegradability, ecological acceptability and low toxicity [7]. Biosurfactants also called green surfactants which play a key role in agriculture and these compounds (hydrophobic), which makes biosurfactants a good agent for cleaning up the environment [6]. These biosurfactants operate their work by emulsifying the non-aqueous phase liquid contaminants and increase their solubility. These features of biosurfactants facilitate contaminants export from the solid phase and allow the microorganisms adsorbed on the soil particles to access and remove the contaminant molecule [8–10].
They also have the capacity to generate a renewable source of energy from cheaper substrates [11]. Biosurfactants produced by microbial genera have been studied extensively for their role as potent surfactants, such as in the degradation of engine oil [12]. They also help in reducing the load of environment polluting agents [3]. Biosurfactants bioactive molecules are especially important due to their unique structural and biological active properties and applications [13]. Surfactin and iturin are already known to be efficient biosurfactants for degrading hydrocarbons containing engine oil [14]. The important properties that make these biosurfactants special molecules are biodegradability, lower toxicity, bioavailability, high foaming, high selectivity and specific activity at extreme temperature, pH and salinity, making them special active biomolecules [15–16].
Here, the objective of this study is to report a cost-effective solution of engine oil pollution via engine oil degradation by B. velezensis KLP2016 (Gram + ve, endospore forming; Accession number KY214239) bacterial strain efficiently and producing a biosurfactant in a suitable same medium. The biosurfactant was further purified and characterized, and engine degradation was investigated by GC-MS [17]. This study will be useful from both the environmental and the industrial point of view, and the outcomes of this study will be useful for cleaning environment via engine oil degradation and also will be helpful in reducing soil water contamination [18].
Production of biosurfactant by B. velezensis KLP2016 cells
A fresh loopful culture of B. velezensis KLP2016 was inoculated in 100 mL of Luria bertani broth and incubated at 200 rpm under shaking at 30ºC to get 1.0 OD of cells at 620 nm. Bacterial growth was monitored regularly and 1.0 OD cells obtained at 9 hrs incubation. For production of biosurfactants, 1000 mL of LB broth was prepared in which 4 % (v/v) of bacterial inoculum (1.0 O.D cells) was inoculated and the flasks were incubated for 72 h at 30ºC at 200 rpm. After incubation, the culture broth was centrifuged at 10,000 rpm for 10 min at 4ºC [18]. Biosurfactants containing supernatant/ cell free broth was collected for further experiments.
Measurement of emulsification index, surface tension and critical micelle concentration
Biosurfactant containing culture broth was evaluated by measuring the emulsification index (E24%) using various chosen organic hydrocarbon compounds (benzene, pentane, cyclohexane, xylene, n-hexane, toluene and engine oil) as the substrate. In a test tube, 1.5 mL of each hydrocarbon was added to 1.5 mL B. velezensis cell-free broth. This combination was mixed by using a vortex for 2 min, and the content was left undisturbed for 24 h. The percentage of the emulsification index (E24%) was calculated by using the following equation [12].
The surface tension of cell-free broth of B. velezensis strain was determined by the drop weight method at 25°С and 35°C temperatures; and Luria bertani broth and Minimal Salt medium (MSM) [19]. Cell-free broths of B. velezensis KLP2016 grown in Luria Bertani (LB) and Minimal Salt Medium (MSM) for 72 h incubation, were used to measure the surface tension. The uninoculated LB and MSM broth (g/l) (KH2PO4, 1.4; Na2HPO4, 2.2; (NH4) SO4, 3; MgSO4, 0.6; NaCl, 0.05; yeast extract, 1; CaCl2 0.02) was taken as negative control. Critical micelle concentration (cmc) is the concentration of biosurfactant above which micelle form and further no reduction in surface tension occurs. The surface tension (γ) and critical micelle concentration (cmc) was calculated by using the following equation [19];
Where γ0 is surface tension, n0 is number of drops and ρ0 is density of uninoculated broths, while γ is surface tension, n is number of drops and ρ is density of cell-free fermentation broth.
