Development of Bio-Fenton System for Non-Specific Degradation of Organic Pollutants by a Newly Isolated H2O2 Producing Bacterium Desemzia sp. Strain C1

Abstract

Fenton reaction has commonly employed to degrade various organic pollutants because of its simple catalytic of hydrogen peroxide (H2O2) with cyclic reaction of Fe(II) and Fe(III), resulting generation of hydroxyl radical (•OH). Among the reactive oxygen species, •OH exhibited the highest oxidation potential at 2.8 eV. Therefore, wide ranges of organic pollutants such as herbicides and plastics can be degraded by •OH through the non-specific degradation mechanism. Although the operation of Fenton reaction is simple, the continuous H2O2 supply is a critical restriction due to the high cost of H2O2 production and transportation. Recently, Bio-Fenton reaction has been studied to operate Fenton reaction in-situ environments using the H2O2 producing enzymes and microbes. The in-situ generation of H2O2 can prevent the accident during H2O2 storage and reduce the energy consumption associated with H2O2 production and transportation. In this study, a promising H2O2 producing bacterium, Desemzia sp. strain C1 newly isolated from oil- contaminated soil was applied to Bio-Fenton degradation of chloroacetanilide herbicides and sulfonated polyethylene at neutral conditions. Strain C1 produced at 1.8 mM H2O2 using 10 mM lactate as an optimal substrate, which was the highest capability of H2O2 production compared to well-known H2O2 producing bacteria such as Streptococcus oralis and Aerococcus in the same conditions. The magnetite-driven Bio-Fenton reaction was employed to degrade chloroacetanilide herbicides in the presence of 0.5% (w/v) magnetite, 10 mM lactate, and resting cells of strain C1 (O.D600 = 1.0) at pH 6.8. The Bio-Fenton degradation of chloroacetanilide showed about 40-50% degradation efficiency, leading to the generation of degradation metabolites and Cl- ions through non-specific degradation mechanism by •OH. Enhanced Bio-Fenton reaction was applied to degrade sulfonated polyethylene at an optimal condition to produce H2O2 by strain C1 in the presence of 25 mM lactate, and resting cells of strain C1 (O.D600 = 2.5) at pH 6.0. As a result, 6.6 mM of acetic acid and fine chemicals were produced from the Bio-Fenton degradation of sulfonated polyethylene. Interestingly, the Bio-Fenton degradation of sulfonated polyethylene showed higher acetic acid production and •OH generation compared to abiotic Fenton degradation through the enhanced Fenton reaction efficiency by siderophore production from strain C1. Therefore, the application of strain C1 in Bio- Fenton system is prospective to degrade recalcitrant organic substrate in-situ environments under neutral conditions. The biochemical physiology of strain C1, leading to high H2O2 production was characterized based on whole genome sequencing and proteomic analysis. The native gel-based enzyme assay revealed strain C1 utilized lactate oxidase as a major enzyme to produce H2O2. The proteomic analysis of strain C1 depending on different culture conditions showed a correlation with the amounts of FMN and up-regulation of MsrB. Therefore, MsrB and lactate oxidase were expressed in Escherichia coli to evaluate the effect of MsrB on lactate oxidase of strain C1. MsrB treatment on lactate oxidase enhanced the H2O2 producing activity with an increase in the homo-tetramer ratio and reduction of oxidized methionine in lactate oxidase. Based on proteomic analysis, the possible mechanism of strain C1 leading to high H2O2 production was suggested by restoration of lactate oxidase to act stable under oxidative stress conditions by coupled reaction with MsrB, thioredoxin family protein, and thioredoxin reductase. In addition, an efficient lactate oxidase secretion system in B. subtilis RIK 1285 was developed by screening the 173 types of signal peptides and expression of lactate oxidase using an IPTG-inducible vector (pHT01). The highest efficiency of lactate oxidase secretion was achieved by B. subtilis harboring ywjE signal peptide in the IPTG-inducible pHT01 vector. The lactate oxidase secreting system using B. subtilis has the advantage of endospore-forming which can applied for the continuous Bio-Fenton reaction. This study must shed light to advance the Bio-Fenton reaction for the oxidative degradation of organic pollutants and valorization of plastics.