Biochemical Study of Bacillus stearothermophilus Immobilized Lipase for Oily Wastewater Treatment

Abstract

Traditional wastewater treatments involve expensive mechanical and physiochemical methods, so researchers have been developing cost-effective, sustainable technologies that use enzymes to produce higher quality effluents and recover more energy and nutrients from wastewater. A thermostable, alkaline, and solvent-tolerant lipase was partially purified from thermophilic Bacillus stearothermophilus. The lipase displayed maximum activity at 50 °C and pH 11.0 and catalyzed both short- and long-chain triacylglycerols at similar rates. B. stearothermophilus lipase also exhibited high stability when incubated at 40 °C for 1 h with anionic and non-ionic surfactants. Studies show that thermostable enzymes can be improved through immobilization and modification of other reaction conditions. Therefore, B. stearothermophilus lipase was immobilized through adsorption on CaCO3, Celite 545, and silica gel with the CaCO3 support producing the best adsorption rate (89.33%). The optimal initial lipase activity was approximately 4500 U.g−1 after 60 min. Interestingly, 93% of the initial lipase activity was retained after six cycles, and almost 50% of the initial activity remained after 12 cycles. Furthermore, immobilization improved storage stability with 98.85% of the initial lipase activity retained after 60 days of storage at 4 °C. The biochemical characteristics of immobilized lipase shifted toward a slightly alkaline region, reaching maximum activity at pH 12. The optimal temperature of immobilized lipase was 60 °C. Immobilization also improved enzymatic stability by widening the pH range from 5–9 (for free lipase) to 4–11, and thermostability by reaching 65 °C. The application of immobilized lipase in wastewater treatment was observed through oil layer biodegradation. Notably, treating wastewater for 10 days with immobilized lipase almost removed the chemical oxygen demand (COD) from 1950.1 down to 4.04 mg.L−1. Similarly, lipid content was almost removed from 15,500 ± 546 mg.L−1 down to 12 mg.L−1. All results highlight the potential value of CaCO3-immobilized lipase as an effective biocatalyst for hydrolyzing wastewater.

Combined immobilized lipases for effective biodiesel production from spent coffee grounds

“…From the perspective of alternative energy and sustainability, several waste-to-energy conversion approaches are extensively gaining importance. Various no-cost organic wastes including gutter oil, noodle waste, and food waste have been studied for biofuel synthesis ( Karmee, 2016 , Karmee, 2017 , Karmee, 2018 , Binhayeeding et al, 2020 , Linganiso et al, 2022 , Ben Bacha et al, 2022b , Weldeslase et al, 2023 , Yusuf et al, 2023 ). Green energy production from spent coffee grounds is currently considered as an emerging and promising field of scientific research (Sanjib et al 2018; Sarno and Iuliano, 2018 , Oliveira et al, 2008 ).…”

“…This could be attributed to the lipase specificity depending on the substrate and the enzyme origin ( Ramos et al, 2019 , Sánchez-Bayo et al, 2019 , Roy and Gupta, 2004 ). In a recent investigation, the influence of CaCO 3 -immobilized single and combined BSL and SAL on the transesterification rate of waste cooking oil to produce biodiesel has been evaluated ( Ben Bacha et al 2022b ). The highest biodiesel yield was obtained when 1% of mixed immobilized enzymes at a ratio of 1:1 (50% of each enzyme) was used.…”

“…Candida rugosa lipase (CRL), CRL-B immobilized on an acrylic resin (Novozyme-435), porcine pancreatic lipase-II (PPL-II), and Rhizomucor miehei lipase (RML) were products of Sigma Aldrich (St. Louis, USA). Staphylococcus aureus (SAL) and Bacillus stearothermophilus (BSL) lipases were produced, partially purified and immobilized on CaCO 3 as described in previous works ( Ben Bacha et al, 2022b , Bacha et al, 2019 , Ben Bacha et al, 2016 , Bacha et al, 2015 ).…”

“…Following two 20 mL washes with hot deionized water, the recovered biodiesel was dried for 24 h at 105 °C ( Zheng et al 2006 ). The percentage conversion to methyl ester was estimated by using the GC–MS analysis of the samples as previously described ( Ben Bacha et al, 2022b , Hadaruga et al, 2008 ). Methyl ester content was evaluated in the obtained product using EN14214 as described by Sarno & Iuliano (2019) .…”

“…To overcome the drawbacks of chemical catalysts, lipases could be utilized as biocatalysts to produce biodiesel ( Karmee, 2016 , Karmee, 2018 ;). Interestingly, lipase enzymes are widely employed under moderate conditions as well as in conventional and nonconventional solvents, free fatty acid and moisture tolerant, and easy to recover ( Weldeslase et al, 2023 , Zou et al, 2023 , Ben Bacha et al, 2022a , Ben Bacha et al, 2022b , Binhayeeding et al, 2020 , Bacha et al, 2019 ). Microbial lipases, in particular, attracted a lot of attention with the recent advances in enzyme technology because they displayed numerous advantages such as the absence of seasonal changes, the continuous supply, high yield production in economically advantageous media besides the extensive variety of their catalytic activities.…”