Cellulose is the major constituent of lignocellulosic biomass and thus the most abundant and renewable resource for bioenergy production (Ja’afaru 2013). Chemically, it is simple repeating molecules composed of D-glucopyranose that are covalently linked together to form linear chain via β-1,4-glucan (Khianngamn et al. 2014). Cellulosic can be broken down either enzymatically or chemically into glucose which can be fermented to liquid fuel such as ethanol. Therefore, the utilization of the cellulosic biomass is an important and promising alternative energy production (Devendran et al. 2016).

The basic enzymatic hydrolysis process of cellulose requires three types of enzymes. Endoglucanases (EGs) hydrolyzes the β-1,4 glucan chain of cellulose internally, thus generating new free ends in the polymer. Cellobiohydrolases (CBHs) release consecutive cellobiose from either the reducing or non-reducing end of oligosaccharide chains (Devendran et al. 2016). β-glucosidases (BGLs) hydrolyze soluble cellobiose molecules to glucose (Teugjas and Väljamäe 2013). These three components are contained in most of the fungal cellulases at different ratios and act synergistically for complete hydrolysis of cellulose (Singhania et al. 2013).

Although the cellulolytic enzymes are synthesized by many microorganisms, higher enzyme quantities are produced by fungi (Wang et al. 2012). Coughlan (1985) reported a list of the cellulase-producing microorganisms, which established or potential commercial use. According to the report, fungi are good producers of cellulolytic enzymes compared to actinomycetes and bacteria. Although fungi have trouble in mass transfer compared to yeast or bacterial growth, they made technological success for enzyme production (Singhania et al. 2010). Trichoderma reesei is among the best protein secretors known which makes it has been widely used in bioprocessing for cellulase production (Berlin et al. 2007). Other fungal species such as Aspergillus, Penicillium, Mucor, Phanerochaete, Fomitopsis, and Humicola are also widely known to have prominent high cellulolytic activities (Saha 2004; Baldrian and Valaskova 2008; Imran et al. 2016).

In the industrial-scale cellulosic ethanol process, the cost of cellulases represents significant operational cost (Ellilä et al. 2017). Recent molecular biology technique aims developing at microorganisms which can produce large amounts and more efficient cellulases. Therefore, obtaining high titer cellulase-producing microorganisms is positively necessary process. The present study aims at screening of indigenously isolated potent cellulase-producing filamentous fungi for further biotechnological applications.

Cultures of filamentous fungi (n=64) were isolated from various sites and substrates in Korea from 2000 to 2013 (Table 1). The cultures were deposited in the Korea University Culture Collection (KUC, Seoul, Korea) and selected for screening. The phylogenetic analysis of the fungi was performed by using ribosomal internal transcribed spacer (ITS) region. The fungal nucleotide sequences were compared to those in the GenBank database using BLAST search. In addition, Bayesian analysis was performed with Markov chain Monte Carlo (MCMC) analysis by MrBayes 3.1.2. All 64 strains of fungi were grown and maintained on plates containing 2% malt extract agar (MEA; BD Difco, USA) medium at room temperature.

Filamentous fungal isolates were screened for cellulases production in liquid cultivation using the small-scale method described by Lee et al. (2011). Two agar plugs with mycelium collected from MEA plates were inoculated in 10 mL of Mandels’ medium (Juhász et al. 2005) containing 1% (w/v) cellulose, 0.3 g L^-1 urea, 1.4 g L^-1 (NH4)2SO₄, 2.0 g L^-1 KH2PO₄, 0.3 g L^-1 CaCl₂, 0.3 g L^-1 MgSO₄, 0.25 g L^-1 yeast extract, 0.75 g L^-1 peptone, 5 mg L^-1 FeSO₄·7H₂O, 20 mg L^-1 CoCl₂, 1.6 mg L^-1 MnSO₄, and 1.4 mg L^-1 ZnSO₄.

Cultures were incubated aerobically at 25°C with shaking at 150 rpm for 7 days. The cultures were prepared in triplicate. After incubation, the fermentation broth was harvested by centrifugation at 4000 rpm for 25 min at 4°C. The supernatants were collected by filtering through 0.45 μm membranes (Sartorius Stedium Biotech, Germany) and used as crude enzyme to measure the cellulase activities.

