Cellulolytic ability of a promising Irpex lacteus ( Basidiomycota : Polyporales ) strain from the subtropical rainforest of Misiones province , Argentina

The cellulolytic activity of fungi growing in the subtropical rainforest of Misiones (Argentina) represents a challenge in the technological development of the production of cellulosic bioethanol in the region using native sources. These fungi are promising to obtain sustainable enzyme cocktails using their enzymes. Cellulolytic ability of 22 white-rot fungi isolated from the subtropical rainforest of Misiones-Argentina in agar medium with two types of cellulosic substrates, carboxy-methylcellulose or crystalline cellulose, were comparatively analyzed, and the activity of two cellulolytic enzymes was evaluated in liquid medium. Although all isolates were able to grow and degrade both substrates in agar medium, and to produce total cellulase Filter paper (FPase) and endo-β-1,4-glucanase (EG) activities in broth, the isolate Irpex sp. LBM 034 showed the greatest enzymatic levels (FPase, 65.45 U L-1; EG, 221.21 U L-1). Therefore, the ITS sequence of this fungus was sequenced and analyzed through a phylogenetic analysis. These results indicate that the isolate LBM 034, corresponding to Irpex lacteus, has a promising cellulolytic ability and enzymes such as EG useful in sustainable saccharification of cellulosic materials in the region. Rev. Biol. Trop. 66(3): 1034-1045. Epub 2018 September 01.

Cellulose is considered to be the most abundant organic compound derived from plant biomass.Perpetual renewal of plant biomass via the process of photosynthesis ensures an inexhaustible supply of such material.The worldwide production of this biopolymer is estimated to be between 10 10 and 10 11 tons each year (Lynd, Weimer, van Zyl, & Pretorius, 2002).Cellulose is a linear homopolysaccharide of β-1.4-linked anhydro-D-glucose units with a degree of polymerization of approximately 10 000 for cellulose chains in nature and 15 000 for native cellulose cotton (Habibi, Lucia, & Rojas, 2010;Siqueira, Bras, & Dufresne, 2010).
In Argentina, subtropical and tropical rainforests are restricted to small areas that do not reach 2 % of the land area, and yet, are home to more than 50 % of the biodiversity (Brown, Grau, Lomáscolo, & Gasparri, 2002).Specifically these forests belong to two phytogeographic regions of the Amazon Domain: Yungas rainforests, located on the eastern slopes of the foothills in the Northwest of the country; and the Paranaense rainforest located in the province of Misiones and Northeast of Corrientes (Cabrera, 1994).Currently, studies of wood-decaying fungi diversity are in an active process of analysis and dissemination (Rajchenberg & Robledo, 2013;Gallo, Robledo, Romero, & Catania, 2014;Robledo, Giorgio, Franco, Popoff, & Decock, 2014).
However, knowledge about the cellulolytic enzyme production of these fungi is very much restricted.The forest environments in the province of Misiones in Northeast Argentina, are a rich reservoir of biodiversity (Zuloaga, Morrone, & Rodríguez, 1999).Exploration and description of new fungi from Paranaense rainforest can reveal innovative abilities for potential biotechnological applications such as plant biomass conversion (Saparrat, Martínez, Cabello, & Arambarri, 2002;Giorgio et al., 2012;Daâssi et al., 2016).Moreover, the relevance of screening of new isolates in addition to culture collection strains has been already highlighted (Tekere, Mswaka, Zvauya, & Read, 2001).In this sense, although most reports available are related to the Iguazú National Park (Wright, Lechner, & Popoff, 2008), there is no data for the rest of the region that remains still underexplored (Niveiro, Popoff, & Albertó, 2010).
A high forestry activity is found in the province of Misiones-Argentina due to availability of a lot of plant sources.Therefore, there is an increase of woody wastes (Teza et al., 2012).In spite of the huge amount of this plant material, there are still burning practices for its use, however the law XVI N°106 since 2012 prohibits this activity and promotes their utilization in sustainable processes (Sistema Argentino de Información Jurídica, 2012).This has lead to the search for innovative biotechnological strategies such as the production of second generation biofuels, which is also depending upon reactive and enzymes for the process.In this sense, the availability of enzyme cocktails from autochthonous fungal sources is also a priority (King, Donnelly, Bergstrom, Walker, & Gibson, 2008).
In the present study, we analyzed and compared the cellulolytic ability of 22 white-rot fungi, isolated from woody materials collected from the subtropical rainforest of the province of Misiones, Argentina, grown on agar medium supplemented with either sodium carboxymethylcellulose (CMC) or crystalline cellulose (CC).Furthermore, submerged cultures of these fungi in a liquid medium with CMC were conducted to select the isolate with the highest level of filter paper assay (FPase) and endo-β-1,4-glucanase (EG) activities, which was subsequently identified at molecular level.
