Reutilization of citrus industry wastewater for the production of fungal laccases

MILLÁN ESTEBAN ROGELIO; BENITEZ SILVANA FLORENCIA; CHELALICHE ANIBAL SEBASTIÁN; ACOSTA GABRIELA ALEJANDRA; ZAPATA PEDRO DARIO; FONSECA MARÍA ISABEL

Congreso; LIX Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research (SAIB); 2023

Citrus-processing industries produce large volumes of wastewater (WW) characterized by a highcontent of organic matter. An alternative for their management is their reuse to obtain value-addedproducts. In this sense, white-rot fungi (WRF) can be applied for enzyme biosynthesis. However, WW can inhibit fungal growth, hence, fungal tolerance and strategies to improve it should be evaluated. The aim of this work was to evaluate Trametes sanguinea BAFC 2126 tolerance towards citrus WW, and laccase (Lac) production in solid and liquid media using Luffa cylindrica as immobilization substrate. Citrus WW and L. cylindrica were kindly provided by Cooperativa Citrícola Agroindustrial de Misiones Ltda. (Leandro N. Alem, Misiones) and Espudela (Jardín América, Misiones), respectively. Tolerance was evaluated in 85 mm ø Petri dishes in triplicate containing 12 mL of MEA (malt extract 12.7 g.L-1, agar 17 g.L-1) supplemented with filtered citrus WW (25, 50, 75 and 100 %); pH was adjusted to 5.7 ± 0.1 with 1 M NaOH. One agar plug (7 mm ø) was inoculated; fungal radial growth was measured daily and analyzed using predictive mycology (Sadañoski et al. 2018) to determine τ (time to attain half the plate) and κ (growth rate) values. Lac production was revealed at the moment the mycelium covered 50 % of the plate by adding 10 mL of 2,6-dimethoxyphenol (DMP) 5 mM in sodium acetate buffer 0.1 M (pH 3.6) and incubating in gentle agitation in darkness for 20 min. For Lac production in liquid media, experiments were carried out in triplicate in 250 mL Erlenmeyer flasks containing 1 g of L. cylindrica (1 cm3) and 50 mL of liquid media. Culture media consisted in different concentrations of citrus WW (0, 25, 50, 75, and 100 % v.v-1), concentrations were adjusted with distilled water. Aditionally, the effect of CuSO4 (0.5 mM) was evaluated. Initial pH was adjusted to 5.7 ± 0.1 with 1 M NaOH. Autoclave-sterilized flasks (105 °C, 20 min) were inoculated with three agar plugs (~7 mm ⌀) of the strain and incubated for 19 d at 28 ± 1 °C in static or agitation condition (100 rpm), samples were taken periodically. Lac activity was evaluated in spectophotometer using DMP in a sodium acetate buffer 0.1 M (pH 3.6, 30 °C) as substrate. Tolerance assays showed that the strain BAFC 2126 was able to grow and produce Lac in all the concentrations tested, with an average τ and κ of 3.17 ± 0.08 d and 0.92 ± 0.03 mm.d-1, respectively. As for Lac production in liquid media, the highest yield was obtained with 100 % v.v-1 citrus WW after 15 d of incubation in agitation without the addition of CuSO4 (2539.18 ± 443,44 U.L-1); followed by cultures with 50 % v.v-1 after 9 d of incubation in agitation with CuSO4 (1272.6 ± 4.16 U.L-1). These results suggest that citrus WW can be reused to produce enzymes of biotechnological interest.