نوع مقاله : کاربردی
نویسندگان
1 استادیار گروه مهندسی بیوسیستم، دانشگاه اراک، اراک، ایران
2 کارشناسی ارشد علوم و صنایع غذایی، مدیر سیستم ها و روش ها، شرکت گلنان پوراتوس، تهران، ایران
کلیدواژهها
عنوان مقاله English
نویسندگان English
Introduction: Biodiesel is a renewable, biodegradable, and nontoxic fuel made from fatty acid methyl esters produced via triglyceride transesterification. This reaction is accelerated by various catalysts, including chemical types (alkali, acid, and solid) and biocatalytic lipase enzymes. Lipase enzymes are particularly favored for their mild reaction conditions, reduced formation of soap byproducts, tolerance toward free fatty acids, ease of product recovery, eco-friendliness, and lower overall energy and raw material demands. However, free enzymes suffer from limited operational stability and high cost, motivating the development of immobilization strategies. Enzyme immobilization enhances the catalyst's reusability, thermal and pH stability, and specific activity, thereby enabling frequent cycles without significant activity loss. The process relies on cost-effective supports with ample surface areas and pore structures to maximize enzyme loading and accessibility. Among these, silica/iron oxide magnetic nanoparticles stand out for their rapid separation capabilities under an external magnetic field, which simplifies downstream processing and reduces wastewater generation. Covalent binding methods, such as glutaraldehyde coupling, are employed to securely attach the enzyme to the support, improving overall performance and preventing enzyme leaching. In this research, we aim to optimize biodiesel production from canola waste cooking oil using response surface methodology and assess the operational stability and reusability of the immobilized lipase under repeated batch cycles.
Materials and Methods: Key reagents—including analytical-grade methanol, tetraethyl orthosilicate (TEOS), ferric and ferrous salts, and glutaraldehyde—along with Burkholderia cepacia lipase (≥50 U/mg), were sourced from Sigma-Aldrich. Superparamagnetic mesoporous silica/iron oxide nanoparticles were synthesized via a two‑step approach: iron oxide core formation by coprecipitation, followed by a sol–gel process using TEOS with CTAB as a pore-directing agent. Resulting particles were calcined to remove surfactants, yielding a stable mesoporous shell. These magnetic nanoparticles were used as supports for enzyme immobilization. Lipase was covalently attached using a glutaraldehyde coupling process in phosphate buffer (pH 7.0) at 4 °C, with protein loadings varied between 10 and 40 mg/g support. Immobilization efficiency and activity recovery were determined by Bradford assay and olive oil hydrolysis assays, respectively. Biodiesel production assays were performed by transesterifying pretreated canola waste cooking oil with the immobilized lipase under gentle stirring. Reaction parameters (lipase concentration, methanol-to-oil ratio, temperature, and time) were systematically varied according to a central composite design, and FAME content was quantified by gas chromatography (GC-FID). All experiments were carried out in triplicate to ensure reproducibility.
Results and Discussion: SPION-silica nanoparticles, averaging 90–110 nm with a uniform mesoporous shell (pore diameter ~8 nm), offered a high surface area (~420 m²/g) for enzyme immobilization and pronounced superparamagnetic behavior (saturation magnetization ~45 emu/g). Upon lipase binding, saturation magnetization decreased to ~40 emu/g, confirming successful enzyme attachment without compromising magnetic responsiveness. Immobilization efficiency reached up to 78%, with an optimal activity recovery of 68.4% attained at 30 mg enzyme/g support and 12 h coupling time. The RSM-based second-order polynomial model exhibited strong predictive power (R² ≈ 0.96, adjusted R² ≈ 0.94), with significant linear and quadratic effects of lipase concentration, reaction time, methanol-to-oil ratio, and temperature (p < 0.01). Interaction terms—particularly between methanol ratio and time—highlighted enzyme inhibition by excess alcohol. Surface response plots revealed that FAME yield increased with enzyme loading (20–40 mg/g) and prolonged reaction time (15–30 h), but declined at methanol/oil ratios above 6:1 and temperatures exceeding 40 °C. Under optimal conditions (36 mg/g lipase, 25 h, 6:1 methanol/oil, 34 °C), the desirability function predicted a maximum biodiesel yield of 92%. Experimentally, three consecutive cycles yielded 92%, 89%, and 84% conversion, indicating only ~8.7% cumulative activity loss after three uses, demonstrating robust reusability for practical applications.
Conclusion: Burkholderia cepacia lipase was successfully immobilized on mesoporous silica/iron oxide magnetic nanoparticles. The immobilized enzyme effectively catalyzed the transesterification of waste cooking oil to produce biodiesel. VSM analysis confirmed that the magnetic properties of the nanoparticles remained largely unchanged after immobilization. Key process variables—immobilized lipase concentration, reaction time, methanol‑to‑oil molar ratio, and temperature—were optimized. A quadratic response surface model, developed using RSM, accurately predicted the biodiesel (FAMEs) yield. The desirability function analysis forecasted a maximum FAMEs content of 92% at optimal conditions. Statistical validation showed the model was both adequate and precise in predicting biodiesel yield. The immobilized lipase demonstrated good reusability, with only an 11% activity loss after three cycles. These findings underscore the potential for scalable, eco-friendly biodiesel production using enzyme‑catalyzed processes. The magnetic recovery of the biocatalyst not only simplifies downstream processing but also contributes to reduced overall production costs. Future work will focus on pilot‑scale trials and the evaluation of long‑term operational stability under continuous‐flow conditions to assess commercial feasibility.
کلیدواژهها English
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