Enzymatic bioremediation of polycyclic aromatic hydrocarbons (PAHs) in contaminated soil: a study on the recombinant laccase TVL.

Polycyclic aromatic hydrocarbons (PAHs) are pervasive and persistent pollutants in contaminated soil, posing a severe health and environmental threat. Enzymatic bioremediation presents a viable solution for the remediation of PAH-contaminated soil. In this study, a recombinant laccase with the encoding gene originating from Trametes villosa and recombinantly expressed in Aspergillus oryzae, designated as TVL, was discovered to possess strong PAH reduction capabilities. The specific enzyme activity of TVL was 73485 and 5102 LAMU/g enzyme protein at pH 5.0/7.0 and 37°C. Furthermore, it exhibited significant benzo[a]pyrene degradation, with 100% and 90.48% degradation at pH 5.0/7.0 after 24 h in the liquid phase. The degradation process of benzo[a]pyrene in soil was thoroughly investigated. Optimal conditions were identified as 15 mg/g NK-BSoil-3 and 1.35 mg/g HBT, resulting in a removal rate of 37.54% within 7 days when 0.01 U/g of TVL was applied. The potential mechanisms were investigated using molecular docking simulation. The binding energy between benzo[a]pyrene and TVL protein is notably robust, suggesting a higher propensity for enzyme binding. The TVL protein pocket contains nine amino acids that can interact most strongly with benzo[a]pyrene. Consequently, the recombinant laccase TVL holds considerable practical significance in bioremediation.

Removal of benzo[a]pyrene from the soil by adsorption coupled with degradation on saponin-modified bentonite immobilized crude enzymes.

Bentonite is a non-metallic mineral with montmorillonite as the main component. It is an environmentally friendly mineral material with large reserves, wide distribution, and low price. Bentonite can be easily modified organically using the surfactant saponin to obtain saponin-modified bentonite (Sap-BT). This study investigates the immobilization of crude enzymes obtained from Trametes versicolor by physical adsorption with Sap-BT. Thus, saponin-modified bentonite immobilized crude enzymes (CE-Sap-BT) were developed to remove benzo[a]pyrene. Immobilization improves the stability of free enzymes. CE-Sap-BT can maintain more than 80% of activity at 45 °C and after storage for 15 d. Additionally, CE-Sap-BT exhibited a high removal rate of benzo[a]pyrene in soil, with 65.69% after 7 d in highly contaminated allotment soil and 52.90% after 6 d in actual soil contaminated with a low concentration of benzo[a]pyrene at a very low laccase dosage (0.1 U/3 g soil). The high catalytic and removal performance of CE-Sap-BT in contaminated sites showed more excellent practical application value.

Bioremediation of PAHs-contaminated site in a full-scale biopiling system with immobilized enzymes: Removal efficiency and microbial communities.

Bioremediation of PAHs-contaminated soil by immobilized enzymes is a promising technology. Nevertheless, the practical implementation of highly efficient enzymatic remediation remains confined to laboratory settings, with limited experience in full-scale applications. In this study, the extracellular enzymes from white rot fungi are fully applied to treat sites contaminated with PAHs by combining a new hydrogel microenvironment and a biopiling system. The full-scale project was conducted on silty loam soil contaminated with PAHs. In line with China's guidelines for construction land, 7 out of the 12 PAHs identified are considered to be a threat to the soil quality of construction sites, with benzo[a]pyrene levels reaching 1.50 mg kg-1, surpassing the acceptable limit of 0.55 mg kg-1 for the first type of land. After 7 days of remediation, the benzo[a]pyrene level decreased from 1.50 mg kg-1 to 0.51 mg kg-1, reaching the remediation standard of Class I screening values, with a removal rate of 66%. Microbiomes were utilized to assess the microbial biodiversity and structure analyses for PAHs biodegradation. The remediation enhanced the abundance of dominant bacterium (Marinobacter, Pseudomonas, and Truepera) and fugin (Thielavia, Neocosmospora, and Scedosporium). The research offers further insights into the exploration of soil remediation on the full-scale of the immobilized enzyme and biopiling technology.