Purification and identification of active compound extracted from culture broth of B. velezensis KLP2016
Ammonium sulfate (NH4SO4) mediated protein precipitation and dialysis
The cell-free broth was introduced with 0-20, 20-40, 40-60, 60-80 and 80-100% saturation of NH4SO4 at 4°C, further mixed and kept overnight at 4°C. Thereafter, the precipitates were deposited after centrifugation at 12,000 rpm for 15 min. The precipitates were reconstituted in 1 mL of 20 mM sodium phosphate buffer at pH 7.5 and checked for emulsification activity against engine oil. One unit of emulsifying activity was explicated as the quantity of emulsifier that yielded an absorbance (600 nm) of 0.1 in the assay mixture [20].
Ion exchange chromatography
The DEAE cellulose packed glass column (height 10 cm; diameter 1.5 cm) was equilibrated with 20 mM sodium phosphate buffer (pH 7.5) after activation by 0.5 M NaOH. Five mL of dialyzed biosurfactant preparation (4.0 mg protein) was loaded on the matrix in the column [21]. Column was equilibrated with 20 mM sodium phosphate buffer (pH 7.5). Unbound proteins were eluted with low ionic strength buffer (sodium phosphate buffer; pH 7.5) at a flow rate of 1 mL/min and discarded. The bound biosurfactant molecules eluted with the stepwise gradient of 0.5 M NaCl, 1 M NaCl and 1.5 M NaCl in sodium phosphate buffer (pH 7.5; 20 mM), respectively [22]. Emulsification activity and A280 values were evaluated against the engine oil.
Size exclusion chromatography
Sephadex G-25 packed matrix was washed off with several column volume of 20 mM sodium phosphate buffer (pH 7.5). Pooled active fraction of the DEAE was loaded on the bed surface of Sephadex G-25 column and eluted with the sodium phosphate buffer (20 mM; pH 7.5) and fractions were collected [21]. Absorbance at 280 nm and emulsification activity was evaluated against the engine oil. Active fractions were further checked with UV-vis spectrophotometer and TLC, as detailed below.
TLC and UV-VIS spectrophotometry
The fractions obtained from size exclusion chromatography, were analysed and mixed on the basis of their OD. A solvent system of chloroform: methanol: water (39:15:3; v/v) was prepared, and 5 µl sample of mixed biosurfactant fractions was applied at the point of origin of the TLC plate [23]. Lipid moiety of the molecule was detected by TLC plate sprayed with water and thereafter kept for drying. The Rf values of the biosurfactant spot on the TLC plate were evaluated using the following formula and results recorded accordingly.
The purified biosurfactant was also analysed for ultraviolet absorbance spectrum [24] at range of 190-800 nm (UV-VIS Spectrophotometer, CARY, VARIAN).
High performance liquid chromatography analyses
The presence of biosurfactant in the purified molecule was confirmed by HPLC using an HPLC pump (Waters, USA) by a reverse phase column (Lichrosorb C18-5 µm; Merck, Germany) and 2998 photodiode assay detector [18]. The mobile phase contained acetonitrile (ACN): ammonium acetate (10 mM) in the ratio of 40: 60 (v/v) and mobile phase flow rate was adjusted at 2 mL / min. Biosurfactant sample 5 µl was injected each time and analysed at 254 nm wavelength with comparing standard biosurfactants, i.e., surfactin and iturin.
ESI‑MS of purified biosurfactant
A mass spectrometer (Q-TOF micro Waters 2795 UK) was used to find the molecular weight of the purified biosurfactant. The conditions for used MS were temperature source, 100οC; 3000 V in positive mode; capillary voltage, cone voltage, 30 V; current source, 80.0 A and capillary voltage of 7.0 V in positive mode [21]. About 20 μl of purified biosurfactant was injected into the MS and gently ionized with CH3OH and H2O (80:20) using electrospray (ESI) with flow rate of 1.0 mL/min. ESI-MS results were compared with the authentic surfactin biosurfactant molecule to identify the molecular mass of the purified biosurfactant of B. velezensis.
