Screening of different Serratia marcescens strains for biosurfactant production
Biosurfactant production screening was carried out in 250 mL Erlenmeyer flasks with 100 mL of production medium containing 1% peptone and 6% clean Mix frying oil withmedia were adjusted to pH 7.0 and autoclaved at 121 °C for 15 min. Then, they were inoculated at 2% inoculum size and incubated for 13 days at 28 °C, under orbital agitation (150 rpm)
Inoculum preparation of 5 Serratia marcescens strains (MN2 (KX601268,), MN3(KX601278), MN4 (KX601721), MN5(KX601170) , N2 Bioproject ID PRJNA525074, Biosample ID SAMN11041520, and WGS accession SPSG00000000 Loop from plate in 10 ml Luria Bertani broth for 1 hour OD600 adjustment then 2 ml from inoculum into flask. Surface tension of the collected cell-free metabolic cultures were obtained by centrifugation at 12,000×g for 20 min, and membrane filtration of culture media 0.22mm. Analyses were performed at 25 °C in a Kruss tensiometer (K20Kruss GmbH, Germany) using the Du Nouy ring method with Milli-Q water with surface tension of 72mN/m was used to calibrate the tensiometer (Araújo et al., 2019). Oil spreading techniques was performed as follows: 50 ml distilled water to petri dish followed by 100ml of vegetable oil to surface of water then 10ml of cell free biosurfactant was added slowly and detected in light after 30 s (Ghasemi, Moosavi-Nasab, Setoodeh, Mesbahi, & Yousefi, 2019).
Optimization of production using factorial design
Serratia marcescens N2 was the strain with the lowest surface tension was used in the upcoming experiments, this strain was previously deposited at DDBJ/ENA/GenBank under Bioproject ID PRJNA525074, Biosample ID SAMN11041520, and WGS accession SPSG00000000 with the annotated genome of S. marcescens N2 deposited in the PATRIC database under genome number 615.1488 (Elkenawy et al 2021).. Biosurfactant production experimental design was done using Minitab 18 software (USA) for 2 factors, 3 levels. The design of the experiment is represented in Table S1. The cultivation was carried out in 250 mL Erlenmeyer flasks with 100 mL working volume. The production medium consisted of 1% peptone and carbon sources of 5, 10 and 20 % vol/vol of pre-used frying oil from local restaurant in Heliopolis area, Cairo. Cultivation media were adjusted to pH 7.0 and autoclaved at 121 °C for 15 min. Flasks were inoculated with inoculum sizes 2, 4 and 8% and incubated for 6 days at 28 °C, under orbital agitation (150 rpm). Surface tension was estimated as previously described and wet biosurfactant weight was calculated as the biosurfactant weight at the end of the production process in g/l.
Extraction and characterization of S. marcescensN2 biosurfactant
The biosurfactant produced by S. marcescens N2 after 6 days fermentation was isolated from cell-free metabolic liquid obtained by centrifuging (12,000×g for 20 min) the culture. The metabolic liquid was subjected to precipitation using conc. HCl to get pH 2.0 and kept at 4∘ C overnight. It was then centrifuged at 15,000×g for 15 min and the cell-free metabolic supernatant was collected and centrifuged at 5000×g for 15 min. The supernatant obtained was discarded and the crude biosurfactant was extracted three times with a chloroform-ethanol (2 : 1 v/v) mixture with vigorous shaking, The precipitate was collected, oven dried and used for analysis (Eraqi, Yassin, Ali, & Amin, 2016).
Fourier Transform Infrared Spectroscopy (FT-IR)
The identification of functional groups in the isolated biosurfactant was carried out using Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). The obtained biosurfactant in its dry form was scanned within the range of 4000–400 cm−1 using BRUKER VERTEX 70 device at NCRRT. The spectrum of the biosurfactant under study was compared to literature.
Emulsification Index
1ml of cell free metabolic liquid 1 ml used frying oil – paraffin oil control tween 80 and water vortex 2 min and leave on bench for 24 hrs measure height of emulsion layer divided by total height(Tripathi, Gaur, Dhiman, Gautam, & Manickam, 2020).
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)
Zeta potential
The electrokinetic of potential zeta of biosurfactant aqueous solution was analyzed using PSSNICOMP Zeta Potential/Particle Sizer 380ZLS (PSS-NICOMP, Santa Barbara, CA, USA)with 2 mg of crude biosurfactant dissolved in 1mL of water (Araújo et al., 2019).
