Understanding the role of soil microbes and their associated extracellular enzymes in long-term grassland experiments presents an opportunity for testing relevant ecological questions on grassland nutrient dynamics and functioning. Veld fertilizer trials initiated in 1951 in South Africa were used to assess soil functional microbial diversity and their metabolic activities in the nutrient-poor grassland soils. Phosphorus and liming trials used for this specific study comprised of superphosphate (336 kg ha⁻¹) and dolomitic lime (2250 kg ha⁻¹) (P + L), superphosphate (336 kg ha⁻¹) (+ P) and control trials. These soils were analyzed for their nutrient concentrations, pH, total cations and exchange acidity, microflora and extracellular enzyme activities. The analysed soil characteristics showed significant differences except nitrogen (N) and organic carbon (C) concentrations showing no significant differences. P-solubilizing, N-cycling and N-fixing microbial diversity varied among the different soil treatments. β-glucosaminidase enzyme activity was high in control soils compared to P-fertilized and limed soils. Alkaline phosphatase showed increased activity in P-fertilized soils, whereas acid phosphatase showed increased activity in control soils. Therefore, the application of superphosphate and liming influences the relative abundance of bacterial communities with nutrient cycling and fixing functions which account for nutrient bioavailability in acidic and nutrient stressed grassland ecosystem soils.

Broad-spectrum biocatalysts enzymes, Laccases, have been implicated in the complete degradation of harmful pollutants into less-toxic compounds. In this study, two extracellularly produced Laccases were purified to homogeneity from two different Ascomycetes spp. Trichoderma lixii FLU1 (TlFLU1) and Talaromyces pinophilus FLU12 (TpFLU12). The purified enzymes are monomeric units, with a molecular mass of 44 kDa and 68.7 kDa for TlFLU1 and TpFLU12, respectively, on SDS-PAGE and zymogram. It reveals distinct properties beyond classic protein absorption at 270–280 nm, with TlFLU1's peak at 270 nm aligning with this typical range of type II Cu site (white Laccase), while TpFLU12's unique 600 nm peak signifies a type I Cu²⁺ site (blue Laccase), highlighting the diverse spectral fingerprints within the Laccase family. The K_m and k_cat values revealed that ABTS is the most suitable substrate as compared to 2,6-dimethoxyphenol, caffeic acid and guaiacol for both Laccases. The bioinformatics analysis revealed critical His, Ile, and Arg residues for copper binding at active sites, deviating from the traditional two His and a Cys motif in some Laccases. The predicted biological functions of the Laccases include oxidation–reduction, lignin metabolism, cellular metal ion homeostasis, phenylpropanoid catabolism, aromatic compound metabolism, cellulose metabolism, and biological adhesion. Additionally, investigation of degradation of polycyclic aromatic hydrocarbons (PAHs) by purified Laccases show significant reductions in residual concentrations of fluoranthene and anthracene after a 96-h incubation period. TlFLU1 Laccase achieved 39.0% and 44.9% transformation of fluoranthene and anthracene, respectively, while TpFLU12 Laccase achieved 47.2% and 50.0% transformation, respectively. The enzyme structure–function relationship study provided insights into the catalytic mechanism of these Laccases for possible biotechnological and industrial applications.

The potential of Pleurotus pulmonarius and Pleurotus ostreatus on the degradation of PAHs in spent and fresh cutting fluids (SCF and FCF) contaminated soils was investigated. Different weights of soil samples were contaminated with varying composition (10, 20 or 30%) of spent and fresh cutting fluids separately then inoculated each sample with P. pulmonarius and P. ostreatus separately and incubated at 28 ± 2°C for two months. The samples were analyzed in triplicates for 16 priority polycyclic aromatic hydrocarbons (PAHs) by gas chromatography after extracting with hexane and dichloromethane (3:1). The initial PAHs in the cutting fluids were mainly composed of 2 to 6 fused benzene rings. Significant reductions in PAHs concentrations for SCF and FCF were observed after two months of incubation. The percentage total PAHs remaining in FCF soil ranged from 71.7 to 73.6% when inoculated with P. pulmonarius and 0.93 to 31.0% when inoculated with P. ostreatus. Similarly, the percentage total PAHs remaining in SCF soil ranged from 42.6 to 72.6% when inoculated with P. pulmonarius and 54.9 to 62.2% when inoculated with P. ostreatus. Overall range of PAHs degradation by P. pulmonarius inoculated on FCF contaminated soil was 17.3 to 27.3%, while for P. ostreatus inoculated soil was 69.0 to 99.07% at different contamination levels. In contrast, overall PAHs degradation for P. pulmonarius and P. ostreatus inoculated on SCF ranged from 27.4 to 57.4% and from 37.8 to 45.2%, respectively. Thus, P. ostreatus is found more effective as a biodegradation agent for PAHs in contaminated soils when compared with P. pulmonarius. Key words: Biodegradation, polycyclic aromatic hydrocarbons (PAHs), cutting fluids, Pleurotus ostreatus, Pleurotus pulmonarius.

Two indigenous ascomycetes fungi, Trichoderma lixii strain FLU1 (TlFLU1) and Talaromyces pinophilus strain FLU12 (TpFLU12), were isolated from benzo(b)fluoranthene-enriched activated sludge and tested for bio-catalytically degrade fluoranthene as a sole carbon source. TlFLU1 and TpFLU12 degraded 98 and 99% of 400 mg/L of fluoranthene after 16 and 12 d incubation period, respectively. Degradation correlated with the upregulation of expression of ligninolytic enzymes. The GC-MS and FTIR analysis of the degradation products suggest that the degradation is initiated at the C1-C2 position of the compound ring via oxygenation and ring cleavage to form 9-oxo-9H-fluorene-1-carboxylic acid before undergoing ring cleavage to yield fluorenone, which then proceeds through the ß-Ketoadipate pathway via benzene-1,2,3-tricarboxylic acid. The degradation rate is better fitted in the first-order and zero-order kinetic model for TlFLU1 and TpFLU12, respectively. The metabolites from the TlFLU1 degradation media are shown to be toxic in Vibryo parahaemolyticus after 6 h of exposure with effective concentration (EC₅₀) and toxicity unit (TU) values of 14.25 mg/L and 7.018%, respectively, while also being observed as non-toxic from TpFLU12 degradation media with an EC₅₀ and TU values of 197.1 mg/L and 0.507%, respectively. Results from this study show efficient metabolism of fluoranthene into an innocuous state by TlFLU1 and TpFLU12.