Hydrocarbon degradation activity of B. velezensis KLP2016
Biodegradation of engine oil (K 15W-40) by B. velezensis KLP2016 in a biometric system
For the biodegradation of engine oil, 5% (v/v) starter inoculum of 7 h of B. velezensis KLP2016 culture was inoculated in the 250 mL capacity sterilized flasks each containing 100 mL MSM and LB broth. Hydrocarbon substrate (K 15W-40 Engine oil) was added at 1% (v/v) concentration in each of the sterilized flasks. One test tube containing fresh KOH (10 mL; 0.05 M) was placed in each of the flasks, and these flasks were incubated at 30°C under shaking (100 rpm) from 5 to 20 days. Inoculated and uninoculated broths were observed for their absorbance (A600) and CO2 content at 5-day intervals up to 20 days. The CO2 gas trapped in the KOH solution was titrated by introducing 100 µl of barium chloride (w/v; saturated) and three drops of phenolphthalein with 0.05 M HCl until the appearance of the end point as the colourless solution. The difference in millilitres of HCl used to titrate KOH containing solution of control (placebo) and B. velezensis KLP2016 inoculated media was converted into ppm of fixed carbon dioxide as per method [17, 25]. Hydrocarbon degradation of engine oil facilitated by B. velezensis KLP2016 was also confirmed by Gas chromatography-mass spectrometry (GC-MS) analysis of the engine oil treated with bacterial cells.
Hydrocarbon analysis by GC-MS of K 15W-40 engine oil treated with B. velezensis KLP2016
In order to analyse the hydrocarbon products of engine oil broken down by B. velezensis KLP2016, the culture broth (5, 10, 15 and 20 days) was centrifuged at 10,000 rpm, at 4°C for 10 min. From the supernatant, the upper layer was collected, filtered with syringe filter (0.22 µm) and the filtrate was analysed using GC-MS to evaluate engine oil products. The GC-MS analyses were performed using an MS5973 spectrometer with a ULBON HR-1 column (25 mm x 50 mm), with thickness of 0.25 micron, 1 mL/min flow rate of the sample injected (5 µL) with the carrier gas helium, ion source temperature 230οC at 18.5 psi pressure and 20% split ratio [25]. Results were observed and recorded accordingly.
Statistical analysis: All methods are statistically analysed.
Emulsification index, surface tension and critical micelle concentration of biosurfactant containing cell-free broth of B. velezensis
An emulsification index of ≥30% was considered as significant emulsification activity. The reported results showed that B. velezensis cell-free broth showed E24 % marked 65.7%, 59.0%, 56.1%, 61.0%, 52.3%, 65.2% and 56.2% with benzene, pentane, cyclohexane, xylene, n-hexane, toluene and engine oil, respectively. The surface tension of biosurfactant containing cell-free broth at 35οC was reduced from 55 mN.m-1 to 40 mN.m-1 at 17.2 µg/mL (cmc) and surface tension at 25°C was reduced from 62 mN.m-1 to 48 mN.m-1 at 17.4 µg/mL of critical micelle concentration by using LB broth as a medium for cell-free broth production (Fig 1a). The surface tension at 35οC was reduced from 58 mN.m-1 to 43 mN.m-1 at 17.6 µg/mL (cmc) and at 25οC, surface tension was reduced from 65 mN.m-1 to 50 mN.m-1 at 18.1 µg/mL (cmc) of cell-free broth of B. velezensis grown in MSM broth (Fig. 1b).
Purification of biosurfactant by DEAE- cellulose and size exclusion chromatography
On the basis of emulsification activity against the engine oil, an ammonium sulphate cut in the range 20-40% showed 24.0±1.54 U/mL emulsification activity or ~60% E24%, was selected for further purification. A total of 15 fractions were collected (1.5 mL each) by elution with 0.5 M, 1 M and 1.5 M NaCl (Fig. 2a). Fractions that were eluted were checked for emulsification activity against engine oil, and the maximum activity was recorded in the case of fraction number 9 (33 U/mL). The active fractions from the DEAE column were collected and further loaded on Sephadex G-25 column for further purification. A total of 28 fractions were collected (1.5 mL each) after elution with sodium phosphate buffer. Emulsification activity against engine oil was observed in 9-17 fractions. The fractions (9-17) were checked separately then pooled for further investigations (Fig. 2b). The polled fractions of B. velezensis KLP2016 yielded absorbance maxima at 221 and 210 nm (Fig. 3c), which corresponded to the characteristic absorption of peptide bonds of surfactin. These results showed that the B. velezensis KLP2016 strain might be a producer of a biosurfactant belonging to the ‘iturin or surfactin family’, possessing emulsification activity against engine oil.
