Biodegradation of fluoranthene and anthracene by indigenous fungi isolated from wastewater activated sludge.

Abstract

Environmental pollution by polycyclic aromatic hydrocarbons (PAHs) poses a significant threatowing to their persistence and toxicity. This thesis investigates the potential of indigenous fungi isolated from wastewater-activated sludge to biodegrade PAHs fluoranthene and anthracene. This study also advances the understanding of the degradation mechanisms, enzymatic pathways, optimization strategies, and catalytic efficiency of purified Laccases as the predominant enzymes utilized by these fungi. Empirical information regarding the characterization, structures, catalytic efficiency, and biological process functions of the purified Laccases is also presented. Two indigenous ascomycete fungi, Trichoderma lixii strain FLU1 (TlFLU1) and Talaromyces pinophilus strain FLU12 (TpFLU12), were isolated from benzo(b)fluoranthene-enriched activated sludge and tested for their ability to degrade fluoranthene and anthracene as sole carbon sources. TlFLU1 and TpFLU12 degraded 98% and 99% of 400 mg/L fluoranthene, respectively, after 16 and 12 d of incubation. This degradation was associated with the upregulation of ligninolytic enzymes (Laccase, Lignin peroxidase, and Manganese peroxidase). GC-MS and FTIR analyses of the degradation products indicated that degradation was initiated at the C1-C2 position via oxygenation and ring cleavage to form 9-oxo-9H-fluorene-1-carboxylic acid before progressing through the β-ketoadipate pathway via benzene-1,2,3-tricarboxylic acid. The degradation kinetics followed first order and zero-order models for TlFLU1 and TpFLU12. Metabolites from TlFLU1 degradation media showed toxicity to Vibrio parahaemolyticus after 6 h of exposure, with effective concentration (EC50) and toxicity unit (TU) values of 14.25 mg/L and 7.018%, respectively. In contrast, TpFLU12 degradation media was non-toxic, with EC50 and TU values of 197.1 mg/L and 0.507% respectively. For anthracene, both isolates tolerated exposure of up to 1000 mg/L, with increased ligninolytic enzyme expression (Laccase, Lignin peroxidase, and Manganese peroxidase). The degradation of anthracene was growth-linked and mediated by ligninolytic and intracellular enzymes, resulting in 56% and 38% degradation of 400 mg/L by TlFLU1 and TpFLU12, respectively, after a 24 d incubation period, with pH changes from 5 to 4 (TlFLU1) and 6.2 (TpFLU12). GC-MS and FTIR analyses indicated the formation of 9,10- anthracenedione and benzoic acid as the metabolic products in the TlFLU1 medium and anthrone and 9,10-anthracenedione in the TpFLU12 medium. The degradation by TlFLU1 and TpFLU12 followed a first order kinetic model, with both degradation media metabolites showing no toxicity to V. parahaemolyticus after 6 h of exposure, with EC50 and TU values of 266.1 mg/L and 0.4% respectively, for TlFLU1 and 262.3 mg/L and 0.4% for TpFLU12. Under optimized conditions using response surface methodology (RSM) for anthracene degradation, 100% degradation efficiency was achieved for TlFLU1 and TpFLU12 on days 8 and 12, respectively, at pH 4 and 5 and temperatures of 30°C and 25°C, with 20 mm biomass and 200 mg/L anthracene. Acute toxicity tests revealed reduced media toxicity, as evidenced by the increased survival rate (log CFU/mL) of V. parahaemolyticus after 6 h of exposure. Despite reduced toxicity, both strains degradation media were classified as harmful based on EC50 and TU values of 20.92 ± 1.32 mg/L and 4.78% or TlFLU1, and 35.29 ± 1.55 mg/L and 2.83% for TpFLU12. However, the biocatalytic potential of purified laccases from TlFLU1 and TpFLU12 revealed a reduction in residual fluoranthene concentrations by 46.1% and 38.6% respectively, at 3 U/mL after 96 h. Higher enzyme concentrations (8 U/mL) further reduced the fluoranthene levels to 33.1% and 37.4%, with complete degradation observed at 10 U/mL of either