Rhamnolipids production from citrus waste cellulose extract using oil adapted soil microbiota as biocatalysts : a bioprocess engineering approach

Abstract

The increasing demand for sustainable counterparts to synthetic surfactants has sparked interest in microbial biosurfactants, particularly rhamnolipids, due to their biodegradability, low toxicity, and strong surface activity. However, large-scale production is limited by high substrate costs, pathogenicity of common producer strains, and inefficient process optimization. This study addresses these challenges by valorising orange peel waste (OPW) as a renewable carbon source and employing an oil-adapted soil microbiota.

Cellulose was extracted from OPW via sequential maceration, alkaline, bleaching, and organic acid pretreatments, yielding 42.49% (w/w) with a saccharification efficiency of 42.22%. Structural integrity and purity were confirmed using FTIR, SEM, XRD, and NMR. Oil-adapted soil microbiota was screened for biosurfactant production, leading to the selection of E. hormaechei. Preliminary fermentation utilizing the orange peel-derived cellulose hydrolysate (OPDC) produced 1.1 g/L rhamnolipids and a microbial density (OD₆₀₀) of 0.66. This yield represents a substantial performance increase, outperforming prior waste oil-based media by nearly 30%.

Initial fermentation results revealed modest rhamnolipid production, necessitating statistical optimization to enhance yield and process efficiency. A Plackett–Burman design was employed to screen ten influential variables, followed by OVAT validation and Response Surface Methodology using Central Composite Design (RSM–CCD). Statistical optimization increased the yield to 5.94 ± 0.11 g/L under optimal conditions (3 g/L OPDC, 0.5 g/L MnSO₄·7H₂O, 0.1 g/L CaCl₂·2H₂O, pH 7.0), representing a 5.5-fold improvement. The biosurfactant exhibited strong surface activity, reducing surface tension from 72 to 27.73 mN/m and achieving an emulsification index (E24) of 69.57%. TLC, FTIR, NMR, and LC–MS confirmed the predominance of mono-rhamnolipid congeners, particularly Rha-C10-C10, which enhance emulsification and hydrophilicity.

Kinetic modelling further validated the optimized rhamnolipid yield, with the hybrid Logistic–Luedeking-Piret model accurately predicting a production value of 5.77 g/L, closely matching the experimental yield of 5.94 ± 0.11 g/L. High correlation coefficients for biomass growth (R² = 0.996), substrate utilization (R² = 0.994), and biosurfactant synthesis (R² = 0.996) confirmed the robustness of the model, while substrate yield coefficients (Yₓ/ₛ = 0.070 g/g, Yₚ/ₛ = 0.8 g/g) demonstrated efficient conversion of OPDC into Rhamnolipids. This modelling approach offers a reliable framework for scaling up rhamnolipid production using solid Agro-waste substrates. This integrated approach, combining non-pathogenic microbes, citrus waste valorisation, and advanced modelling, establishes a scalable and environmentally sustainable platform for rhamnolipid production, supporting applications in remediation, industrial formulations, and the circular bioeconomy.