Identification of purified biosurfactant by TLC, HPLC and ESI-MS
A white spot was observed when the TLC plate was sprayed with water, indicating the lipophilic nature of the compound (Fig. 3d). Thus, a peptide without free amino groups (cyclic structure) might be present, as assumed after TLC results. The standard preparation of surfactin also showed a value 0.94 Rf, which was similar to the value of 0.90 Rf recorded for the biosurfactant, indicating the presence of a surfactin-like biosurfactant. The biosurfactant of B. velezensis KLP2016 showed retention time (RT) 1.130 min (Fig. 4c), while the authentic surfactin and Iturin A showed a RT 1.27 min and 6.066 min respectively (Fig. 4a & 4b). Thus, it appeared that the purified biosurfactant appeared to be a ‘surfactin-like’ biosurfactant molecule. The MS/ MS values of the peak (1058.60, 1044.62 and 1030.63 m/z) of the purified biosurfactant of B. velezensis KLP2016, were found similar to that present in commercial grade surfactin (Fig. 5a &b). On the basis of literature analysis [26], it was safely concluded that the purified biosurfactant produced by B. velezensis KLP2016 was identified as surfactin with Mr (~1.0 Dalton) by ESI-MS spectral analysis.
Biodegradation of Engine oil (K 15W-40) using CO2 stoichiometry analysis in a biometric system
Bacterium-inoculated MSM and LB broth gave optical density 1.762 and 2.901, respectively, after 15 days of incubation (Table 1). Engine oil degradation was confirmed by the GC-MS analysis, which indicated disappearance of prominent peaks detected in engine oil (positive control). Results showed that B. velezensis KLP2016 cells degrade engine oil efficiently after 15 days of incubation when grown in LB broth rather than in MSM broth (Fig. 6c). The maximum carbon dioxide content trapped in the KOH solution after 15 days of incubation in LB and MSM broth showed values of 1980×10-2 ppm and 825×10-2 ppm, respectively (Table 1). Thus, LB broth was found to be a better nutrient source for bacterial growth in context to engine oil degradation because a higher amount of CO2 was released then got trapped in KOH.
- 2KOH + CO2 = K2CO3 + H2O
- K2CO3+ 2 HCl = H2O + CO2+ 2KCl
One molecule of K2CO3 contains one molecule or 44 g of CO2 . To calculate the CO2 trapped by the KOH solution, K2CO3 was titrated with HCl. As per reaction, it was observed that 2 molecules of HCl are required to neutralize one molecule of K2CO3. CO2 trapped after the 5th, 10th, 15th and 20th days of incubation in LB broth was observed as 880× 10-2, 1320× 10-2, 1980× 10-2 and 1969× 10-2 ppm, respectively.
Hydrocarbon analysis of engine oil (K 15W-40) by GC-MS
The uninoculated LB-broth containing engine oil exhibited more peaks than B. velezensis KLP2016 inoculated/ treated engine oil, after 5 and 15 days treatment (Fig. 6b& 6c). Engine oil was broken down into methylsulfonyl, borane, pyridine, piperazine, octanamide, ethylene, diethyl propyl and benzenenamine, as can be seen in Fig. 6, on the basis of variation in the peaks generated by the GC-MS.
Due to the hazardous effects of engine oil and associated hydrocarbons, it is urgent need to find methods of controlling and biodegrading them to safeguard the environment and human welfare. Biosurfactants have been successfully used in cleaning up polluted areas at low cost and high efficiency [27]. Biosurfactant-mediated remediation of hydrocarbons containing engine oil is an eco-friendly approach, which is able to transform toxic substances into nontoxic compounds, and this technique is an effective technology for the treatment of soil and water contamination [28]. In earlier reports, many methods for screening biosurfactants have been discussed, such as the haemolytic assay, BATH assay, oil spreading, drop collapse and surface tension measurement [29]. In earlier reports, these methods have been noted as screening methods, excluding surface tension measurement which is the key parameter for detecting surfactant activity [30]. Oil spreading is a widely used and effective biosurfactant screening method to detect the potential biosurfactant-producing microbes in the mixtures [31]. This method is a rapid detection method, which can be applied when the activity/quantity of biosurfactant is low in the respective fermentation medium [32].
