Background Commercialization of biosurfactant production is a big challenge due to high production cost. Biosurfactant production can be made economic by using low cost agro-industrial wastes or byproducts as media supplement. It also solves the problem of environmental pollution through waste management. In the present study attempt was made to produce biosurfactant from Sphingobacterium thalpophilum DP9 using various agro-industrial wastes or byproducts at optimum fermentation conditions evaluated by traditional and statistical methods. Partial characterization of biosurfactant was also carried out through qualitative chromatographic techniques; quantitative spectroscopic method and functional group identification by Fourier transform infrared spectroscopy. Results One factor at a time optimization experimentation showed highest emulsification in media supplemented with potato peel powder (43.12 ± 3.41%), urea (46.00 ± 3.09%), pH 7 (41.74 ± 1.15%) at 40% aeration (47.41 ± 1.62%). 1% Inoculum (O.D.600=1.00) size favored highest emulsification (26.39 ± 1.60%). Plackett-Burman design experimentation showed carbon source (potato peel powder), nitrogen source (urea) and temperature significantly affect on biosurfactant production. Response surface experimentation by central composite design showed interaction between nitrogen source and temperature significantly influenced on biosurfactant production. Validation of model was showed increase in emulsification from 43.62 ± 2.55% to 86.11 ± 3.47% on fourth day of incubation. Partial characterization showed biosurfactant contains 439.58 ± 0.0129 µg/ml carbohydrate and 507.41 ± 0.0064 µg/ml of protein. Lipid was absent in it. Fourier transform infrared spectroscopy chromatogram showed peaks at 1652.17 cm− 1, 1541.01 cm− 1, 1463.71 cm− 1 and 722.32 cm− 1 which indicated presence of peptide bond, peptide moiety, carbohydrate protons and primary as well as secondary amines. Conclusion Potato peel powder, urea and temperature have significant influence on biosurfactant production from Sphingobacterium thalpophilum DP9. When potato peel powder and urea supplied at optimum level, biosurfactant production was increased by two fold. Biosurfactant produced by S. thalpophilum DP9 was belongs to glycoprotein class and as per our best knowledge, this is the first report on glycoprotein biosurfactant produced from Sphingobacterium genus.

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