Enzyme production by white-rot fungi immobilized in low-cost lignocellulosic materials, employed in citrus wastewater treatment

BENITEZ, S.F.; SCHRODER, M.; ZAPATA, P. D.; SADAÑOSKI, M. A.; LEVIN, L. N.; FONSECA, M. I.

Workshop; Third Meeting & First Workshop of the Argentine Network of Enzymatic Technology; 2021

Citrus-processing industries produce large volumes of waste waters (WW) characterized by high content of organic matter, color, presence of pesticides and terpenes. Although several strategies were developed for their treatment, current research focuses on their valorization as a source of bioactive and value-added compounds. In this context, white-rot fungi (WRF) are considered promising organisms for combined processes of bioremediation and enzyme biosynthesis. WW can inhibit fungal growth due to the presence of toxic compounds and/or bacterial proliferation, therefore fungal immobilization on low-cost lignocellulosic materials is proposed as an effective approach to increase their stability. The aim of this work was to evaluate enzyme production after citrus WW treatment under non-sterile conditions with WRF immobilized on lignocellulosic materials. The strains Irpex lacteus LBM 037, Phlebia brevispora LBM 036, Pleurotus pulmonarius LBM 105 and Trametes sanguinea LBM 023 were immobilized on sugar cane bagasse (SB), Panicum sp. seeds (PS) and Luffa cylindrical (LC). SB and PS were kindly provided by Coop. Ingenio Azucarera (San Javier, Misiones) and Clean Up (Posadas, Misiones), respectively. LC was purchased from El Cacique Limpieza (CABA, Buenos Aires). These materials were washed with tap water, rinsed with distilled water and oven-dried at 40 ± 2 °C. LC was cut into 1 cm3 pieces while SB was sieved and portions >1 mm were reserved for use. All the experiments were carried out in triplicate in 250 mL Erlenmeyer flasks containing 50 cm3 of substrate. Initial moisture content was adjusted to 75 % w/w with Czapek medium (sucrose 30 g/L; K2HPO4 1 g/L; KCl 0.5 g/L; MgSO4 7H2O 0.5 g/L; NaNO3 20 g/L). Autoclave-sterilized flasks were inoculated with three agar plugs (~7 mm diameter) of each strain and were incubated 14 days at 28 ± 1 °C under static conditions. Then, 50 mL of filtered citrus WW was added to the cultures and flasks were incubated for three extra days. Control consisted in WW without immobilized fungi. Supernatants were obtained by centrifugation at 4600 g for 10 min and enzyme activities were evaluated by spectrophotometric assays. Laccase (EC 1.10.3.2) activity was determined using 2,6-dimethoxyphenol (5 mmol/L) as substrate. Endoxylanase (EC 3.2.1.136), endoglucanase (EC 3.2.1.4) and amylase (EC 3.2.1.1) activities were assayed using beechwood xylan (1 % w/v), carboxymethylcellulose (2 % w/v) and soluble starch (2 % w/v) as substrates respectively; reducing sugars were measured by 1,3-dinitrosalicylic acid assay. β-glucosidase (EC 3.2.1.21) and cellobiohydrolase (EC 3.2.1.91) activities were determined using p-nitrophenyl-β-D-glucopyranoside (1 mmol/L) and p-nitrophenyl-β-D-cellobioside (1 mmol/L) as substrates, respectively. Laccase activity was detected in P. brevispora LBM 036, P. pulmonarius LBM 105 and T. sanguinea LBM 023 cultures immobilizedon PS, respectively: 1121.02 ± 41.04, 1022.51 ± 31.32 and 446.73 ± 27.00 U/L. Laccase activity was also measured in P. brevispora LBM 036 cultures immobilized on SB (331.27 ± 29.59 U/L). Endoxylanase, cellobiohydrolase and amylase activities were observed in alt he assays with immobilized fungi and in the control. The highest endoxylanase and amylase activities were produced by T. sanguinea LBM 023 immobilized on SB and PS, respectively 1571.43 ± 92.60 and 2178.87 ± 151.71 U/L. The highest cellobiohydrolase activity was produced by P. pulmonarius LBM 105 (60.30 ± 3.09 U/L). Low quantities of β-glucosidase activity were detected in all the assays (