In our study, the bacterium B. velezensis KLP2016 cell-free broth showed excellent biosurfactant properties, as was evident on the basis of data of emulsification activity, surface tension measurement and critical micelle concentration. All these methods strongly detected the biosurfactant nature of B. velezensis KLP2016, as it reduced surface tension up to 40 mN.m− 1 in an in vitro assay at 35οC after using cell-free broth of B. velezensis grown in LB broth. The critical micelle concentrations (cmc) of cell-free broth of B. velezensis grown in LB at 35οC and 25οC were 17.2 µg/mL and 17.4 µg/mL, respectively while in MSM broth at 35οC and 25 οC were 17.6 µg/mL and 18.1 µg/mL respectively. E24 % of the cell-free broth of B. velezensis was observed as 65.7%, 59.0%, 56.1%, 61.0%, 52.3%, 65.2% and 56.2% against benzene, pentane, cyclohexane, xylene, n-hexane, toluene and engine oil, respectively.
Bacterial biosurfactants are generally peptides containing a small lipidic moiety and gel permeation, hydrophobic interaction and ion exchange methods are generally employed for the purification from cell-free fermentation broth of B. velezensis KLP2016. In previous studies, ion exchange chromatography has been reported for the purification of biosurfactants [33]. Another lipopeptide-like biosurfactant was purified by using DEAE anion exchanger chromatography, followed by an HPLC [34] or a HiTrap Q system [35]. In the earlier reports, molecular sieve chromatography was also used to resolve the low molecular mass biosurfactant by using Sephadex as the matrices [35]. Ion exchange chromatography is also very effective in eliminating coloured contaminating molecules from the biosurfactant fraction, and this technique resolved the antibiotic biosurfactant peak from other chromatographic peaks [24].
UV-Visible spectrophotometry (210 nm) and thin layer chromatography (Rf 0.90) confirmed the purity of a biosurfactant molecule. A dense white spot in the TLC of the purified biosurfactant molecule at Rf 0.90 confirmed the presence of a lipid moiety in the purified molecule. In our study, the purity check and confirmation of the biosurfactant molecule was further detected by HPLC. Detection of a prominent single peak during HPLC indicated the purity of the surfactin type biosurfactant produced by B. velezensis KLP2016. Furthermore, the ESI-MS data confirm the Mr ~ 1.0 kDa of the purified surfactin-type biosurfactant. The purified surfactin biosurfactant molecule from this strain (B. velezensis KLP2016) was found to be a strong degrader of engine oil, as compared to previous reports [17].
The adaptation of microbial communities to hydrocarbons increases their hydrocarbon degradation rates [36]. In the present study, B. velezensis KLP2016 growing cells were observed to be a potent degrader of engine oil. In this approach, engine oil degradation occur by the B. velezensis KLP2016 bacterium strain in which engine oil was used by the bacterium as a substrate for its growth on the basis of previous studies and a surfactin biosurfactant was released by the B. velezensis KLP2016 bacterium in the production medium [37]. The highest value of CO2 was recorded to be 1980 × 10− 2 ppm, which was trapped in the KOH solution after 15 days of incubation of B. velezensis grown in LB broth containing engine oil (1%). Engine oil degradation in our study was found to be ~ 1000 times higher than the previously reported value of 656 µmol [17], which shows the efficiency of the B. velezensis strain.
The high efficiency of the B. velezensis strain for engine oil degradation may be due to the high production of the biosurfactant molecule, which degrades by binding hydrophobically to the engine oil. LB broth appeared to be the best nutrient source to sustain bacterial growth as well as providing an efficient adjustment of engine oil for the degradation, which was qualitatively and quantitatively analysed by GC-MS on the 5th and 15th days. Engine oil degradation by B. velezensis KLP2016 was very efficient, and it indicated a potential for using B. velezensis KLP2016 in the treatment of oil-spills. This approach to hydrocarbon degradation, which achieves a valuable compound, surfactin, as a by-product, can be used to solve problems such as oil spills and soil water contamination.
