Adaptation of the tween 80 assay with a resolution V fractional factorial design and its application to rank Ophiostoma fungi with wood extractive degrading capabilities

Wood extractives in radiata pine are the source of the observed pitch problems in the pulp and paper industry. Various methodologies have been studied and used to reduce or eliminate their negative effects. A biological treatment with albino fungi of the Ophiostoma genus, fungi that degrade the lipidic components of extractives, has been proposed as a more environmentally friendly alternative for pitch degradation. The current methods used to search for Ophiostoma albino strains with the highest degradation rates of wood extractives are labor intensive and require a large amount of resources. The Tween 80® Opacity test, an assay designed to measure lipolytic enzyme activity in filamentous fungi, was studied to verify the feasibility of its application as a methodology to rank Ophiostoma albino strains with deresination capabilities. The Tween 80® Opacity Test was first characterized by implementing a non-replicated 2k-p fractional factorial design of resolution V for a 5 factors with 16 treatments and then to study the effect of the fungus species on the lipolytic enzyme activity; a randomized one factor general factorial design was conducted. The incubation temperature; antibiotics presence; and Tween 80®, CaCl2, and peptone concentrations were investigated in the first experiment. The Ophiostoma species effect was studied in the second experiment. In both experiments, the halo area size, which was formed by the fatty acid-calcium complex precipitate, was the response variable. The results demonstrated the effectiveness of the Tween 80® opacity test to measure the lipolytic enzyme activity of Ophiostoma albino strains. Similarly, the incubation temperature and the concentrations of Tween 80®, CaCl2, and bacteriological peptone had the highest statistically significant effect on the response variable. In addition, our data demonstrated that the albino strains from the specie O. floccosum exhibited the highest rate of lipolytic enzyme production.