Biosurfactant identification of serrewettin gene
The WGS of Serratia marcescens N2 was used to identify the biosurfactant gene and the results were compared to other genes deposited in NCBI database repository. Data was represented as identity percentage.
Oil displacement activity (ODA) and Critical Micelle Concentration (CMC) of biosurfactant
ODA was performed by adding 10 µl of cell free broth on 100 µl of engine oil that was added to a petri dish containing 40 ml distilled water. The zone of displacement was recorded as positive. CMC of biosurfactant was determined using crude biosurfactant. Different concentrations (0.2-4 mg/mL) were prepared and surface tension was measured for each concentration. The value of CMC was obtained from graph (Araújo et al., 2019)
Gamma radiation for biosurfactant biorecovery and assessment of the structural and functional changes
At the end of cultivation period, the content was divided into four equal portions in clean sterile containers for gamma irradiation to test effect gamma radiation on biosurfactant biorecovery and yield. Irradiation process was carried out in Cobalt-60 (Co60) 220 gamma cell, Canada Co. Ltd. located at the National Centre for Radiation Research and Technology (NCRRT), Atomic Energy Authority, Cairo, Egypt at doses 500, 1000 and 2000 Gy. The dose rate was 1.119 kGy/ h at the time of experiment. At the end of the gamma irradiation process, the culture filtrate was collected and biosurfactant extracted as previously described. Surface tension, emulsification index, FTIR and Zeta potential were performed as previously described. Gravimetric analysis was performed by obtaining the dry weight for the biosurfactants after oven drying.
Oil valorization assessment
The whole content of 300 ml flask with 1% peptone, 20% oil content and 8% inoculum size cultivated for 13 days at 150 rpm and 28⁰C. The remaining oil content at the end of the experiment was measured to evaluate valorization of using the used frying oil into produced biosurfactant. The dry cell weight was measured to assess bacterial ability to valorize oil into biosurfactant. The consumed oil was divided by the initial oil to obtain the valorization percentage.
Application of biosurfactant as washing detergent
Washing experiment
Pieces of dry cotton cloth were cut into 2x2 cm pieces and each piece was stained with 0.3mL used frying oil. The pieces were left for 15 days to stabilize the stain as a challenge for stain removal, and their precise weights were recorded before staining, after staining and after washing.