enzyme. The addition of a mediator ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) significantly enhanced the degradation with a degradation kinetics of vmax values, 7.73 ± 0.23 mg/L/h and 7.97 ± 0.18 mg/L/h and Km value of 54.8 ± 0.27 mg/L and 26.6 ± 0.21 mg/L for TlFLU1 and TpFLU12 respectively. Without ABTS, vmax values of 1.35 ± 0.02 mg/L/h and 1.29 ± 0.02 mg/L/h with higher Km values of 119.2 ± 0.02 mg/L and 170.8 ± 0.03 mg/L for TlFLU-1 and TpFLU-12. GC-MS analysis of the degradation products identified a distinct degradation pathway different from that of whole fungal cell with TlFLU1 generated products such as 9-oxo-fluorene-1-carboxylic acid, 9H-fluoren-9-one, and phthalic acid through dioxygenation at the C1-C2 bond. In contrast, TpFLU12 produces 9,10- phenanthrenedione and benzene-1,2,3-tricarboxylic acid via C2-C3 bond cleavage. Ecotoxicity assessments of the degradation products using V. parahaemolyticus and cytotoxicity using the HT- 22 cell line indicated potential toxicity of TlFLU1 products to V. parahaemolyticus, whereas TpFLU12 products were primarily non-toxic. However, complete detoxification was achieved with the degradation products containing ABTS. Also, for anthracene, purified Laccases from TlFLU1 and TpFLU12 degrade anthracene with vmax values of 3.51 ± 0.06 mg/L/h and 3.44 ± 0.06 mg/L/h respectively, and Km values of 173.2 ± 0.06 mg/L and 73.3 ± 0.07 mg/L respectively. The addition of ABTS as a mediator increased degradation by up to 2.9-fold in vmax values and reduced Km values by up to threefold. GC-MS analysis suggested a unique pathway involving hydroxylation and carboxylation at C-1 and C-2 to form 3-hydroxy-2-naphthoic acid, leading to the formation of chromone and subsequent benzoic acid and CO2. This pathway is in contrast with the dioxygenation route observed in whole fungal cells. Furthermore, toxicity tests using V. parahaemolyticus and HT-22 cells demonstrated the nontoxic nature of laccase-ABTS-mediated metabolites. Intriguingly, analysis of the expression level of Alzheimer’s-related genes in HT-22 cells exposed to degradation products revealed no induction of neurotoxicity, unlike untreated cells. Furthermore, characterization of TlFLU1 and TpFLU12 laccases revealed that they have molecular masses of 44 kDa and 68.7 kDa, respectively. These enzymes differ in their optimum pH and temperature, with one laccase being more active at a lower pH (TlFLU1) and the other being more active at a higher pH (TpFLU12). Their activity was enhanced by a broad range of metal ions and organic solvents, and sodium azide was a potent inhibitor of its activity. Both laccases have similar kinetic properties, with a better affinity for ABTS as a substrate compared to other phenolic substrates. Based on structural predictions, the difference in the optimum pH is likely due to the different arrangements of copper atoms in the active site. In addition, oxidation reduction, lignin metabolism, phenylpropanoid catabolism, biological adhesion, cellular metabolism, cellular metal ion homeostasis, aromatic compound metabolism, and cellulose metabolism have been linked to laccase biological functions. The catalytic efficiency of laccases has been observed to depend primarily on their structural conformation and stability. The Laccaseamino acid conjugates were predicted to be stable, with an instability index ranging from 33.43 to 39.45, thermophilic, with an aliphatic index of 76.58 to 77.50, and hydrophilic, with a grand average of hydropathicity between -0.508 and -0.578. The predicted biological functions of the protein include oxidation-reduction, lignin metabolism, cellular metal ion homeostasis, phenylpropanoid catabolism, aromatic compound metabolism, cellulose metabolism, and biological adhesion. These insights into the enzyme structure-function relationship enhance our understanding of its catalytic mechanism, paving the way for improved bioremediation strategies.