Characterization of cellulase producing from Aspergillus melleus by solid state fermentation using maize crop residues

The production of cellulase by the yellowish orange sclerotia producing species, Aspergillus melleus UPAR01 on lignocellulosic material by solid state fermentation (SSF) was investigated. The first experiment was conducted to find out the colony radial growth rate (Kr) of fungus on solid medium using potato dextrose agar (PDA) and incubated at 30°C in the dark. The result shows that the average Kr value of A. mellues strain approximately was 0.77±0.03 cm/day. When the fungus was used to produce cellulase using maize crop residues as the sole carbon source by SSF at 30°C for seven day, the values of FPase, endoglucanase, -glucosidase, and xylanase were achieved at 0.284±0.04, 9.45±0.33, 1.20±0.12, 12.58±0.08 U/mg protein, respectively. The optimal pH and temperature (°C) for the enzymatic activities was expressed by response surface methodology (RSM). The data shows that the optimum pH range was between 5.5 and 5.8 and the optimum temperature ranged from 53 to 59°C. In addition, none of the metal ions and ethylene-diaminetetra-acetic acid (EDTA) induced cellulase and xylanase activities.

Solid state fermentation (SSF) of lignocellulosic material for production of cellulase and xylanase is an attractive means to produce enzymes because of its *Corresponding author.E-mail: khanchai.da@up.ac.th.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License lower capital investment and lower operating cost.Several studies have indicated that agricultural residues including maize crop residues can be used as lignocellulosic carbon source for the production of cellulase (Liming and Xueliang, 2004;Zhu et al., 2009;McMillan et al., 2011) including solid state fermentation of castor bean (Ricinus communis L.) for cellulase production by Aspergillus japonicas URM5620 (Herculano et al., 2011).Both bacteria and fungi are known to produce cellulase, however, fungal enzymes are generally complete comprising all the cellulosic activities (Stockton et al., 1991).Aspergillus species are well known to be good for producing cellulases and xylanase and many species have been studied, including A. terreus (Gao et al., 2008), A. niger (Ncube et al., 2012), A. fischeri (Senthikumar et al., 2005), A. niveus and A. ochraceus (Betini et al., 2009).
The aims of this experiment were to investigate the enzymes kinetics including optimal pH, temperature and kinetics based on the optimization by Response Surface Methodology (RSM).

Fungal cultures
The fungus Aspergillus melleus UPAR01 used in this investigation was originally isolated from maize crop area, Phayao province, Thailand (2010) using modified Aspergillus flavus and Parasiticus Agar (AFPA) (Cotty, 1994).The culture was transferred and preserved in Potato Dextrose Agar (PDA) slants at 4°C in the culture collection of the Biotechnology program, School of Agriculture and Natural Resources, University of Phayao, Thailand.To prepare spore suspension, A. melleus fully grown spores (approximately seven days incubation on PDA at 30°C) were suspended in 0.85% (w/v) sodium chloride to prepare the homogenous spore suspension (10 6 spore/ml).

Substrate preparation
Maize crop residues (stalk, stover, and hub) were collected from Dok Kham Tai district, Phayao province, Thailand.Sample was dried in a hot air oven at 60°C for 72 h and then ground and passed through 2 mm sieve.The sample was analyzed for lignocellulosic compositions by the detergent method (Goering and Van Soest, 1970) and was used as substrate for enzyme production.

