Halotolerance, Ligninase Production and Herbicide Degradation Ability of Basidiomycetes Strains
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Brazilian Journal of Microbiology 44, 4, 1207-1214 (2013) Copyright © 2013, Sociedade Brasileira de Microbiologia ISSN 1678-4405 www.sbmicrobiologia.org.br Research Paper Halotolerance, ligninase production and herbicide degradation ability of basidiomycetes strains R.L. Arakaki, D.A. Monteiro, M. Boscolo, R. Dasilva, E. Gomes Laboratório de Bioquímica Aplicada, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista “Júlio de Mesquita Filho”, São José do Rio Preto, SP, Brazil. Submitted: August 13, 2012; Approved: April 4, 2013. Abstract Fungi have been recently recognized as organisms able to grow in presence of high salt concentration with halophilic and halotolerance properties and their ligninolytic enzyme complex have an unspe- cific action enabling their use to degradation of a number of xenobiotic compounds. In this work, both the effect of salt and polyols on growth of the basidiomycetes strains, on their ability to produce ligninolytic enzyme and diuron degradation were evaluated. Results showed that the presence of NaCl in the culture medium affected fungal specimens in different ways. Seven out of ten tested strains had growth inhibited by salt while Dacryopinax elegans SXS323, Polyporus sp MCA128 and Datronia stereoides MCA167 fungi exhibited higher biomass production in medium containing 0.5 and 0.6 mol.L-1 of NaCl, suggesting to be halotolerant. Polyols such as glycerol and mannitol added into the culture media improved the biomass and ligninases production by D. elegans but the fungus did not reveal consumption of these polyols from media. This fungus degraded diuron in medium control, in presence of NaCl as well as polyols, produced MnP, LiP and laccase. Key words: basidiomycetes, halotolerance, ligninases, diuron degradation. ing the growth and activities of microorganisms (Petrovic Introduction et al., 2002; Abou-Elela et al., 2010). In this way, the utili- The ability to growth and the mechanisms involved in zation of salt-tolerant organisms in biological treatment adaptation to low water activity (aw) has been well under- could be an interesting approach. A successful degradation stood for prokaryote groups. However, only in the last de- of xenobiotics by halotolerant Penicillium spp, Trametes cade fungi were isolated from saline and hypersaline envi- and Flavodon flavus (Leitão et al., 2005; Cantrell et al., ronments (Moubasher et al., 1990; Cantrell et al., 2006) but 2010) has been described. their adjustment mechanisms to halophile conditions is still The increasing in the use of agricultural chemicals re- not clear. Two proteins, in the membrane and in the cyto- sults in the accumulation of these compounds and their de- plasm (HwHhk7 and HwSho1) have been reported to act as rivatives in soil and water. Diuron is a phenylurea herbicide osmosensors in halophile fungus which is a signal for cas- (N-(3,4-dichlorophenyl)-N,N-dimethylurea; CAS 330- cade of the MAPKs, leading to phosphorylation of Hog1 54-1) and it is one of the most often employed agrochemi- that is translocated into the nucleus, regulating the expres- cals for controlling weeds in sugarcane, citrus and coffee sion of a number of genes involved in salt tolerance crops. It has high mobility in soils, low susceptibility to nat- (Gostinc et al., 2011). Lenassi et al. (2011) described dif- ural attenuation and strong toxicity. Besides, more toxic ferences among adaptive, obligate halophiles and salt-sen- metabolites with genotoxic and teratogenic actions such as sitive fungi at the molecular level involving enzyme as 3,4-dichloroanilines, N-3,4-dichlorophenylurea and N- glycerol-3-phosphate dehydrogenase Gpd1. (3,4-dichlorophenyl)-N-methylurea can be generated from Sometimes, high salinity of the medium containing diuron by biotic and abiotic reactions (Tixier et al., 2000; xenobiotics can difficult the biodegradation process, affect- Dellamatrice and Monteiro, 2004; Giacomazzi and Cochet, Send correspondence to E. Gomes. Laboratory of Biochemistry and Applied Microbiology, Ibilce, Universidade Estadual Paulista “Júlio de Mesquita Filho”, São José do Rio Preto, SP, Brazil. E-mail: [email protected]. 