Eco-friendly treatment of municipal wastewaters by means of fungal enzymes

Since global consumption of fresh water doubles every 20 years, and water availability is increasingly limited due to pollution, the purification of water is crucial. In Italy, many efforts have been done to remove pathogens and pollutants but, due also to the lack of specific law restriction, the removal of persistent micropollutants is not a primary objective. Nowadays, the integration of conventional biological processes (mainly using bacteria) with chemical and physical ones does not provide satisfactory results, causing the progressive and constant accumulation of these recalcitrant pollutants into the environment, free to enter again into the water cycle. In the past years, the massive exposure to EDCs has posed institutional and scientific concerns: even at low doses, they can interfere with the endocrine system in mammals and aquatic organisms (Frye et al. 2012). Several household and industrial products show estrogenic activity, raising the social, economic and environmental interest for their removal (Benotti et al. 2009). An enzymatic process opens up new eco-friendly scenarios for the treatment of contaminated wastewaters: they are versatile, robust and indeed able to transform a wide spectrum of xenobiotics. In particular, due to their versatility and non-selectivity, fungal laccases are considered promising catalysts in micropollutants degradation. In the present study, laccases were obtained by a focused stimulation of Trametes pubescens MUT 2400, which previously demonstrated to express a powerful enzymatic pathway. The laccase-mediated treatment was used towards real municipal wastewaters containing recognized EDCs, pharmaceuticals, PCPs, pesticides, etc. A multishot stir bar sorptive extraction with targeted in-situ derivatisation and a gaschromatography-mass spectrometric was used for analytes detection. This analytical method represents an innovative approach, enabling the contemporary detection of several target analytes in complex matrix effect and their quantification to ppt level (ng/l). Once the enzymatic treatment was optimized by using model spiked solution, few water samples were collected in real municipal wastewater treatment plant of the district area of Torino and used to assess the capability of laccases to work also in extreme environments. Laccases degraded most of the detected compounds in a range of 50-96% within 24 h, slightly reducing also COD values. The enzymatic treatment was able to operate towards samples collected in different point of the plant and during repeated collection time, varying then for the quality the detected compounds and their actual concentration. It is important to underline that the final effluent of the plant was less polluted but several compounds were still detected at μg/l. The proposed system seems to better respond to the chemico-physical properties of this sample: due to the less disturbing solution, the final recovery of laccase activity was higher than the other wastewaters (60% vs 40%). More in detail, the initial pollutants concentration of the untreated effluent (120-15700 ng/l) was considerably lessened by the enzymatic treatment and the amount of five compounds decreased even below 100 ng/l. Besides, chemical analyses were coupled with a wide ecotoxicological assessement, in order to evaluate the deleterious effects of target compounds and their oxidized intermediates and ultimately predict the environmental impact associated to municipal wastewaters on the whole ecosystem. Two ecotoxicologycal tests (the plant Lepidium sativum and the algae Pseudokirchneriella subcapitata) were taken into consideration detecting a mild toxicity of the untreated wastewaters. Laccases treatment detoxified the samples. As regard the estrogenic activity (estradiol equivalent quantity, EEQ, through E-screen test and MELN assay), similar results were obtained, showing that the enzymatic treatment successfully operated towards this parameter, too, resulting in EEQ concentrations lower than those at which these compounds have been reported to chronically affect the endocrine systems of living organisms. According to these results, the degradation of at least a fraction of the chemical components of the samples significantly reduced its potential impact on the aquatic ecosystem and human beings. Further studies are in progress to optimize the operative parameters of the treatment (i.e. working pH, laccase concentration, enzyme immobilization), in order to enhance the stability and the efficiency of the laccase-mediated system.