Evolution and stabilization of environmental persistent free radicals during the decomposition of lignin by laccase

Highlights

• The mechanism of interaction between lignin and laccase from the perspective of EPFRs was described.

• EPFRs and ROS were detected during the decomposition of lignin by laccase.

• ROS concentration had a strong correlation with the EPFRs concentration.

• Laccase induced the demethylation and oxidation of lignin.

Abstract

Soil microbial enzymes may induce lignin decomposition, accompanied by generation of free radicals. The evolution of environmentally persistent free radicals (EPFRs) and reactive oxygen species (ROS) during laccase-catalyzed lignin decomposition remains unclear. Characterization by electron paramagnetic resonance spectroscopy revealed gradually increased concentration of EPFRs, with maximum levels within 6 h that remained constant, accompanied by the increase in g-factor from 2.0037 to 2.0041. The results suggested the generation of oxygen-centered radicals on lignin. The EPFRs produced on solid samples slowly decreased by 17.2% over 17 d. ROS were also detected to have a similar trend as that of the evolution of EPFRs. Scanning electron microscopy, attenuated total reflectance-Fourier transform infrared spectroscopy, gel permeation chromatography and nuclear magnetic resonance analyses suggested the demethylation and oxidation of lignin. We clarify the biogeochemical transformation of lignin and potential contributions to the generation of EPFRs and ROS in soil.

Introduction

Lignin is an amorphous, three-dimensional, and highly branched polyphenolic macromolecule with a complex structure consisting of three types of aromatic units, including syringyl, guaiacyl, and p-hydroxyphenyl (Li et al., 2015). Wood and herbaceous plants are the main sources of lignin, which finally present in natural phases, especially soil and sediments (Mishra et al., 2016). After entering the soil, lignin is inevitably decomposed. Soil microbial communities play a significant role in this process (Crawford et al., 1977). As reported previously, approximately 53.75% of lignin in tobacco stalks could be degraded by Phanerochaete chrysosporium in 15 d (Su et al., 2016). Fungi may induce the complete decomposition of lignin during the same length of time (Davis and Sello, 2010).

Microbe-mediated depolymerization of lignin is a complex process involving various types of enzymes, which include lignin peroxidase, manganese peroxidase, and laccase (Higuchi, 2004). These enzymes can directly attack lignin, cellulose, and hemicellulose of the plant cell wall, which often results in the structural modification of lignin (Orpin, 1984). Laccase is a type of oxidases that may promote cleavages in lignin molecules, including opening of the aromatic ring, alkyl-aryl disruption, and increase in phenolic hydroxyl group (Kawai et al., 1988a). During these processes, bond cleavage and redox reaction via electron transfer always participate in lignin decomposition. As a consequence, a large variety of free radical species may be produced within the lignin matrix (Kudanga et al., 2011). The polymer-associated free organic radicals may be stable and detected by electron paramagnetic resonance (EPR) spectroscopy under natural condition, and have been termed environmentally persistent free radicals (EPFRs) (Perna et al., 2019).

EPFRs have attracted a lot of attentions recently due to their unique properties, which include the potential to induce the electron transfer reaction (Jia and Wang, 2013) and promote the generation of ROS (Jia et al., 2018), such as peroxide, superoxide, and hydroxyl radicals, which have benefits and drawbacks concerning the environmental effects of EPFRs (Khachatryan et al., 2011). On the one hand, EPFRs-induced ROS may eliminate the organic contaminants in natural phases (Chen et al., 2017; Jia et al., 2015; Wang et al., 2015). On the other hand, the potential formation of ROS may have a negative effect on human health by the oxidative stress (Ray et al., 2012; Zhang et al., 2019). Much work has been performed to understand the formation and fate of EPFRs associated with various matrices, such as fly ash, carbonaceous materials, and superfund sites contaminated by organic contaminants (Cruz et al., 2011; Liao et al., 2014; Zhao et al., 2019). Only a few studies have been conducted to examine the evolution and potential generation of ROS of lignin-EPFRs induced by enzymatic treatment (Silva et al., 2011).

The objectives of this work were (1) to probe the evolution of EPFRs during the decomposition of lignin by laccase, (2) to observe the generated ROS in the lignin/laccase system using the chemical spin trap, and (3) to clarify the underlying mechanisms for the generation of lignin-EPFRs using scanning electron microscopy (SEM), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), nuclear magnetic resonance (NMR), and gel permeation chromatography (GPC). The findings clarify the biogeochemical transformations of lignin in the soil environment, and provide a new insight into the environmental significance of the generation of EPFRs and ROS during the decomposition of lignin by laccase.