This bacterium is, therefore, reported as an engine oil degrader which is highly efficient in LB medium. ~75% more engine oil was degraded by B. velezensis KLP2016 cells using LB medium than MSM medium. The released bioactive biosurfactant is considered to be a surfactin-like molecule after the purification and characterization studies. Thus, B. velezensis KLP2016 has been proved to be an efficient engine oil degrader which also generates valuable compounds as a by-product, such as surfactin. Therefore, use of B. velezensis strain can be a better solution organism for engine oil degradation than the conventional one.
The bacterium Bacillus velezensis KLP2016 (Accession number KY214239) showed excellent biosurfactant properties checked by surface tension and critical micelle concentration measurement. Surfactin type biosurfactant was found after the characterization by ESI-MS and HPLC. In this study, hydrocarbon(s) containing engine oil was degraded by the Bacillus velezensis KLP2016 (Accession number KY214239) strain where hydrocarbons probably might be used as a carbon source for bacterial growth and additionally, a biosurfactant released by the bacterium may be used further for its wide applications. This biosurfactant-based approach to engine oil degradation is highly promising and may play a key role in the reduction of soil and water pollution in the near future.
Acknowledgements
Authors are thankful to Dr. M. N. Jha for kind support and for provide technical assistance. They are also grateful to the Department of Biotechnology, HPU, Shimla and Department of Microbiology, CBS&H, Dr. Rajendra Prasad Central Agricultural University, Pusa, Samastipur, India for providing laboratory facilities.
Author contribution
SSK, KRM, RD, AS designed the experiments while KRM, RD, KS and SK performed the experiments in the laboratory. KRM wrote paper and SD, AKM and OLF helped in writing and reviewing the research paper and provided valuable suggestions.
Funding
This work was supported by DBT, Govt. of India, by providing a DBT-Junior Research Fellowship grant [Letter No. DBT-JRF/2011-12/270] awarded to one author (KRM).
Research involving animal and human rights
There are no human participants and/or animals involved in this study.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Availability of data and material
All data generated or analyzed during this present study are included herewith this article.
Conflict of interest
The authors declare that they have no competing interests.
- Akintunde WO, Olugbenga OA, Olufemi OO. Some adverse effects of used engine oil (common waste pollutant) on reproduction of male sprague dawley rats. Maced J Med Sci 2015; 3:46-51
- Gontikaki E, Potts LD, Anderson JA, and Witte U. Hydrocarbon-degrading bacteria in deep-water subarctic sediments (Faroe-Shetland Channel). J Appl Microbiol. 2015; 125:1040-1053.doi: 10.1111/jam.14030. Epub 2018 Jul 24.
- Rafael VD. Environmental impact of used motor oil. Sci Total Environ. 1989; 79: 1-23.
- Sujata SJ, Sanket JJ, Geetha SJ. Lipopeptide production by Bacillus subtilis R1 and its possible applications. Braz J Microbiol. 2016; 47:955-964.doi: 13005/msri/070133
- Moghimi H, Heidary Tabar R, Hamedi J. Assessing the biodegradation of polycyclic aromatic hydrocarbons and laccase production by new fungus Trematophoma UTMC 5003. World J Microbiol Biotechnol 2017; 33:136. doi:10.1007/s11274-017-2304-8.
- Marchut-Mikolajczyk O, Drożdżyński P, Pietrzyk D et al.Biosurfactant production and hydrocarbon degradation activity of endophytic bacteria isolated from Chelidonium majus Microb Cell Fact 2018; 17: 171. doi.org/10.1186/s12934-018-1017-5
- Das P, Yang XP, Ma LZ. Analysis of biosurfactants from industrially viable Pseudomonasstrain isolated from crude oil suggests how rhamnolipids congeners affect emulsification property and antimicrobial activity. Front Microbiol. 2014. org/10.3389/fmicb.2014.00696 .
- Bustamante M, Durán N, Diez MC. Biosurfactants are useful tools for the bioremediation of contaminated soil: a review. J Soil Sci Nutr. 2012; 12: 667–87. org/10.4067/S0718-95162012005000024.
