Efficient degradation and detoxification of structurally different dyes and mixed dyes by LAC-4 laccase purified from white-rot fungi Ganoderma lucidum.

The purpose of this study is to evaluate the decolorization ability and detoxification effect of LAC-4 laccase on various types of single and mixed dyes, and lay a good foundation for better application of laccase in the efficient treatment of dye pollutants. The reaction system of the LAC-4 decolorizing single dyes (azo, anthraquinone, triphenylmethane, and indigo dyes, 17 dyes in total) were established. To explore the decolorization effect of the dye mixture by LAC-4, two dyes of the same type or different types were mixed at the same concentration (100 mg/L) in the reaction system containing 0.5 U laccase, and time-course decolorization were performed on the dye mixture. The combined dye mixtures consisted of azo + azo, azo + anthraquinone, azo + indigo, azo + triphenylmethane, indigo + triphenylmethane, and triphenylmethane + triphenylmethane. The results obtained in this study were as follows. Under optimal conditions of 30 °C and pH 5.0, LAC-4 (0.5 U) can efficiently decolorize four different types of dyes. The 24-hour decolorization efficiencies of LAC-4 for 800 mg/L Orange G and Acid Orange 7 (azo), Remazol Brilliant Blue R (anthraquinone), Bromophenol Blue and Methyl Green (triphenylmethane), and Indigo Carmine (indigo) were 75.94%, 93.30%, 96.56%, 99.94%, 96.37%, and 37.23%, respectively. LAC-4 could also efficiently decolorize mixed dyes with different structures. LAC-4 can achieve a decolorization efficiency of over 80% for various dye mixtures such as Orange G + Indigo Carmine (100 mg/L+100 mg/L), Reactive Orange 16 + Methyl Green (100 mg/L+100 mg/L), and Remazol Brilliant Blue R + Methyl Green (100 mg/L+100 mg/L). During the decolorization process of the mixed dyes by laccase, four different interaction relationships were observed between the dyes. Decolorization efficiencies and rates of the dyes that were difficult to be degraded by laccase could be greatly improved when mixed with other dyes. Degradable dyes could greatly enhance the ability of LAC-4 to decolorize extremely difficult-to-degrade dyes. It was also found that the decolorization efficiencies of the two dyes significantly increased after mixing. The possible mechanisms underlying the different interaction relationships were further discussed. Free, but not immobilized, LAC-4 showed a strong continuous batch decolorization ability for single dyes, two-dye mixtures, and four-dye mixtures with different structures. LAC-4 exhibited high stability, sustainable degradability, and good reusability in the continuous batch decolorization. The LAC-4-catalyzed decolorization markedly reduced or fully abolished the toxic effects of single dyes (azo, anthraquinone, and indigo dye) and mix dyes (nine dye mixtures containing four structural types of dyes) on plants. Our findings indicated that LAC-4 laccase had significant potential for use in bioremediation due to its efficient degradation and detoxification of single and mixed dyes with different structural types.

Effective Decolorization and Detoxification of Single and Mixed Dyes with Crude Laccase Preparation from a White-Rot Fungus Strain Pleurotus eryngii.