The stained cotton cloths were washed using biosurfacatant released from cells after culture exposure to 500, 1000 and 2000 Gy. The results were compared to non-irradiated biosurfactant producing culture at 1 g/litre tap water under both static and stirring in water bath at 50rpm and 30⁰C and 60 ⁰C for 1 hour. After washing, the pieces were rinsed in water for half an hour, and dried at ambient temperature to a constant weight. The removal percentage of each stain was calculated using the precise weights of the pieces before and after washing (Khaje Bafghi & Fazaelipoor, 2012; Tripathi et al., 2020).
![](data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAN0AAABGCAYAAABIZZGaAAALOklEQVR4nO2dz2sbx/vH3/rwuQazUo4hB60KLS0Y2rEMxc2tWje51pUwPQRaCFJzaUt6WCWn2gEthUJpXcunUtJIxT2UUKWVDjZ4FYOtbVjRhhziXUoIOe3WDf0D5nvQdwatflmWpXWUz/OCPXh3dmZ2d947zzzPo3WEc85BEERo/Oe0O0AQ/2uQ6AgiZEh0BBEyJDqCCBkSHUGEDImOIEKGREcQIUOiI4iQIdERRMiQ6AgiZEh0BBEyJDqCCBkSHUGEDImOIEKGREccG9/3kcvlEI1GEYlEEI1Gkc/nx97O4uIiIpFIYEskErh79+7Y2woTEh1xLFzXxUsvvYS///4blmXB8zyoqoqZmZmxt/XDDz8AACqVCjjn8DwPqVQK77///tjbChMSHXEscrkckskkyuUy4vE4YrEYlpaWcP78+bG39ezZMwDA/Pw8ACAWi+HChQtjbyds/nvaHSCmh3q9jmq1Ctu2A/s/++yzibS3v78PTdMQi8UAAHfv3kUul8Pa2tpE2gsLEh0xNLdv3wZjDLOzs6G0t7Ozg2q1ikgkAgBQFAW3bt3CxYsXQ2l/UpB5SQxNrVbD0tLS0OXr9XqXI6RzW1xcHNhe53ruq6++GselnCo00xFD4zjOsdZuCwsLGPW7V77vw3EcvPzyywBa67mrV6/irbfeGqm+5wma6Yjnkr29Paiqing8Lvf9+uuvUBTlFHs1Hkh0xNBomobvvvsOvu8DaDk2otHoRNq6d+8eGGPy73K5jJs3byKbzU6kvVDhBDEktm1zTdM4AA6AK4rCdV2fSDuKosh2AHDGGF9fXx97W6dBhHP62Oxp47ouvvjiC1y4cAGZTObY5+fzeczMzEzMdU+MlxOZl4ZhdHmjMpkMms3muPr3wtNsNpFKpXDlyhUpuHw+L9OrOlOeXNdFNBqVJh4ArK6u4vz585ibmwu179OIYRgwDKNrf7PZlGln0WgU5XI5cFykvolxfqK0t5NOlYwxaWI4jsPT6TRXFIU7jnOsOkzTPGlXppLOa7dtm6uqyj3P45VKhauqGiivaRovlUo968pms7xQKEy0v9OK4zicMcYB9LxHjDGezWY555yXSiUOIPBcdF3njDHueZ40f0e91ycWnaqqvFKpyL89z+t7Yb2oVCocAPc876RdmTp6iWp9fT1w79rfi6Zpck3T+tbnOA5XFOV/8l4OwnEcns1mued5XNO0rrFp2zYHEJgoVFWVIuScc0VRAi+7bDbb9eyG5UTmpeu6gVgK0IqnaJqG+/fvy32dWelias7n87h06RIA4OzZs4hEInBdd+A5zWYTiURCmlLCFGs3GdpNgWg0CsMwutZKhmEgkUjIAK3v+33r7gzgdpoaom6xr90kFGXazUHBL7/8gnQ6Hdgn8g17cfnyZRQKhb7H4/E4otEo9vb2+pZ5EWk2m3Kc5HI5AJDPtlgsIh6PY21tTaaTdVKtVgEgEJ5gjMmxWK/XcXh4iHPnzsnjs7OzcBxnpP6eSHT7+/tdsRSBqqoAWgNUDNpHjx6hUqng5s2bAFprkUKhgHQ6Dd6adRGPxweeUywWUavVYFkW8vk83nvvPei6HgjaLi4uotFowHEcPHr0CBsbG3j99dfl8Vwuh83NTdRqNXieh2q1iocPHwbqNgwD77zzDiqVinwondfjeR4qlYpcey0vLyObzQayJsT19XrgjUajK9isaRo2Njbg+z7q9bp0mxuGgVQqdWQKViKRwJ9//tn3+FEZIiLlapqYnZ3F9vY2FEXB8vIyACCVSqFUKuHKlSsj1dk+Xnrx6quvjlQvgJOt6bLZbGAKFgCQ7t1CoSBNItu2eTqdDphImqZ1uYKPOsc0zS6bW1AqlbpMLFVVZVnTNOVxx3F4oVAIlO+sW5Rv79sgs0KYywJd1/uuV/tdg67rHABXVZXbts0dx5HrPHF96GPC9zKfxgHa3PfPy9aJuDeFQqHvurfX/SkUCl31tY/BXuNN7BuFE6WBNRoNfPrpp4F9wuvz9ttvAwA2NzdhWRYikQgYY1haWsIHH3wgy1er1S6T6ahzdnd3wRjDwsJCV59+/vlnZDIZObMIE/iVV14B0MpqODw8xNmzZ6GqKlKpFCzLkuU7697d3Q2Yppubm7h27VrfeyJMbdd1MTMzA8dxevZzEKurq1hdXZV/ZzIZXLt2DbFYDLlcDtvb2zhz5gwYY3j33Xd7Whrjhk9BZCmTyWBnZwcbGxsnNrH/+uuvgcefPHkyct0ji873fViWhWQyGdiXy+Wg67ocCJZlwTTNngOvXq8