Application of white-rot fungi for the biodegradation of natural organic matter in wastes

摘要

Natural organic matter (NOM), a complex mixture of organic compounds, influences drinking water quality and water treatment processes. The presence of NOM is unaesthetic in terms of colour, taste and odour, and may lead to the production of potentially carcinogenic disinfection by-products (DBPs), as well as biofilm formation in drinking water distribution systems. Some NOM removal processes such as coagulation, magnetic ion exchange resin (MIEXTM) and membrane filtration produce sludge and residuals. These concentrated NOM-containing sludges from alum precipitation, membrane treatment plants and MIEX regeneration must therefore be treated prior to disposal. The white-rot fungi possess a non-specific extracellular oxidative enzyme system composed of lignin peroxidase (LiP), manganese-dependent peroxidase (MnP) and laccase (Lac) that allows these organisms to mineralise lignin an d a broad range of intractable aromatic xenobiotics. Rojek (2003) has shown the capability of Phanerochaete chrysosporium ATCC 34541 to remove 40-50% NOM from solution, however, this was found to be mainly due to adsorption and to be a partially metabolically linked activity. Consequently, the bioremediation of NOM wastes by selected white-rot fungi was further investigated in the present study. The P. chrysosporium seemed to preferentially remove the very hydrophobic acid (VHA) fraction, and so was most effective for a NOM preparation with a high proportion of hydrophobic content (and so high in colour and specific UV absorbance (SUVA)). The extent of NOM decolourisation by P. chrysosporium in three growth media with different C:N ratios followed the trends: Waksman (C:N = 6) & Fahy (C:N = 76) & Fujita medium (C:N = 114), such that the lower the C:N ratio, the greater NOM removal. This was consistent with the findings of Rojek (2003), who used a different NOM preparation and demonstrated that the removal of NOM increased with decreased C:N ratio (1.58-15.81). As removals of NOM with P. chrysosporium ATCC 34541 were low, and little biodegradation occurred, this organism was compared with P. chrysosporium strain ATCC 24725, Trametes versicolor ATCC 7731, and three strains of yeast (Saccharomyces species arbitrarily denoted 1, 2 and 3). T. versicolor gave the greatest removal (59%) which was attributed largely to degradation, whereas the NOM removal by the two strains of P. chrysosporium (37%) and the yeast was predominantly due to adsorption as indicated by the deep brown colouration of the biomass. Saccharomyces sp. 1, 2 and 3 removed 12%, 61% and 23% of the colour, respectively. Although Saccharomyces sp. 2 had similar high colour reduction to T. versicolor, the specific removal values differed markedly: 0.055 compared to 0.089 mg NOM/mg biomass, respectively. The low level of the ligninolytic enzymes secreted by both strains of P. chrysosporium corresponded with the low degree of NOM removal by biodegrada tion as shown by high performance size exclusion chromatography (HPSEC). The high NOM removal attained by T. versicolor was attributed to the activities of the ligninolytic enzymes, especially laccase. The NOM removal was attributed to the breakdown of the high molecular weight compounds to form a pool of low molecular weight materials, which were then most likely utilised by the T. versicolor. Growth of T. versicolor cultures at 36oC caused inhibition or denaturation of the activity of the phenoloxidase enzymes compared to those grown at 30oC. The low activity of LiP in both cultures suggested that this enzyme may not play much of a role in NOM removal. The higher levels of MnP and Lac activities at 30oC were responsible for the greater NOM removal (73% vs. 59%) and thus the cleavage of aromatic rings, conjugated and C-Cβ αbonds in phenolic moieties, as well as catalysing alkyl-aryl cleavage in the NOM structures. T. versicolor cultured in Waksman medium with higher initial glucose (5 g/L cf. 2 g/ L) led to lower ligninolytic enzyme activities and a lower degree of NOM removal (25% less colour reduction), probably due to preferential use of glucose over NOM as carbon source. NOM removal (mg removed) increased linearly with NOM concentration up to 600 mg C/L (62 mg (A446); 31 mg (A254)), above which removal decreased markedly. This trend coincided with increasing total ligninolytic enzyme activity, where the level of Lac increased up to 600 mg C/L NOM although MnP decreased gradually across the range while LiP was only detected for 100 and 300 mg C/L NOM. Hence, the removal of NOM from solution by T. versicolor was associated with high oxidative enzyme activity, particularly of laccase. Laccase was the major extracellular enzyme secreted by T. versicolor and by deduction, played a major role in NOM removal. The optimum temperature for Lac activity secreted by T. versicolor cultured in Waksman medium supplemented with 4.5 g/L wheat bran plus 0.5% Tween 80 was determined to be 50oC. The optimum pH for th e Lac activity for guaiacol and NOM was identified as pH 4.0-4.5. Although the optimum enzyme activity occurred at 50oC, 30oC was recommended for enzymatic removal of NOM as the phenoloxidase enzyme activity may be denatured if the NOM removal process were considered to run for long period at high temperature. Although agitation led to apparent enzyme denaturation, fermentations with continuous agitation promoted enzyme activity faster than those with occasional agitation (agitated every 6 hours for 30 minutes at 130 rpm and 30oC) as it provides better mass transfer. However, it seemed that continuous agitation had an adverse effect on the fungal growth and enzyme production over extended fermentation periods. Addition of 4.5 g/L wheat bran to modified Waksman medium in the absence of NOM led to high production of Lac activity compared with LiP and MnP activities, showing its great potential as a laccase inducer. Addition of Tween 80 alone to the cultures led to a small improvement in Lac activity; however, with the presence of wheat bran it caused marked increases in LiP, MnP and Lac activities. When NOM was added to cultures of T. versicolor with the two supplements, it led to markedly reduced Lac activity, but increased LiP and MnP activities, and no improvement in NOM removal compared with the cultures in the absence of supplements (12 mg (or 61%) cf. 15 mg (or 73%) for 100 mg C/L after corrected for colour from and adsorption by wheat bran).