Characterization of A. melleus
A. melleus was identified according to taxonomic schemes proposed by Klich (2002).Macroscopic characteristics, including colony diameter, colony colors, colony texture, conidial color, sclerotia, reverse color, and soluble pigment, were observed by naked eye.Fine structural characteristics, including seriation, vesicle shape, stipe length, and the shape, size and texture of conidia, were observed under a dissecting microscope.
The molecular investigation was examined in the laboratory test. A. melleus was inoculated into Malt Extracted Agar (MEA) plate at 25°C for 7 days in the dark, and then its DNA was extracted from the culture using MoBio-UltracleanTM Microbial DNA Isolation Kit.Fragment containing part of the -tubulin gene was amplified using the primers Bt2a (GGTAACCAAATCGGTGCTGCTTTC) and Bt2b (ACCCTCAGTGTAGTGACCCTTGGC) (Glass and Donaldson, 1995).The PCR fragments were sequenced with the ABI Prism  Big DyeTM Terminator v. 3.0 Ready Reaction Cycle sequenced Kit.Samples were analyzed on an ABI PHISM 3700 Genetic Analyzer and contigs were assembled using the forward and reverse sequences with the programme SeqMan from the LaserGene package.For storage, isolates were grown on V8 medium (5% V8 juice and 2%agar) (Cotty and Misaghi, 1984), from which 3-mm plugs of sporulating culture were taken and placed in vials containing 5 ml of 0.01% Triton X-100 (TX).
Measurement of colony radial growth rate (Kr), mycelia agar plug (0.2 cm) of A. melleus was inoculated onto the center of 20 ml of PDA in Petri dish and then incubated at 30°C for 7 days in the dark.Fungus was measured for the radial growth of colonies (cm) by a ruler with repeated 3 times for 3 days.Typically, the fungal mycelia had grown and reached into the end of the plate approximately in 6 to 7 days.After the last measurements, the Kr was determined according to the method described by Morrison and Righelato (1974).The linear regression was Kr = dr/dt in which dr/dt was colony radial growth rate (cm) obtained during time o (observed time) and time i (initial time), respectively.

Production of enzymes
Solid State Fermentation (SSF) process was used in this study.The maize crop substrate (5.0 g) was weighed and put into a 250 ml Erlenmeyer flask with 40 ml of the Semi-synthetic medium (approximately 63% moisture content using Extech SDL550 Moisture Content Meter, Datalogger, USA).Culture medium was inoculated with 2.0 ml of A. melleus UPAG01 spore suspension (10 6 spore/ml) and incubated at 30°C for 7 days.After incubation, crude enzymes were extracted by adding 50 ml of 0.01% (w/v) Tween 80 in 50 mM sodium citrate buffer pH 5.0 and mixed for 1 h on a rotary shaker at 150 rpm.The suspension was then centrifuged and the same buffer was added to the clarified supernatant to make a total volume 100 ml.The suspension contained the crude enzymes used for further studies.

Determination of enzyme activities
Cellulase (FPase, endoglucanase and-glucosidase) and xylanase activities were assayed according to the method described by Ghose (1987) and Ghose and Bisaria (1987).The reaction mixtures were determined by incubating 0.5 ml of enzyme solution with 50 No.1 filter paper for FPase, 0.5 ml of 2.0 %(w/v) carboxymethylcellulose (CMC) for endoglucanase and 0.5 ml of 1.0% (w/v) xylan for xylanase, respectively.The reaction mixtures were incubated at 50°C for 60 min (FPase) and 30 min (endoglucanase andxylanase).-glucosidase activity was assayed according to the method described by Sternberg et al. (1977).The reaction mixture was determined by incubating 0.5 ml of enzyme solution and 0.5 ml of 4 mg/ml D-salicin, followed by incubation at 50°C for 30 min.
After incubation, all reactions were assayed by estimating the reducing sugar liberated using DNS reagent (Miller, 1959), and were calculated in international units of enzyme activity.One unit of cellulase (FPase, endoglucanase, and β-glucosidase) is defined as the amount of enzyme that liberates 1.0 µmol of glucose per 1.0 min under the assay conditions.One unit of xylanase is defined as the amount of enzyme that liberate 1.0 µmol of xylose per 1.0 min under the assay conditions.
Lowry method (Lowry et al., 1951) was used in this study for protein determination.The amount of total proteins (mg/ml) was performed by means of standard curve obtained from solutions containing (0.0-2.0 mg/ml) bovine serum albumin (BSA).

Kinetic characterization (pH and temperature) by response surface methodology effect of pH and temperature
Sodium citrate (50 mM) buffer pH 3.172, 4.0 and 6.0 and 50 mM sodium phosphate buffer pH 8 and 8.8 were examined in this study.The statistical approach with Response Surface Methodology (RSM) was used to determine the optimal conditions to obtain the highest activity of enzymes.The experiments were based on 2 2 full factorial Central Composite Design (CCD) with augmented points in two independent variables including  1 : pH, and  2 : temperature values, while enzyme activity (; U/ml) was the dependent variable as described in Table 1.
All measurements were performed in three replicates, and data were expressed as average values, and analyzed using Minitab 5.1 software.The lower p-value and insignificant lack of fit was obtained with a quadratic model that suggested good fit.Higher coefficients of regression (95%) suggested that there was good agreement between predicted and estimated enzymatic activities.The results of RSM were used to fit a second-order polynomial equation that represents the behavior of the system;

Km and Vmax
Four types of substrates, Avicel ® , CMC, salicin and xylan were also chosen to test the substrate specificity of FPase, endoglucanase, glucosidase and xylanase, respectively.After the selection of the optimum pH for enzyme activities, the Michalis-Menten for Km and Vmax values were determined by the investigation using varying substrate concentrations ranging from 0 to 0.25 %(w/v), and the assays were conducted in the same manner of cellulases and xylanase determination.

