Elsevier

Bioresource Technology

Volume 271, January 2019, Pages 360-367
Bioresource Technology

A promising laccase immobilization approach for Bisphenol A removal from aqueous solutions

https://doi.org/10.1016/j.biortech.2018.09.129Get rights and content

Highlights

  • Laccases from T. pubescens were produced by semi solid-state fermentation.

  • Laccase was successfully immobilized by cross-linking plus entrapment.

  • Cross-linking prior to entrapment diminished laccase leakage from the beads.

  • Immobilized laccase removed almost completely 20 mg L−1 BPA in 2 h.

  • BPA (20 mg L−1) was efficiently removed in 10 successive batches.

Abstract

The immobilization of crude laccase from Trametes pubescens by glutaraldehyde crosslinking prior to entrapment into Ca-alginate beads increased the immobilization yield by 30% and reduced the leaking by 7-fold compared to the immobilization with no crosslinking. The performance of the newly developed biocatalyst to degrade Bisphenol A (BPA) from aqueous solutions was tested. Thus, operating at optimal conditions (i.e. pH 5, 30 °C, 20 mg L−1 BPA and 1500 U L−1 laccase), a BPA removal higher than 99% in 2 h was achieved. This value is higher than those reported to date for BPA removal by immobilized laccases. In addition, the biocatalyst was able to remove BPA in 10 successive batches with an efficiency higher than 70% at the end of the last batch. BPA adsorption on the alginate beads was negligible, therefore BPA removal was only due to laccase action. Moreover, Fourier-transform Infrared (FTIR) spectroscopy suggested BPA transformation by laccase.

Introduction

For some decades, endocrine disrupting compounds (EDCs) or xenoestrogens have been widely introduced into the environment mainly through the anthropogenic activities. Among these compounds, Bisphenol A (BPA) (2,2-bis (4-hydroxyphenyl) propane), which is mainly used in the production of polycarbonates and epoxy resins, is produced in large amounts worldwide with an annual production exceeding 3.8 million tons (Michałowicz, 2014). The US Environmental Protection Agency (EPA) identified BPA as a compound that may present an unreasonable risk of injury to the environment (Bisphenol A Action Plan, 2010). Recently, in June 2017, the European Chemical Agency (ECHA) has included BPA in the list of “substances of very high concern” (ECHA, 2017). More recently, BPA has been pointed as a potential risk factor in breast cancer development (Shafei et al., 2018). Hence, the presence of BPA in the aquatic environment poses a serious threat to the environment and public health (Arboleda et al., 2013).

Biological processes have been considered as a viable alternative as compared to the conventional physicochemical ones which present a high cost and lead to the formation of hazardous by-products (de Freitas et al., 2017). Instead of microorganisms, the use of enzymes separated from their cells presents several advantages such as no toxicity, high catalytic efficiency, high substrate specificity, shorter reaction time and mild reaction conditions (Sharma et al., 2018). White-rot basidiomycetes secret individual and multiple ligninolytic enzymes, such as laccases, lignin peroxidases (LiPs) and manganese-dependent peroxidases (MnPs), under suitable environmental conditions, with high BPA degrading ability (Bilal et al., 2017). Among the above-mentioned enzymes, laccases (p-diphenol oxygen oxidoreductases, EC 1.10.3.2) are the most frequently studied (Ba and Vinoth Kumar, 2017) due to, unlike peroxidases, laccases use molecular oxygen as the final electron acceptor which is usually available in the environment (Baldrian, 2006). However, the use of enzymes in their soluble form limits their practical application due to their non-reusability, sensitivity towards denaturing agents and low stability (Gasser et al., 2014, Songulashvili et al., 2012). To overcome these drawbacks, immobilization of enzymes is a suitable technique that can increase the stability, facilitate the efficient recovery and re-use of enzymes, thus, enabling their cost-effective use in continuous processes (Gasser et al., 2014, Sheldon and van Pelt, 2013).

Among the reported immobilization techniques (Barrios-Estrada et al., 2018, Gasser et al., 2014), enzyme entrapment in alginate beads is an eco-friendly, low-cost and easy immobilization technique (Chaudhari et al., 2015). Nevertheless, this technique is limited by the porosity of the gel which can lead to enzyme leakage (Brandi et al., 2006). Thus, in the present study, a new strategy consisting in crosslinking laccase enzymes prior to their entrapment into calcium alginate beads was investigated. Further, this approach was applied to BPA removal from aqueous solutions. To the best of our knowledge laccase from Trametes pubescens has not been used for BPA removal before this study.

Section snippets

Chemicals

ABTS (2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid), sodium alginate, glutaraldehyde (50% v/v), succinic acid, potassium ferricyanide (K3Fe(CN)6), 4-amino-antipyrine and other standard laboratory grade solvents and chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA). Bisphenol A (>97%) used for the degradation studies was provided by Alfa Aesar (Karlsruhe, Germany). All reagents were used as received without further purification.

Microorganism

The fungus Trametes pubescens MB89 was

Crude laccase production

Laccase activity peaked on the 18th day (17907 U L−1). No other ligninolytic enzymes were detected in the culture broth. The enzymatic activity and the specific activity of the crude laccase after ultrafiltration in Amicon were 40,446 U L−1 and 12.3 U mg−1, respectively. This crude laccase was used to perform the subsequent experiments.

Laccase immobilization and leakage

Laccase immobilized by crosslinking prior to entrapment showed a higher immobilization yield (>72%) than that of the laccase immobilized only by entrapment

Conclusion

Crude laccase from T. pubescens was successfully immobilized by entrapment and crosslinking prior to entrapment in alginate beads. In addition, the crosslinked-entrapped laccase reduced laccase leakage from the alginate beads by 7-fold and removed BPA (20 mg L−1) efficiently in 10 successive batches with a removal percentage higher than 70% at the end of the last batch. Also, FTIR results suggested BPA biotransformation by laccase enzymes. Moreover, laccase treated BPA solution showed no

Acknowledgements

We gratefully acknowledge the financial support by Ministry of Higher Education and Scientific Research (MESRS) of Algeria for funding provided under the PNE scholarship Program. Dr. Oihane Beldarrain from the BioMEMS group at Ceit-IK4 (Donostia-San Sebastian, Spain) is gratefully acknowledged for her assistance in the ATR-FTIR analyses.

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