- Khan MSA, Singh B, Cameotra SS. Biological applications of biosurfactants and strategies to potentiate commercial production biosurfactants. Prod Util Processes Technol Econ. 2015;1: 269–95. org/10.1201/b17599-16y.
- Hausmann R, Syldatk C. Types and classification of microbial surfactants, biosurfactants. Prod Util Processes Technol Econ. 2015. org/10.1201/b17599-3.
- Mohan PK, Mukhla G, Yanful EK. Biokinetics of biodegradation of surfactants under aerobic, anoxic and anaerobic conditions. Water res 2006; 40: 533-540. doi: 1016/j.watres.2005.11.030
- Lai CC, Huang YC, Wei YH, Chang JS. Biosurfactant-enhanced removal of total petroleum hydrocarbons from contaminated soil. J Hazard Mater 2009; 167: 609-614. doi:10.1016/j.jhazmat.2009.01.017
- Cameotra SS and Makkar RS. (2004). Recent applications of biosurfactants as biological and immunological molecules. Curr OpinionMicrobiol. 2004; 7:262–266. doi: 1016/j.mib.2004.04.006
- Banat IM, Franzetti A, Gandolfi I, Bestetti G, Martinotti MG, Fracchia L, Smyth TJ, Marchant R. Microbial biosurfactants production, applications and future potential. Appl Microbiol 2010; 87: 427–444. doi: 10.1007/s00253-010-2589-0
- Desai JD and Banat IM. Microbial production of surfactants and their commercial potentials. Microbiol Molecular Biol Rev. 1997; 61: 47-64. PMCID: PMC232600
- Faria et al. Purification and structural characterization of fengycin homologues produced by Bacillus subtilis LSFM-05 grown on raw glycerol. J Ind Microbiol Biotechnol 2011; 38:863–871. doi: 1007/s10295-011-0980-1
- Pathak H, Bhatnagar K, Jaroli P. Serratia-The 4T Engine oil degrader. Sci Rep 2012; 1: 117. doi: 10.4172/scientificreports.117
- Meena KR, Sharma A, Kumar R, Kanwar SS. Two factor at a time approach by response surface methodology to aggrandize the Bacillus subtilis KLP2015 surfactin lipopeptide to use as antifungal agent. J King Saud Univ-Sci 2018; 32:337-34. doi: 10.1016/j.jksus.2018.05.025
- Chauhan S, Chauhan MS, Sharma P, Rana DS, Umar A. Physico-chemical studies of oppositely charged protein–surfactant system in aqueous solutions: sodium dodecyl sulphate (SDS)–lysozyme. Fluid Phase Equilibr 2013; 337: 39–46.doi: 1016/j.fluid.2012.09.003
- Pathak KV and Keharia HK. Application of extracellular lipopeptide biosurfactant produced by endophytic Bacillus subtilis K1 isolated from aerial roots of banyan (Ficus benghalensis) in microbially enhanced oil recovery (MEOR). 3 Biotech 2014; 4: 41-48.doi:10.1007/s13205-013-0119-3
- Meena KR, Sharma A, Shamsher SS. (2019). Antitumoral and antimicrobial activity of surfactin extracted from Bacillus subtilis Int J Peptide Res Therapeutics 2019; 26:423–433. doi:10.1007/s10989-019-09848-w
- Moyne AL, Shelby R, Tuzun S, vleveland TE. Bacillomycin D: an Iturin with antifungal activity against Aspergillus flavus. J Appl Microbiol 2001; 90: 622–629.
- Xiao-Hong C, Zhen-Yu L, Chun-Ling W, Ping C, Wen-Yan Y, Mei-Fang L. Guo-Wei H. Purification and antitumour activity of a lipopeptide biosurfactant produced by Bacillus natto TK-1. Biotechnology and Applied Biochemistry 2009; 52: 97–106.