Screening for fungal growth and cellulolytic ability on agar medium: Cellulolytic ability of each fungal isolate was determined by using plate screening medium containing Czapek's mineral salt solution (NaNO 3 2 g, KH 2 PO 4 1 g, KCl 0.5 g, MgSO 4 .7H 2 O 0.5 g, FeSO 4 .7H 2 O 0.01 g, H 2 O 1 000 mL, adjusted to pH 5.0 with glacial acetic acid) and agar (20 g L -1 ) supplemented with 1 g L -1 of CMC (Sigma-Aldrich, USA) or with 1 g L -1 of CC (Sigma-Aldrich, USA) as sole carbon source.Agar block (6 mm in diameter) from five days old fungal colony grown on MEA plates was cut and inoculated in the center of the plates.Inoculated plates were incubated at 28 ± 2 °C for 4 days to estimate the growth extension of the colony (Guigón-López et al., 2010;Andrino, Morte, & Honrubia, 2011) and the degradation of carbon substrate supplemented through the procedure of Mangelli & Forchiassin (1999), calculated as the Cellulolytic Efficiency Index (CEI) according to Martínez, Chiocchio, & Godeas (2001).All analyzes were performed in duplicate.
Fungal screening for selected cellulolytic enzymatic activities under submerged conditions: Thirty mL Czapek's mineral salt solution (adjusted to pH 5.0) supplemented with 1 g L -1 of peptone and 5 g L -1 of CMC in 250 mL Erlenmeyer flask was inoculated with each fungus, and incubated at 28 ± 2 ºC under static condition for 14 days.After 4, 9 and 14 days of incubation the culture medium was collected, centrifuged at 10 000 xg for 15 min, and the clarified supernatant was used to estimate total cellulose and endo-β-1,4-glucanase activities.FPase and EG activities were determined according to Ghose (1987) using Whatman no.1 filter paper and CMC as substrates.The released reducing sugars were estimated as glucose by the DNS method (Miller, 1959).One unit of FPase and EG activities were defined as the amount of enzyme required to liberate 1 μmol/mL min of glucose from the particular substrate under the assay conditions.
Molecular identification of the fungal isolate LBM 034: DNA extractions and PCR amplifications: Genomic DNA was extracted from a three day old mycelium grown on 12.7 g L -1 malt extract and corn extract 0.5 % v v -1 according to Fonseca et al. (2016).PCR was performed in 20 µL reactions, using 1 Unit of Taq DNA polymerase (Thermo Scientific, Fermentas), 2 µL of 10X Taq polymerase buffer, 2 µL of 25 mM MgCl 2 , 1.4 µL of 2.8 mM dNTPs, 1 µL of each 10 µM primer and 1 µL of 60 ng template DNA.The oligonucleotide primer set ITS1 and ITS4 was used to amplify the ITS region of the rDNA (White, Bruns, Lee, & Taylor, 1990).Amplifications were performed on a Labnet thermal cycler model MultiGene TM II Personal Thermal Cycler using the following parameters: 4 min step at 94 °C, followed by 35 cycles of 40 s at 94 °C, 40 s at 55 °C and 40 s at 72 °C, and a final 10 min extension step at 72 °C.
DNA sequencing was performed on both strands in triplicate using the primers ITS1 and ITS4.Sequencing assays were analyzed on an automated DNA Sequencer ABI3730XL by Macrogen Korea.The nucleotide sequences were aligned using the Bio-Edit software.
Phylogenetic analysis: For primary sequence analyses, the software tools on the Fungal Barcoding (http://www.fungalbarcoding.org)and NCBI (http://www.nih.ncbi.gov)servers were used.The ITS sequence with the highest identity was obtained by nucleotide BLAST searches (Altschul et al., 1997).For nucleotide sequence phylogeny, maximumparsimony (MP) method was adopted.Phylogeny analysis was carried out using the T.N.T version 1.1 software (Goloboff, Farris, & Nixon, 2008).To assess the support for the identified groups, we performed a Bootstrap test.Bootstrap analyses included 1 000 resampled matrices.For each resampled matrix, we performed 100 Random Addition Sequences (RAS) + Tree Bisection Reconnection (TBR) cycles.Sequences were aligned using T-Coffee Multiple Sequence Alignment.All alignment gaps were treated as missing data.The alignments were manually trimmed (overhangs were removed and gaps were corrected) prior to phylogenetic calculations.The ITS region of 5.8S ribosomal DNA sequences was submitted to GenBank (accession LN606596).
