Bio-based and cost effective method for phenolic compounds removal using cross-linked enzyme aggregates

https://doi.org/10.1016/j.jhazmat.2020.124021Get rights and content

Highlights

  • A new source of peroxidase was immobilized for phenolic compounds treatments.

  • Crosslinked peroxidase showed better physical and thermal stability compared to the free enzyme.

  • Immobilized peroxidase retained 100% activity after storage at 4 °C for 8 weeks.

  • RSVNP-CLEAs were able to remove 92% of phenol and 98% of p-cresol.

  • More than 71% degradation of phenols was achieved after 3 successive reuses.

Abstract

This work aimed at presenting a green method using a new source of peroxidase isolated from Raphanus sativus var. niger (RSVNP) in immobilized form, for the treatment of wastewater. To ensure stability and enzymatic activity in the biodegradation process, RSVNP was immobilized as a cross-linked enzyme aggregate (CLEAs). With more than 29% of recovered activity and 85% aggregation yield, acetone was selected as the best precipitating agent. The formed protein aggregates required 2% (v/v) of glutaraldehyde (GA) concentration and a ratio of 9:1 (v/v) enzyme (E) amount to cross-linker (E/GA). Compared to the free enzyme, RSVNP-CLEAs were found more chemically and thermally stable and exhibited good storage stability for more than 8 weeks. In addition, RSVNP-CLEAs were evaluated for their ability to remove phenol and p-cresol from aqueous solution by varying several operating conditions. A maximal yield (98%) of p-cresol conversion was recorded after 40 min; while 92% of phenol was degraded after 1 h duration time. The reusability of RSVNP-CLEAs was tested, displaying 71% degradation of phenol in the third batch carried out and more than 54% was achieved for p-cresol after four successive reuses in the presence of hydrogen peroxide at 2 mM concentration.

Introduction

Modernization and economic development have led to changes in the various industrial sectors to ensure the comfort and well-being of humans. On the other hand, they participated negatively and irreversibly to the deterioration of our environment (Bhandari, 2015). Intensive industrialization also has a direct effect on ecosystems and severe climate changes ([Chopra, 2016], [Jhariya et al, 2019]). Industrial activity releases numerous toxic chemicals compounds into water, air and soil (Mielke and McLachlan, 2020). This hazardous waste represents a significant risk to human health and can be in some cases an additional source of cancer development (Jagai et al. 2017). The effluents generated by the petrochemical industry are characterized by a strong concentration of toxic organic products, as well as oil and grease ([Abd El-Gawad, 2014], [Bahri et al, 2018]). Indeed, this industry uses a large volume of water for cooling systems and refining of crude oil; thus causing, a continuous discharge of pollutant-laden wastewater ([Bustillo-Lecompte et al, 2018], [Tian et al, 2019]). Generally, wastewater from petrochemical refineries contains sulfides, hydrocarbons, phenols, oils and greases ([Abdelwahab et al, 2009], [Altaş and Büyükgüngör, 2008]; Dat et al., 2017). The US Environmental Protection Agency (EPA), Agency for toxic Substances and Disease Registry (ATSDR) and the National Institute for Occupational Safety and Health (NIOSH) consider phenols as priority pollutants and dangerous for health or life at a concentration of 250 mg L−1 (Tandjaoui et al. 2019). Due to their solubility, phenolic compounds are widely present in sewage treatment plants discharged by petroleum refineries. Indeed, the concentration of phenolic compounds varies from 40 mg L−1 to 185 mg L−1 in the wastewater of oil refinery (Naguib and Badawy, 2019). For example, they can reach a concentration of 185 mg L−1 for raw outlets sour water, 50 mg L−1 for catalytic cracker and 80 mg L−1 for post-stripping (Al Hashemi et al., 2014). Moreover, several authors have revealed the undesirable effects of phenols and their derivatives on aquatic organisms. It has been reported that exposure of algae and fish to phenol concentrations of 72.29 mg L−1 and 14 mg L−1 respectively, lead to their death ([Duan et al, 2017], [Patra et al, 2007]). The same observations have been reported for exposure to p-cresol at concentrations of 45.8 mg L−1 for algae and 28.6 mg L−1 for fish ([Aruoja et al, 2011], [DeGraeve et al, 1980]). This is why, in order to protect the receiving environment, many wastewater treatment processes are implemented to reduce their impact on the environment (Villegas et al., 2016).