1208 Arakaki et al. 2004). The dissipation of phenylurea herbicides in the envi- also studied. For diuron degradation experiments, a ronment was reported as a predominantly biological pro- commercial formulation of diuron Karmex® (dupont) was cess, suggesting that the restoration of polluted sites employed in culture medium. The pesticide was diluted in through bioremediation is a promising tool (Cameron et al., methanol (12.5 mg.mL-1), homogenized by two sonication 2000; Sørensen et al., 2003). pulses of 15 min and centrifuged at 10,000 g for 10 min at Fungi from basidiomycetes group are referred by rea- 10 °C. Then, the solution was sterilized by filtration son of their noticeable versatility to oxidize a wide range of through a 0.22 mm Millipore membrane. Aliquots were dis- xenobiotic, phenolic or non-phenolic compounds (Cerni- pensed into sterile 125 mL flasks in a laminar flow. After glia, 1997), dyes (Trovaslet et al., 2007), pesticides (Gian- solvent evaporation, each one received 25 mL of sterile liq- freda and Rao, 2004), chlorophenols and organophos- uid culture medium previously described, resulting in final phorus (Keum and Li, 2004). The mechanisms consist of concentration of 5 and 10 mg.L-1 of diuron. Diuron dissipa- carbon bond breakage with aromatic ring fission, aryl-alkyl tion and ligninolytic enzyme production were evaluated for breakage and lateral chains removal. until 10 days in slight agitation at 28 °C, in the dark. At the In spite of their ability to degrade several compounds, end of the cultivation period, cultures were centrifuged at a few strains of basidiomycetes were evaluated in relation 10,000 g for 10 min and the mycelium was dried at 70 °C to to their salt-tolerance. Therefore, the aim of this work was quantification of both biomass and the filtered used for de- to investigate the effect of NaCl concentration on the ability termination of diuron degradation. of basidiomycetes species to grow, produce ligninases and degrade herbicide diuron. HPLC analysis All media samples were centrifuged and afterwards Materials and Methods filtered through a 0.22 mm membrane. Diuron degradation Fungal cultures rates were estimated using an isocratic Jasco HPLC, em- ploying a UV-975 detector adjusted to 240 nm and a Ten basidiomycetes strains, previously isolated from starchromatography 4.0 Varian software. Analyses were decaying wood in the Atlantic Rainforest fragments, at developed with a C18 (Perkin Elmer) column, in an oven “Estação Ecológica do Noroeste Paulista”, northeast of São stated at 40 °C. The pesticides were eluted by a degassed Paulo State, Brazil (Santos et al., 2004; Abrahão et al., mobile phase composed of acetonitrile and water 2008) were used in this work. The fungi were being pre- (60/40 v.v-1), with a flow rate of 1 mL.min-1. As standard served using Castellani’s method, under mineral oil and liq- for calibration curve diuron Ultrachem was employed (99% uid Nitrogen. purity). All samples were injected three times and the re- sults represent the means taken. Culture medium, inoculum and cultivation condition Mycelium was scraped from agar plates, under sterile Enzyme assays conditions, and transferred to 120 mL Erlenmeyer flasks Enzymatic activities in the crude extract of fungal loosely capped with cotton plugs, containing 50 mL of the -1 cultures were determined spectrophotometrically, by reac- basal medium composed of (g.L ): 5.0 glucose, 2.0 yeast tions carried out at 40 °C. Laccase (EC 1.10.3.2) was mea- extract, 1.0 potassium sodium tartrate, 1.0 ammonium sured based on ABTS (Sigma) oxidation (e = 3.6.104 M-1 phosphate monobasic, 0.5 magnesium sulfate, 0.2 potas- cm-1), at 420 nm Buswell et al. (1984). Lignin peroxidase sium chloride and 1.0 mL of micronutrient solution. (EC 1.11.1.14) (LiP) activity was estimated by means of The pH was adjusted to 5.5. After growing for 6-7 days, at veratryl alcohol (Fluka) oxidation to veratraldehyde 28 °C, the medium was removed and the mycelium washed e 3 -1 -1 twice with Knapp buffer (Tixier et al., 2000). After that, ( = 9.3.10 M cm ) at 310 nm Li et al. (2003). Manga- nese-dependent peroxidase (EC 1.11.1.13) (MnP) had its fungal biomass was mixed with the buffer in a sterile mixer, 3+ and cell suspension was diluted to reach the optical density activity determined by generation of lactate-Mn com- e 3 -1 -1 of 0.5 at the wavelength of 550 nm. This suspension was plexes ( = 8.1.10 M cm ), absorbing at 240 nm (Glenn et used as inoculum for experiments using the same medium. al., 1986; Aitken and Irvine, 1990). For all cases, 1 unit of The incubation was carried out for 10 days at 28 °C. enzymatic activity was expressed as the amount of enzyme capable of oxidizing 1 mmol of substrate per minute, under Culture conditions for halotolerance, effect of assay conditions, and expressed as U per gram of dry bio- mannitol and glycerol on growth and diuron mass. degradation Analytical procedures In order to evaluate the tolerance of the fungi to salt concentrations, the culture medium was supplemented with Reducing sugars were determined by DNS method 0.5, 0.6 and 0.8 mol.L-1 of NaCl. The effect of addition of (Miller, 1959), using D-glucose as standard. The separation mannitol and glycerol at 0.5 M to the culture medium was and identification of carbohydrate was carried out by Herbicide degradation ability of basidiomycetes strains 1209 HPAEC-PAD (ICS 3000, Dionex Corporation, EUA), with Demoulin (1997). Phlebia sp tolerated 30 to 50% (26) and AS40 automated sampler and carbopac PA-1 anionic col- Formitopis,47g.L-1 (Miyazaki et al., 2007; Li et al., 2002). umn.