Fast decolorization of azo dyes in alkaline solutions by a thermostable metal-tolerant bacterial laccase and proposed degradation pathways

Abstract

Biocatalytic decolorization of azo dyes is hampered by their recalcitrance and the characteristics of textile effluents. Alkaline pH and heavy metals present in colored wastewaters generally limit the activity of enzymes such as laccases of fungal origin; this has led to an increasing interest in bacterial laccases. In this work, the dye decolorization ability of LAC_2.9, a laccase from the thermophilic bacterial strain Thermus sp. 2.9, was investigated. Its resistance towards different pHs and toxic heavy metals frequently present in wastewaters was also characterized. LAC_2.9 was active and highly stable in the pH range of 5.0 to 9.0. Even at 100 mM Cd +2 , As +5 and Ni +2 LAC_2.9 retained 99%, 86% and 75% of its activity, respectively. LAC_2.9 was capable of decolorizing 98% of Xylidine, 54% of RBBR, 40% of Gentian Violet, and 33% of Methyl Orange after 24 h incubation at pH 9, at 60 °C, without the addition of redox mediators. At acidic pH, the presence of the mediator 1-hydroxybenzotriazole generally increased the catalytic effectiveness. We analyzed the degradation products of laccasetreated Xylidine and Methyl Orange by capillary electrophoresis and mass spectrometry, and propose a degradation pathway for these dyes. For its ability to decolorize recalcitrant dyes, at pH 9, and its stability under the tested conditions, LAC_2.9 could be effectively used to decolorize azo dyes in alkaline and heavy metal containing effluents.

Recent Achievements in Dyes Removal Focused on Advanced Oxidation Processes Integrated with Biological Methods

“…Bacterial treatment Isolation of new strains or consortia from activated sludge, oxidation ditch, palm oil mill effluent or desert soil, alkali-, haloand thermophilic strains implementation, consortium with algae, bacteria immobilization, co-substrate addition, proposal of mechanisms, pathways genome and transcriptome analysis [109][110][111][112][113][114][115][116][117][118][119] Fungal treatment Implementation of microbial consortium (e.g., yeast consortium with ability of lignin valorization dye treatment and biodiesel production), fungi immobilization, isolation of new strains from plant roots or effluent site [120][121][122][123][124][125][126][127][128][129][130] Enzyme treatment Optimization of enzyme production, enzyme immobilization, metabolites and toxicity assessment [131][132][133][134][135][136] Algal treatment…”

“…Among the microorganisms that are able to produce enzymes necessary for dye degradation under aerobic conditions, white-rot fungi are the most efficient [215,231]. However, experiments were conducted on bacteria (especially Streptomyces) [34,119,134], yeast [127], and other fungi [120,124,128,202,203] for the implementation of aerobic dye removal. Although bacterial laccase is less efficient in the azo bond cleavage than fungal laccase, this drawback may be partially overcome by the addition of a natural redox mediator-methyl syringate in 0.5 mM concentration led to the Acid Orange 63 decolorization increase from 0 to 35% [34].…”

“…Although bacterial laccase is less efficient in the azo bond cleavage than fungal laccase, this drawback may be partially overcome by the addition of a natural redox mediator-methyl syringate in 0.5 mM concentration led to the Acid Orange 63 decolorization increase from 0 to 35% [34]. Moreover, bacterial or yeast laccase obtained from halo-or thermophilic microorganisms may act in a wider range of pH and temperatures than white-rot fungi laccase [34,127,134]. However, there is also one literature report concerning the engineering of fungal laccases with a successful shift of optimum catalytic activity to alkaline pH [135].…”

“…As can be seen above, a lot of experiments were conducted with fungi immobilization-this is the result of the morphologic specificity of fungal growth. As for the isolated enzymes implementation, investigations were primarily performed in disposable cuvettes [34,120,134], vials with immobilized peroxidase [132], and Erlenmeyer flasks with free and immobilized laccase [136]. The color removal obtained using free or immobilized enzymes varied between 33 and 98%.…”

Phenothiazines as efficient redox mediators for dye-decolorization at neutral / alkaline pH by bacterial laccases

“…Although decolorization of XYL by different fungal laccases at acidic pH has been reported (Campos et al, 2016;Levin et al, 2005;2012), there is only one recent report of oxidation by bacterial enzymes at alkaline pH. Laccase LAC-2.9 fromThermus sp 2.9 manage to decolorize 98% of XYL after 24 h of incubation at pH 9 and 60°C, without the addition of redox mediators (Navas et al, 2020), being an attractive candidate to be evaluated with PhCOOH. On the other hand, laccase SilA has been successfully used for decolorization of other azoic dyes such as Reactive Black 5 and Orange II (˜90% of decolorization after 24 h at pH 8 and 35°C) in the presence of 0.1 mM acetosyringone or methyl syringate as redox mediators, while for Acid Orange 63 o Tartrazine decolorization was not higher than 22% in the same incubation conditions.…”