Bioremediation of 2,4,6-trichlorophenol by extracellular enzymes of white rot fungi immobilized with sodium alginate/hydroxyapatite/chitosan microspheres.

Hydroxyapatite, a calcium phosphate biomass material known for its excellent biocompatibility, holds promising applications in water, soil, and air treatment. Sodium alginate/hydroxyapatite/chitosan (SA-HA-CS) microspheres were synthesized by cross-linking sodium alginate with calcium chloride. These microspheres were carriers for immobilizing extracellular crude enzymes from white rot fungi through adsorption, facilitating the degradation of 2,4,6-trichlorophenol (2,4,6-TCP) in water and soil. At 50 °C, the immobilized enzyme retained 87.2% of its maximum activity, while the free enzyme activity dropped to 68.86%. Furthermore, the immobilized enzyme maintained 68.09% of its maximum activity at pH 7, surpassing the 51.16% observed for the free enzyme. Under optimal conditions (pH 5, 24 h), the immobilized enzymes demonstrated a remarkable 94.7% removal rate for 160 mg/L 2,4,6-TCP, outperforming the 62.1% achieved by free crude enzymes. The degradation of 2,4,6-TCP by immobilized and free enzymes adhered to quasi-first-order degradation kinetics. Based on LC-MS, the plausible biodegradation mechanism and reaction pathway of 2,4,6-TCP were proposed, with the primary degradation product identified as 1,2,4-trihydroxybenzene. The immobilized enzyme effectively removed 72.9% of 2,4,6-TCP from the soil within 24 h. The degradation efficiency of the immobilized enzyme varied among different soil types, exhibiting a negative correlation with soil organic matter content. These findings offer valuable insights for advancing the application of immobilized extracellular crude enzymes in 2,4,6-TCP remediation.

Performance enhancement of white rot fungi extracellular enzymes via new hydrogel microenvironments for remediation of benzo[a]pyrene contaminated soil.

Organic pollutants with low solubility and high ecotoxicity, mutagenicity, and carcinogenicity, are rapidly entering and accumulating in soil, resulting in soil pollution. Several methods have been investigated for remediation of organic contaminated soil, including enzymatic remediation approach. However, free enzymes are easily deactivated, which hinders their practical application in soil remediation. Immobilization of enzyme improves its stability and catalytic performance, but the immobilized material itself becomes secondary pollutants in soil. In this study, Trametes versicolor extracellular enzyme was immobilized on the degradable calcium alginate hydrogel microspheres. The laccase maintained a high activity. In addition, the addition of cellulose improved the strength of the carrier. Hydrogel microspheres solved the problems of easy inactivation of free enzyme and secondary contamination of immobilized materials. By a novel combination of extracellular enzymes and hydrogel microenvironments, immobilized enzymes not only demonstrate outstanding performance in thermal stability and pH adaptability, but also achieves a significant improvement in biocatalytic activity for benzo[a]pyrene contaminated soil. The thermal stability of immobilized enzyme was much higher than that of free enzyme. When the temperature increased to 50 °C, the activity of immobilized enzyme remained at 93.15% of the maximum enzyme activity, while the activity of free enzyme decreased to 63.76%. At pH 8, the immobilized enzyme activity maintained 74.84% of the maximum enzyme activity, while the free enzyme activity was only 11.86%. Immobilized enzymes can effectively remove 91.40% of benzo[a]pyrene from soil within 96 h. Furthermore, the catalytic oxidation of benzo[a]pyrene by enzymes that have been immobilized ultimately results in the production of 6,12-benzo[a]pyrene-dione. Molecular dynamics simulation showed that the catalytic degradation of benzo[a]pyrene was mainly through the interaction of amino acid residues PRO-391 with the Pi-alkyl of benzo[a]pyrene. This study presents an innovative strategy for designing and developing immobilized enzymes for use in biocatalytic applications related to eco-remediation of soil.