- Bechard J, Eastwell K, Sholberg P, Mazza G, Skura B. (1998). Isolation and partial chemical characterization of an antimicrobial peptide produced by a strain of Bacillus subtilis. J Agri Food Chem 46: 5355-5361. doi: 1021/jf9803987
- Pathak H and Bhatnagar K. Alcaligenes-The 4T engine oil degrader. J Bioremed Biodegrad 2011. 2:1-4.doi: 10.4172/2155-6199.1000124
- You J, Yang SZ, Mu BZ. Structural characterization of lipopeptides from Enterobacter strain N18 reveals production of surfactin homologues. Eur J Lipid SciTechnol 2015; 117: 890–898.doi:10.1002/ejlt.201400386
- Bidoia ED, Montagnolli RN, Lopes PRM. Microbial biodegradation potential of hydrocarbons evaluated by colorimetric technique: A case study. In: Current research, technology and education topics in applied microbiology and microbial biotechnology, Mendez-Vilas, A. (Ed.). FORMATEX, Badajoz, 2010; Spain: 1277-1288.
- Milic JS, Beskoski VP, Ilic MV, Ali SAM, Gojgic-Cvijovic GD, Vrvic MM. Bioremediation of soil heavily contaminated with crude oil and its products: Composition of the microbial consortium. J Serbian ChemSoc 2009; 74: 455-460.doi: 10.2298/JSC0904455M
- Afsar S, Lotfabad TB, Roostaazad R, Najafabadi AR, Noghabi AK. Comparative approach for detection of biosurfactant-producing bacteria isolated from Ahvaz petroleum excavation areas in south of Iran. Ann Microbiol 2008; 58: 555-560.doi: 10.1007/BF03175557
- Thavasi R, Sharma S, Jayalakshmi S. Evaluation of screening methods for the isolation of biosurfactant producing marine bacteria. J Petro & Environ Biotechnol 2011. doi:10.4172/2157-7463.S1-001
- Plaza GA, Zjawiony I, Banat IM. Use of different methods for detection of thermophilic biosurfactants producing bacteria from hydrocarbon contaminated bio remediated soils. J Petrol Sci Eng 2006; 50: 71‑77.doi:1016/j.petrol.2005.10.005
- Youssef NH, Duncan KE, Nagle DP, Savage KN, Knapp RM, McInerney MJ. Comparison of methods to detect biosurfactant production by diverse microorganisms. J Microbiol Meth 2004; 56: 339-347. doi: 1016/j.mimet.2003.11.001
- Mandal SM, Sharma S, Pinnaka AK, Kumari A, Korpole S. Isolation and characterization of diverse antimicrobial lipopeptides produced by Citrobacter and Enterobacter. BMC Microbiol 2013; 13: 152. doi: 10.1186/1471-2180-13-152
- Kim PI, Bai H, Bai D, Chae H, Chung S, Kim Y, Park R, Chi YT. Purification and characterization of a lipopeptide produced by Bacillus thuringiensis J Appl Microbiol 2004; 97: 942–949.doi: 10.1111/j.1365-2672.2004.02356.x
- Moyne AL, Shelby R, Tuzun S, vleveland TE. Bacillomycin D: an iturin with antifungal activity against Aspergillus flavus. J Appl Microbiol 2001; 90: 622–629.doi: 1046/j.1365-2672.2001.01290.x
- Leahy JG and Colwell Microbial degradation of hydrocarbons in the environment. Microbiol Rev 1990; 54: 305–315.
- Parthipan P, Preetham E, Machuca LL, Rahman PKSM, Murugan K, Rajasekar Biosurfactant and degradative enzymes mediated crude oil degradation by bacterium Bacillus subtilis A1. Front Microbiol 2017; 8: 193.
Table 1: Growth of B. velezensis KLP2016 in MSM and LB broth containing engine oil at 30°C in a shake flask culture
|
Incubation time (Days) |
(MSM broth+ Engine oil+ B. velezensis) |
(LB broth+ Engine oil+ B. velezensis) |
||
|
OD600 nm
|
Fixed carbon dioxide (ppm) |
OD600 nm
|
Fixed carbon dioxide (ppm) |
|
|
0 |
0.665 |
- |
0.742 |
- |
|
5 |
1.312 |
550×10-2 |
2.085 |
880×10-2 |
|
10 |
1.421 |
770×10-2 |
2.402 |
1320×10-2 |
|
15 |
1.762 |
825×10-2 |
2.901 |
1980×10-2 |
|
20 |
1.667 |
814×10-2 |
1.720 |
1969×10-2 |