The use of microorganisms, enzymes and immobilized cells of organisms for the degradation of phenol and p-cresol has been the subject of several studies ([Bayramoǧlu and Arica, 2008], [Edalli et al, 2016], [Günther et al, 1995], [Tallur et al, 2009]). However, the treatment of pollutants with microorganisms may show certain limits due to the drastic conditions in which bacterial growth must be ensured (Karigar and Rao, 2011). Therefore, the use of enzymes may be a more attractive approach to overcome these problems. Several studies have demonstrated the potential of peroxidases for wastewater bioremediation by involving free radicals, thus generating polymerized or oxidized products (Bansal and Kanwar, 2013). In addition, through work carried out by researchers on horseradish peroxidase (HRP) and soybean peroxidase (SBP), it turns out that peroxidases are an effective tool in the degradation of phenolic compounds ([Bayramoǧlu and Arica, 2008], [Steevensz et al, 2014]). Despite their certain efficacy in the treatment of these compounds, free enzymes also encounter problems which can lead to their denaturation and a loss of catalytic activity (Brady and Jordaan, 2009). Immobilization or insolubilization of enzymes, can give rise to a more stable biocatalyst, resistant to extreme conditions, recyclable and having the capacity to operate continuously (Brady and Jordaan, 2009). Four traditional methods of enzymes immobilization can be distinguished mainly, including entrapment, encapsulation, fixation on support and cross-linking (Sheldon and van Pelt, 2013). Among them, cross-linking is a promising technique since the immobilization of the enzyme is possible by using bifunctional cross-linking agent to bind the enzyme molecules without using a support (Brady and Jordaan, 2009). In addition, the other techniques show some drawbacks. In the case of entrapment, the molecules of the enzymes are free but the gel which surrounds them limits their movement and acts as a barrier to mass transfer, which impacts reaction kinetics (Brady and Jordaan, 2009). For encapsulation, its use is limited to enzymes with substrates of small molecular size due to the diffusion limits (Hanefeld et al., 2009). As for attachment to support by adsorption, the enzyme seems to retain certain mobility; which allows it to retain its catalytic properties. However, it has a major drawback because the interactions between the enzyme and the support are weak. The enzyme can thus be released over time, especially under industrial conditions (strong agitation). On the other hand, the ionic and covalent bindings are generally stronger but the fact remains that this technique is more expensive due to the use of the support (Sheldon and van Pelt, 2013). Therefore, the synthesis of cross-linked enzyme aggregates (CLEAs) is a simple alternative technique in its implementation; it allows the reduction of the immobilization cost compared to immobilization on support while retaining a considerable rate of initial enzyme activity. Many advantages are associated to CLEAs technology in comparison with soluble enzyme, including stability to denaturation caused by high temperature, organic solvents and proteolysis (Sheldon, 2007). It also offers many benefits, such as volumetric productivities and recoverability, superior operational stability following the formation of a rigid structure which prevents the breakdown of the aggregated enzyme by the multipoint attachment of enzyme molecules ([Park et al, 2012], [Sheldon, 2011]). Despite several advantages, carrier-free immobilized enzymes have some drawbacks which can make its application at industrial scale complicated. The lack of control on their particle size, mass-transfer issues related to recovery operations such as filtration and centrifugation, make the CLEAs particles only partially recycled (Cui and Jia, 2013). To overcome these constraints, several researchers have developed techniques to improve the mechanical stability of CLEAs and facilitate their recovery, by encapsulating, coating and trapping them in polymers and matrices. Penicillin G acylase has been successfully co-aggregated with amino-functionalized superparamagnetic iron oxide nanoparticles and then cross-linked, thus allowing its efficient and rapid magnetic decantation (Kopp et al., 2013). Similarly, by adopting an innovative approach Cui et al. (2016) developed a hybrid magnetic CLEAs (HM-CSL-CLEAs) using lipase and unfunctionalized magnetite nanoparticles. The resulting CLEAs enhanced the enzyme stability to elevated temperature, increased storage stability while allowing their separation in a simple and easy manner. The same observations were concluded in the synthesis of spherical CLEAs with biosilica shell which turned out stable against denaturants, while maintaining 70% of its activity after 13 cycles (Cui et al., 2017). In addition, the combination of traditional techniques with CLEAs technology may prove useful, as in the case of the adsorbed cross-linked phenylalanine ammonia lyase aggregate on the crude-pored microspherical silica core. The immobilized CLEAs can be precipitated naturally without filtration or centrifugation, thus reducing mass-transfer limitations (Cui et al., 2014).