All results are shown as mean ± standard deviation (SD).The differences in fungal growth and CEI between isolates were analyzed by a one-way analysis of variance (ANOVA) and means were contrasted by the Tukey Test, using the InfoStat version 2013 software (Grupo InfoStat, FCA, UNC, Argentina).To establish differences of extracellular total cellulase and endo-β-1,4-glucanase activities among isolates, ANOVA tests were performed.

RESULTS
Screening for fungal growth and cellulolytic ability on agar medium: Screening for fungal growth and cellulolytic ability was carried out by the hydrolysis of CMC or CC supplemented to the basal medium.All fungi showed ability to grow and degrade both substrates.
The cellulolytic ability of all fungi isolates was obtained by calculating the CEI.Trametes sp.LBM 033 isolate showed the most efficient degradation of CMC with a CEI of 3.58 ± 0.13 (P < 0.05), followed by P. martius Cu 40 (with a CEI of 2.15 ± 0.015; P < 0.05), P. martius Cu 57, and P. martius Cu 58 isolates (1.64 ± 0.09, and 1.47 ± 0.02, P < 0.05).CEI values lower than 1.16 were obtained for the rest of the filamentous fungi tested; BAFC 266 isolate presented the lowest CEI with a value of 0.68 ± 0.023.Perenniporia martius Cu 58, and P. martius Cu 57 isolates exhibited the most efficient degradation of CC with a CEI of 4.44 ± 0.12, and 4.38 ± 0.01, respectively.These values were followed by P. martius Cu 40 isolate (with a CEI of 4.00 ± 0.32; P < 0.05, Fig. 1b).CEI values lower than 1.98 were obtained for the other filamentous fungi tested.
All isolates were selected for further studies related to cellulolytic enzymatic activities under submerged conditions.Fungal screening for selected cellulolytic enzymatic activities under submerged conditions: At 4 days of incubation, cellulolytic activities (both FPase, and EG) were found in all the supernatants analyzed.2b).
Therefore, Irpex sp.LBM 034 isolate was selected for its cellulolytic ability showing the highest values of FPase and EG activities in submerged cultures analyzed after 14 days of incubation.
Molecular identification of the fungal isolate LBM 034: Irpex sp.LBM 034, isolated from the Misiones subtropical rainforest, was selected due to its outstanding potential for EG production.It was deposited at the Culture Collection of the Laboratorio de Biotecnología Molecular of the InBioMis.Because of these reasons, the fungus was explored at the molecular level for the first time.
The phylogenetic tree was reconstructed by the maximum parsimony method, based on ITS1-5.8S-ITS2rDNA region sequences.Alignment of all 38 ITS sequences from Basidiomycota yielded a consensus length of 503 characters, of which 338 (67 %) were parsimony informative.Parsimony analysis of combined nucleotide and gap data yielded a single most-parsimonious tree of 2163 steps (CI = 0.41, RI = 0.58).Phylogenetic tree showed that the LBM 034 isolate formed a cluster with species of I. lacteus with 100 % bootstrap of branch support (Fig. 3).Therefore, the isolate selected in this study was assigned to the taxon I. lacteus.

DISCUSSION
Although the diversity of wood-decaying fungi associated to the Paranaense rainforest region has been studied (Rajchenberg & Robledo, 2013;Gallo et al., 2014;Robledo et   al., 2014), only a few researchers have explored the fungal biota of this environment, and the knowledge of cellulolytic enzyme production has been very much restricted.Therefore, the present study was carried out to analyze the cellulolytic ability of 22 white-rot fungi, isolated from woody materials collected from the subtropical rainforest of Misiones, Argentina.
First, the growing ability of filamentous fungi on agar medium supplemented either sodium carboxy-methylcellulose or crystalline cellulose as sole carbon source was scanned.Mycelia growth extension is indicative of the ability to synthesize and produce extracellular hydrolytic enzymes involved in cellulolysis, providing the nutritional requirements of these fungi of biotechnological importance (Nwodo-Chinedu, Okochi, Smith, & Omijidi, 2005).
All isolates showed ability to grow and degrade both substrates in spite of their different nature; one is a soluble derivative (CMC) and the other is an insoluble element (CC).Phlebia brevispora BAFC 633 isolate was the only fungus that showed the faster radial growth rate on CMC in comparison with CC medium, whereas P. sanguineus BAFC 2126, Trametes sp.LBM 031, Trametes sp.LBM 033, and P. sanguineus LBM 106 isolates showed the faster radial growth rate on CC in comparison with CMC medium.