To fully harness the potential of laccase in the efficient decolorization and detoxification of single and mixed dyes with diverse chemical structures, we carried out a systematic study on the decolorization and detoxification of single and mixed dyes using a crude laccase preparation obtained from a white-rot fungus strain, Pleurotus eryngii. The crude laccase preparation showed efficient decolorization of azo, anthraquinone, triphenylmethane, and indigo dyes, and the reaction rate constants followed the order Remazol Brilliant Blue R > Bromophenol blue > Indigo carmine > New Coccine > Reactive Blue 4 > Reactive Black 5 > Acid Orange 7 > Methyl green. This laccase preparation exhibited notable tolerance to SO42- salts such as MnSO4, MgSO4, ZnSO4, Na2SO4, K2SO4, and CdSO4 during the decolorization of various types of dyes, but was significantly inhibited by Cl- salts. Additionally, this laccase preparation demonstrated strong tolerance to some organic solvents such as glycerol, ethylene glycol, propanediol, and butanediol. The crude laccase preparation demonstrated the efficient decolorization of dye mixtures, including azo + azo, azo + anthraquinone, azo + triphenylmethane, anthraquinone + indigo, anthraquinone + triphenylmethane, and indigo + triphenylmethane dyes. The decolorization kinetics of mixed dyes provided preliminary insight into the interactions between dyes in the decolorization process of mixed dyes, and the underlying reasons and mechanisms were discussed. Importantly, the crude laccase from Pleurotus eryngii showed efficient repeated-batch decolorization of single-, two-, and four-dye mixtures. This crude laccase demonstrated high stability and reusability in repeated-batch decolorization. Furthermore, this crude laccase was efficient in the detoxification of different types of single dyes and mixed dyes containing different types of dyes, and the phytotoxicity of decolorized dyes (single and mixed dyes) was significantly reduced. The crude laccase efficiently eliminated phytotoxicity associated with single and mixed dyes. Consequently, the crude laccase from Pleurotus eryngii offers significant potential for practical applications in the efficient decolorization and management of single and mixed dye pollutants with different chemical structures.

First report of leaf blight caused by Alternaria alternata on Lactuca indica in China.

Lactuca indica, an annual or biennial herbaceous plant, is widespread in valleys, shrubland, ditches, hillside meadows or fields (Wang et al. 2003). In China, it is widely used as medicine and high protein feed for herbivorous animal husbandry. In July 2022, leaf blight on L. indica was observed at Zhejiang Normal University (29°8'4″N, 119°37'54″E) in Jinhua City, Zhejiang Province, China. 70% of the 87 plants investigated were infected. Small brown spots with a yellow halos first appeared on the leaves, then became irregular necrotic spots until the entire leaf wilted and fell off. To identify the pathogen, four symptomatic leaves were collected and disinfected according to Wang et al. 2023. Then they were transferred onto potato dextrose agar (PDA), and incubated at 28°C for 7 days. To obtain the pure culture, the marginal mycelium was transferred to a new PDA plate. The colony of the isolated LPB-1 was light gray and regularly round at the early stage, and then changed to dark gray and villous. The back of the culture plate appeared sooty black. The conidia of the isolated fungi (n=50) were in chains, brown, obclavate, ovoid or ellipsoid, with an average size of 29.09 µm long and 6.41 µm wide, with 0 to 3 longitudinal and 1 to 7 transverse septa. These cultural and morphological characteristics were consistent with those of Alternaria alternata (Simmons 2007). To identify the strain, internal transcribed spacer (ITS) region, RNA polymerase Ⅱ second largest subunit (RPB2), and translation elongation factor 1-alpha (TEF1-α) genes were amplified with the primers ITS1/ITS4 (White et al. 1990), RPB2-5F/RPB2-7cR (Liu et al. 1999) and EF1-728F/EF1-986R (Carbone & Kohn 1999). The RPB2 (OP909715), TEF-1α (OP909714), and ITS (OP776880) were 99 to 100% identical to those of A. alternata (GenBank accession nos. MZ170963.1, MK605900.1, and MK605895.1 for RPB2 sequences; ON951981.1, KJ008702.1, and MK672900.1 for TEF-1α sequences; OP850817.1, OP811328.1, and OP740510.1 for ITS sequences). In addition, the phylogenetic analysis also showed that the stain LPB-1 was A. alternata. To complete Koch's postulates, the conidial suspension (1×108 conidia/mL) were spray-inoculated on healthy leaves of three mature L. indica plants with sterile water as a control. All plants were incubated at 28 ℃ in a greenhouse with 12-h-light/12-h-dark photoperiod and approximately 70% humidity (Li et al. 2019). Fourteen days after incubation, the inoculated leaves showed symptoms similar to those of naturally infected leaves, while the controls remained asymptomatic. The pathogen reisolated from the inoculated leaves had the same morphological characteristics and molecular identification results as the original isolate. All the results shown above indicated that A. alternata was responsible for the leaf blight of L. indica. As far as we know, this is the first report of leaf blight caused by Alternaria alternata on Lactuca indica in China. The identification of the pathogen could provide relevant information for the establishment of methods to control the disease.