However, it is important to take into consideration the biochemical properties and protein structure of each enzyme before proceeding to any immobilization (Yamaguchi et al., 2018).

In a circular economy objective, the present study relates to the use of a green method for the degradation of phenolic compounds, whose component is a new source of peroxidase which could succeed to SBP and HRP. In this context, Raphanussativus var. niger peroxidase was extracted and immobilized in the form of cross-linked enzyme aggregates (RSVNP-CLEAs). After preparation, optimization and characterization of RSVNP-CLEAs, the latter were evaluated for their potential in the degradation of phenol and p-cresol while investigating the parameters that can influence their elimination. This approach could provide an ecological and cost-effective alternative for a large-scale enzyme treatment.

Section snippets

Chemical reagents

Phenol (purity 99%), p-cresol (purity 99%), 4-aminoantipyrine, hydrogen peroxide solution (30% w/w) and glutaraldehyde (GA) were supplied by Sigma-Aldrich (Saint Quentin Fallavier, France). Sulfuric acid (purity ≥ 95) was purchased from AcrosOrganics (Thermo Fisher Scientific, Geel, Belgium). All solutions were prepared with ultrapure water and all reagents used for experiments were obtained from Acros and Sigma.

Protocol for RSVNP-CLEAs preparation

The enzyme was isolated from a fresh black radish using a juice extractor. The

Preparation of RSVNP-CLEAs

Results from this study represent two novelties. First, cross-linking and the characterization of the resulting CLEAs of RSVNP, as a new source of plant peroxidase. On the other hand, the capacity of RSVNP-CLEAs to degrade phenols under specified conditions. First step was to implement an aggregation protocol by testing different precipitant agents. Previous studies have shown the importance in the choice of the precipitation solvent which plays an important role in the preservation of enzyme

Conclusion

The efficacy of immobilized RSVNP-CLEAs in the form of cross-linked enzyme aggregates was investigated for the biodegradation of phenolic compounds. The immobilization step consisted in the preparation and optimization of a robust biocatalyst with efficient catalytic performances, as well as great chemical and thermal stability compared to the free enzyme. The cross-linked enzyme showed interesting characteristics, retaining 100% of its activity after 60 days of storage and significant

Ethical statement

We declare that there are no ethical issues form human or animal rights in the work presented here.

CRediT authorship contribution statement

All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Furthermore, each author certifies that this materials or similar material has not been and will not be submitted to or published in any other publication before its appearance in the Journal of Hazardous

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The Ministry of Higher Education and Scientific Research of Algeria and Campus France are gratfully acknowledged for providing K. Sellami with Profas B+ grant (933290A).

Conflict of interest

We declare that we have no conflict of interest.

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