Compared to our results, the Ascomycota Trichothecium roseum (Pers.)Link showed a similar radial growth rate to that P. brevispora BAFC 633, I. lacteus BAFC 1171, andT. villosa LBM 110. On the other hand, Shanmugam, Mani, &Narayanasamy (2008) reported a higher growth in this fungus on a medium using CMC compared to one with cellobiose or filter paper.Kudanga & Mewenje (2005) found a higher growth in some filamentous fungi on CMC medium due to the ability of CMC to act as an indirect inducer of cellulase production.
The differences observed according to the cellulosic sources used may be indicative of a differential ability of these isolates to produce cellulolytic enzymes, possibly involving specific mechanisms of induction (Ganner et al., 2012).However, other characteristics of the enzymes produced in the culture medium, linked to their stability and activation can lead to differences in the extent of the degradation halo observed with regard to the diameter of the fungal colony (Llacza-Ladera, 2012).
To quantitatively evaluate the cellulolytic ability of the 22 isolates, they were scanned for the production of two extracellular hydrolytic enzymes under submerged fermentation.
The reduction in levels of cellulolytic enzyme activity increasing incubation time in some isolates could be attributed to several reasons such as the production and activity of proteases during growth (Ohnishi, Yoshida, & Sekiguchi, 1994), the acidification of the medium according to culture time (Gao & Breuil, 1995) and the low stability of cellulases (Janusz, Rogalski, Barwińska, & Szczodrak, 2006).However, it was found that only the Irpex sp.LBM 034 isolate showed a high cellulase activity towards filter paper and CMC substrates with increasing incubation time.Enzymatic activity values of Irpex sp.LBM 034 are higher than those previously reported by Llacza-Ladera (2012) in species of Paecilomyces sp.(48 U L -1 ) and Aspergillus sp.(48 U L -1 ).However, results here are somewhat lower than those reported by Vilches (2002) in species of Chrysosporium sp., and Fusarium sp. with activities of 120, and 112 U L -1 , respectively.The difference of values obtained in this work with the previously reported values may be explained by the differences in the test conditions, such as incubation time, substrate concentration, temperature, pH and others, which are known to have influence on cellulases production (Li et al., 2013).
The production cost of cellulolytic enzymes has declined over the last decade (Stephanopoulos, 2007), but it is still high enough to seriously affect the lignocellulosic ethanol process (Klein-Marcuschamer, Oleskowicz-Popiel, Simmons, & Blanch, 2012).Most of the cellulolytic enzymes currently available in the market are from fungal sources.The primary industrial use of fungi is the production of enzymes and two species in particular are industrial workhorses: Trichoderma reesei E. G. Simmons and Aspergillus niger Tiegh.However, literally thousands of fungi occupy the same ecological niche as Trichoderma and Aspergillus as decomposers and recyclers of cellulosic biomass and as yet they have not been exploited commercially.
We also demonstrate that LBM 034 isolate produced high EG activity.Therefore, this isolate is a promising fungus for obtaining ethanol from cellulosic biomass.
ITS region sequences are highly used to infer phylogenetic relationships of closely related fungal species, as well as to evaluate the variability within a population and between geographically distant isolates (Hilden, Bortfeldt, Hofrichter, Hatakka, & Lundell, 2008).Based on our phylogenetic analysis (Fig. 3), the LBM 034 isolate belongs to Irpex lacteus.Since there are previous reports about other isolates from this species (Hamada et al., 1999;2001;Novotný, Cajthaml, Svobodová, Šušla, & Šašek, 2009), future studies are still needed using this fungus to evaluate its potential in regional development of bioethanol production.

Fig. 1a .
Fig. 1a.Radial growth of the colony under selective conditions in test plate.Clear (£), and filled (¢) bars represent culture medium containing CMC and CC, respectively.Asterisks (*) denote significant differences (P < 0.05, Tukey Test) in the growth extension of a given isolate on both types of substrates used.Data are mean ± SD of duplicates.b.Cellulolytic Efficiency Index (CEI) of the isolates.Clear (£), and filled (¢) bars represent culture medium containing CMC and CC, respectively.Asterisks (*) denote significant differences (P < 0.05, Tukey test) in the CEI of a certain isolate on the two types of substrates used.Data are mean ± SD of duplicates.

Fig. 3 .
Fig. 3. Phylogenetic tree performed using maximum parsimony method based on the sequence alignment of the ITS1-5.8S-ITS2 of the fungus under study with homologous sequences obtained from the GenBank (NCBI).The tree was constructed with T.N.T. program.Confidence levels were obtained by bootstrapping (1 000 replicates) and indicated at each node.*Represent the collapsed branches below a cutoff of 50.