Enhancing wastewater treatment efficiency through laccase-mediated biodegradation of trace organic contaminants

Abstract

Recently, concerns have been raised about trace organic contaminants (TrOCs) in wastewater and their potential adverse effects on human health and the environment. TrOCs found in wastewater are a cause for concern due to their ability to contaminate drinking water sources and the environment. Consuming contaminated water with TrOCs can pose health risks to humans. It can also harm aquatic life, leading to ecosystem disruptions and potential entry into the food chain. Additionally, certain TrOCs have properties that can disrupt endocrine systems, which can further impact human health and the stability of the environment. Researchers have explored various treatment methods, including fungal treatments and laccase enzymes. However, there needs to be more literature regarding selecting fungal strains and optimizing laccase-based biodegradation mechanisms for efficient TrOC removal. This thesis aims to fill that gap by delving into laccase production, purification, immobilization, and its impact on degrading TrOCs. By exploring various aspects of this process, we can work towards developing more efficient and sustainable methods of reducing the negative impact of TrOCs on our environment. The current study encompasses a comprehensive examination of different fungal strains, including Trametes hirsuta, Pleurotus dryinus, Tramates versicolor, and Coriolopsis polyzona, to evaluate their laccase productivity and response to trace elements. The sorption mechanisms of laccase-mediated fungal treatment on targeted TrOCs were also explored. Furthermore, the study investigated free Laccase's purification and removal efficiency and the potential of laccase mediator systems (LMS) and immobilization techniques for enhanced biodegradation. The findings from this study provided valuable insights into the optimization and cost-effectiveness of laccase-based bioremediation strategies for sustainable wastewater treatment and environmental protection. Throughout experiments, fungal strains have been found to play a crucial role in producing external laccase activities. One of the advantages of using fungi is its high adsorption capacity, thus making it an effective pollutants-removal method. It is interesting to note that the reactions of internal and external enzymes on selected compounds differ from one compound to another. It has been observed that Laccase does not affect all types of TrOCs. It is essential to establish reasonable system control measures to prevent the release of adsorbed contaminants or enhance laccase activities to optimize the fungal treatment. It also highlights the importance of degradation by external enzyme activity and/or adsorption mechanisms as a primary step in removing TrOCs, followed by internal enzyme activities that remove some of the existing compounds after reaching a specific limit of adsorption capacity. Following these experiments, the study focused on the efficiency of laccase-mediator systems in removing recalcitrant organic contaminants. The results showed that laccase-mediator systems efficiently removed recalcitrant organic contaminants such as atrazine (ATZ) and carbamazepine (CBZ) in individual compound solutions, even at environmentally relevant concentrations. The removal efficiency of ATZ and CBZ in single compound solutions sets was observed up to 100% during the first 36h, and no significant difference was observed for all the tested mediators. Regardless of single compound sets, ATZ and CBZ in mixed compound sets removal were less than 65%. Also, the phenolic contaminant acetaminophen (APAP) is examined as a potential mediator candidate for laccase, and the results showed positive results. The study on laccase mediator systems led to a sub-study on "Laccase Catalyzed Iodine Synthesis as Disinfectant." Results showed that acetophenone was a more efficient mediator than APAP for iodine synthesis. Increasing APAP concentration increased iodine synthesis with a maximum of 0.5 mM. High concentrations of KI (>20 mM) inhibited laccase activities. Under 10 U/L, 20 mM KI concentration was most efficient for enzyme activity, chemical consumption, and contaminant removal efficiency. APAP and other phenolic compounds in real wastewater could serve as a mediator and increase the system's efficiency. Laccase-catalyzed iodine demonstrated high disinfection performance in fecal coliform tests, with the optimal concentrations observed at 15 and 20 mM KI. The study examined the effectiveness of laccase immobilization and optimization. The immobilization was achieved by bonding and adsorbing on mesoporous silica-covered packing. The results showed that the immobilized laccase was more efficient and stable in harsh conditions than the free enzyme. It had broader optimal temperature and pH ranges and better storage stability. This study suggests that immobilization can be a promising solution for industries that rely on sensitive biological enzymes. Particularly noteworthy is the use of mesoporous silica-covered packing and laccase-abundant broth instead of pure laccase for the immobilization, which offers substantial financial benefits. This approach facilitates the immobilized enzyme at any desired time, allowing for multiple cycles of re-utilization. Such a strategy holds tremendous potential for enhancing the efficiency and sustainability of enzymatic processes in various industries. The resulting enzyme immobilization yield on the support was between 56-74 %, and the highest immobilization efficiency, 1.8 U/g packing, was achieved in the sets where initial laccase activity was limited (15 U). The unique part of this study was that the silica-covered mesoporous packing was immobilized by contacting with 15000 U laccase-abundant broth without the purifying step, which consumes many chemicals. The immobilization yield was 60%, and the highest activity was 346 U/g packing. The immobilization process remained consistent after 2 days, indicating that most laccase molecules were adsorbed within the pores without undesired lateral interactions. The immobilized laccase exhibited good reusability and retained more than 50 % of its initial activity after being stored at 4ºC for 5 months. It was addressed that the best pH was selected as 7 to condition silica-covered packing with glutaraldehyde, while the optimum pH was 3.5 or 5.5 to immobilize laccase on the silica-covered packing. The optimum glutaraldehyde concentration was also selected as 3%. Unfortunately, it is not enough to immobilize laccase; it requires a high amount of laccase immobilization, so it requires a high amount of laccase production. It examined the potential of non-polar solvent hexane and polar solvents, methanol, and ethanol, as inducers for laccase production and the established inducer copper. Also, the abundant hexane effluent from silica production is tested as an inducer to diminish the ecological footstep of the study. The impact of mesoporous silica-coated plastic packing on free laccase production and concomitant immobilization was evaluated under both sterilized and unsterilized conditions to understand if the costly step-sterilization can be avoided. The study revealed that copper and ethanol were the most effective inducers, with the highest total laccase activity achieved at a copper concentration of 0.5 mM under sterilized conditions. The groups treated with copper and ethanol also showed immobilized laccase activity under unsterilized conditions. Our evaluation of concomitant laccase immobilization showed that the sterilized waste hexane sets had the highest activity of immobilized laccase, with a value of 1.25 U/mg packing. The hexane sets under sterilized conditions had the second-highest immobilized enzyme activity. It was found that even though a small amount of immobilized laccase activity was detected in the packings of the copper and inducer sets in the unsterilized sets, the desired levels of free and immobilized laccase activity were not achieved in comparison to the sterilized sets. These results likely are because the sets were not sterilized, which could have led to other species due to planned -microbial contamination from the laboratory. This presence of several fungal species could have had a significant impact on the reduction of laccase activity. Using pure cultures and ensuring a sterilized system is essential to avoid contamination. This step of the study focuses on one of the most remarkable outcomes of the current research: the successful application of waste hexane as an inducer for concomitant immobilized laccase activity. This innovative approach to laccase production could lead to a promising method for decreasing running costs and improving the cost-efficiency of enzymatic processes in wastewater treatment plants, both industrial and domestic.