DQJfJNOgcALh//37fxNt//vknYBroug7GmBTV77//jkKh0Dem1Vn35uZm4MViWRa+/PLLnucCrXWBoih4+vQpfvrpJ2kW94IxhgcPHgwUZb1eh+M4KJfLaDabSCaT8n4lk0k8ffr0WKIbxnycBoH1QrzwGWP46KOPutz+/XjttdcAtF6U4l42Gg2kUikAkC/sdqH98ccfgYyZ4zDymm5vbw+Kosg1WLlcxvz8PJLJJD755BNZTlVVPHjwAEDrotrjeLu7u1BVFb7vo1gsolgsHnkO0Mo+f/PNN/v2TThxRH3t8at4PC6P+74PwzBkOVG3eAhAS2Tnzp1DPp+H7/uBvtXr9Z7B7GQyia+//lq214+5uTk8fvy473EA+Pjjj6XI//3334FlAeDg4CDQ/074/6+dB23TSL1ex87ODtbW1vDNN9/AsizpVGnH9/2uWezixYtQFAW6rgNopbdZliWtq1gshnQ6jevXr8P3fbiui2+//bbLyhuaUWxSz/NkzENs/dJ0TNPkqqrKNUq7rS1c3ACkS/eoc4R7t59bvFKpcEVR5Hqos1+O48hUJmH/i7p61c0Y44qicNu2Oec8sKZKp9M945GFQoEzxo68j71CBu2sr693rZlFXzzP61q7nkbIwDTNibVn2zY3TTOw9brf4rmJtbeIs6HNt6DruhxTYry2p7CJsSLGXHsYjPPWmE+n04FxMyovTO6leBF0DkJ0xF/CQNO0odtkjHU9YM5b16Oqalc9QvSdcSPOww+OD3JojYNsNitFoGnawOD2NPHCiE4MADFIRQZCL+/qpBBvQzErDoPIQDnOOb0olUpDza7jhDE28UTkTlGvr6+P7DV8XnhhftqzsLCAbDYLxpj0eqZSKXz++eehtD83N4f5+XlcvXr1WJ8zmJ2dRa1WQ7FYHHrh30k+n8fjx4/RaDRGOn8UisUiPvzwQ8RisYEB/ZPQbDahKErA0fTs2TMZA55aTlv1xPQh0qk4n1xckPPWrCZyU03T5LquB9bX0wp9roE4Njdu3MD169ePLOf7Ph4+fDiwzJkzZ/paBtvb2zg8PMTKyor8fMOdO3dC+zDSpCDREcei2Wyi0WhgeXlZxlm3trZ6xj2fPHmClZWVgfW98cYbfUVUq9Vw584daV4ahoHLly/j4ODghFdxutCPWIlj0Zn8fXBwgEQigd9++22s7biuC1VV4Xle4LuXly5dmtpYooBmOmJohKOnXWCGYWBra2vsbe3v7wcyiQDg+++/n34nCkh0xJCIFL9bt27Jfa7rYmtrayLm3s7ODmKxmDRhb9++jR9//BGlUmnsbYXNCxMyICbLjRs3kEwmce/ePblvf38fAMb+n3Sazab8LdvKygpWVlagKAps2x7pGzLPG7SmI4iQoZmOIEKGREcQIUOiI4iQIdERRMiQ6AgiZEh0BBEyJDqCCBkSHUGEDImOIEKGREcQIUOiI4iQIdERRMiQ6AgiZEh0BBEyJDqCCBkSHUGEDImOIEKGREcQIUOiI4iQIdERRMiQ6AgiZEh0BBEyJDqCCJn/A0mCqnB+f4cEAAAAAElFTkSuQmCC)
where A is the weight of a soiled cloth, B is the weight of a white cloth, and C is the weight of a soiled cloth after washing (Fei et al., 2020)
Optical microscopy
Optical images of clean, soiled and soiled clot patches washed woth 500 Gy assisted biosurfactant at 60oC under strring conditions were captured using AX10 Zeiss light microscope coupled with Axiocam 105 color (Germany) at NCRRT. Images representing the fabric threading were captured at 25X and 100X magnification.
Skin irritation test
Acute dermal irritation test was performed Co. based on OECD/OCDE404 method after the approval of Research Ethics Committee at the National research Center or Research and Technology (REC-NCRRT) with serial number 47 A/21, the result was expressed in terms of primary irritation index (PII). Irritation scores for erythema, eschar and edema formation at 1, 24, 48 and 72 hr after patch removal were summed up and divided by the number of observations, to obtain the individual PII. For the calculation of PII, all individual PII’s were summed up and divided by the number of animals used during the test. The detailed information on experimental procedures of each test was described in (OECD, 2015)