Enzyme activity was determined by estimating the released reducing sugar using 3,5-dinitrosalicylic acid (DNS) (Miller 1959) according to the method of Xiao et al. (2004) with modifications. The reaction mixture composed of 40 μL of 50 mM sodium citrate buffer (pH 4.8), 20 μL of enzyme solution, and a 7-mm-diameter filter paper disk (Whatman No. 1) in a 200 μL PCR tube (BR781301, Sigma). The reaction mixture was incubated for 60 min at 50°C, then 120 μL of the DNS was added into each reaction. The reaction was boiled and then cooled for 5 min each. The color was developed and 36 μL of each sample in triplicate was transferred in a 96-well microtiter plate containing 160 μL of distilled water. The absorbance was measured at 540 nm. The EG activity was assayed using carboxymethyl cellulose (CMC) according to the miniaturized enzyme assay of Lee et al. (2011). The released reducing sugar was determined using the DNS method (Bailey 1988), and expressed as glucose equivalent. Each micro PCR tube contained 25 μL of 2% CMC (C5678, low viscosity, Sigma) in 50 mM sodium citrate buffer (pH 4.8) and 25 μL of enzyme solution. After incubation for 30 min at 50°C, 150 μL of DNS reagent was added. The reaction was boiled and then cooled for 5 min each. The color was developed and 33 μL of each sample in triplicate was transferred to individual wells in a 96-well microtiter plate containing 165 μL of distilled water. The absorbance was measured at 540 nm. BGL enzyme activity was carried out using p-nitrophenyl-β-D-glucopyranoside (pNPG, N7006, Sigma) as the substrate according to Lee et al. (2011). Briefly, 20 μL of the appropriate enzyme suspension was mixed with 20 μL of a 1 mM substrate solution in 100 mM sodium acetate buffer (pH 5.0). After incubating the assay mixture for 5 min at 50°C, the reaction was stopped by the addition of 20 μL of 2 M Na₂CO₃. The absorbance obtained at 405 nm with a microplate spectrophotometer (Bio-Tek, PowerWaveXS, VT, USA). One unit per mL of enzyme activity was defined as the amount of enzyme required to release 1 μmol from the substrates per milliliter of culture medium per minute.

Cellulolytic activities of the 64 filamentous fungi belonging to 26 genera and 57 species were determined. The results of the cellulase activities are shown in Fig. 1. The strains investigated in this study had FPase activity ranging from 0 to 0.259 U mL^-1. The highest FPase activity was determined from Trichoderma harzianum KUC1716. Bjerkandera adusta KUC10565, Heterobasidion orientale KUC10556, and Hyphoderma praetermissum KUC10609 showed similar FPase activities with T. harzianum KUC1716. Above the three basidiomycetes are known as white-rot fungi (Wu 1997; Maijala et al. 2003; Jung et al. 2014). Although white-rot fungi effectively degrade cellulose and hemicellulose (Schmidt 2006), they are more known as the best degraders of lignin in nature (Ten Have and Teunissen 2001). For this reason, many studies have focused on the degradation of lignin and environmental pollutants (Bumpus and Aust 1987; Aust 1995; Youn et al. 1995; Ohkuma et al. 2001). Meanwhile, T. harzianum KUC1716 also produced the highest EG activity (0.753 U mL^-1) among the 64 strains, followed by Phanerochaete sp. KUC10530 (0.561 U mL^-1) and T. gamsii KUC1747 (0.568 U mL^-1). The ascomycetous Trichoderma is important fungi used to produce enzymes by fermentation process and has long been considered as the most productive cellulolytic fungi (Valencia and Chambergo 2013). One of the best known cellulolytic organisms is T. reesei. The present commercial cellulase preparations based on mutant strain of T. reesei have been produced on an industrial scale (Ma et al. 2011). However, BGL from T. reesei is produced in very small quantities, which leads to uncomplete biomass hydrolysis and limits its industrial application (Qian et al. 2016). This pattern is also presented in this study (Fig. 1). Despite Marasmiellus candidus KUC10547 was the highest BGL producer, followed by Penicillium oxalicum KUC5242 and T. harzianum KUC1716 in this study, the activities were generally low (0.020 to 0.298 U mL^-1). However, T. harzianum has become a potent microorganism for the secretion of xylanases and cellulases (Theodore and Panda 1995; Seyis and Aksoz 2005; Maeda et al. 2011; Delabona et al. 2012), and the induction of xylanases and cellulases is likely to be under separate regulatory control (Senior et al. 1989). Moreover, the capacity of this fungus to secrete well-balanced cellulase complex, which breakdowns cellulosic compounds into glucose, has been reported in several studies (Kalra et al. 1984; Benoliel et al. 2013; Delabona et al. 2016).

From the screening results for cellulase activities, T. harzianum KUC1716 revealed the highest FPase and EG activities in this study. Although overall activities of BGL were low, T. harzianum KUC1716 possessed high BGL activity among 64 strains. Based on this study, T. harzianum KUC1716 can be recommended as a promising producer of cellulases, and will be further studied for its enzymatic potentials.