RESULTS AND DISCUSSION
The AFPA medium was modified by Pitt et al. (1983).This medium was used for Aspergillus spp.isolation by several authors including Reddy et al. (2009) and Foley et al. (2014).The medium was used to select Aspergillus sp.including A. flavus or A. parasiticus because both fungi produced yellow orange color when viewed from the reversed side of the plate.Yellow orange color of colony was observed because medium help improved color production on the reverse of the plate due to the optimal concentration of a more soluble iron salt and the addition of yeast extract (Pitt et al., 1983).In this study, A. melleus UPAG01 color change of the reverse phase on AFPA was similar as A. flavus or A. parasiticus by appearance of yellow orange color reverse colony.Conidia and mycelium germination of isolates Aspergillus could be observed in these AFPA medium except groups of A. nomius.Damann et al. (2004) showed AFPA medium did not support sporulation by A. flavus.A. melleus showed dark yellow reverse colony similarity as A. flavus and Foley et al. (2014) reported that Aspergillus spp.produced yellow orange color when viewed from the reversed side of the plate.However, A. melleus UPAG01 produced yellow radiate conidia heads (50-150µm) which enable clear differentiation from A. flavus or A. parasiticus (green conidia).The microscopic morphology showed that the hyphae (2-3µm diameter) were septate, hyaline, and biserate with smooth wall globose conidia (2-3 µm diameters) which was reported by Christensen (1982) and Klich (2002).In addition, this strain produced abundantly yellow to brown sclerotia on 5% V8 medium and PDA (Klich, 2002) (Figure 1).When A. melleus UPAG01 DNA was extracted, to identify the species of  Aspergillus was followed according to the method described by Glass and Donaldson (1995).The sequences were compared on GenBank using BLAST and in a large fungal database of CBS-KNAW Fungal Biodiversity Centre with sequences of most of the type strains.The results had a 100% match in all databases with the type strain of A. melleus sample that is a fungus in the Subgenus Circumdati, Section Circumdati.
For the measurement of colony radial growth rate (Kr), several reports showed that Aspergillus sp. had wide range of Kr values that depended on species, strains and culture conditions.The Kr values of Aspergillus strains isolated from honey bee and A. flavus ATTC 16872 were 0.228 and 0.24 cm/day, respectively (Yoder et al., 2008;Lopez-Malo et al., 1998).High or low values of pH and chemical substances which included bicarbonate, vanillin and avermectin (more than 0.01% w/v) affected colonies formation by Aspergillus, and lead to the reduction of the values of Kr (Lopez-Malo et al., 1995;Rodr´ıguez-Urra et al., 2009;Danmek et al., 2011).In this study, PDA with pH at 6.0 was used because several reports discussed PDA as suitable for the culturing of Aspergillus and had no effect in the range between pH at 5.5 to 7.5 (Brancato and Golding, 1953;Cotty, 1988;Thompson, 1990;Yoder et al., 2008).In addition, Gupta et al. (2012) showed that PDA is the most commonly used as laboratory medium for fungi due to its good and balanced nutrient content.A. melleus UPAG01 grew well and produced white mycelia and brown conidia with dark-brown sclerotia until the end of the PDA plate within 6 days at 30°C in the dark same as the other Aspergillus spp.including A. flavus and A. niger (Reddy et al., 2009;Foley et al., 2014).Data showed the beginning of exponential phase at the second day of incubation.Therefore, linear regression of A. melleus UPAG01 was calculated using the relationship between colonies radial growth (cm) versus time (day) that at this time.Average Kr value of all A. melleus UPAG01 for 6 days was 0.770.03cm/day.
Among the selected maize crop residue, it contained the high cellulose (33.15%dw) hemicellulose (44.67%dw) and low lignin content (4.91 %dw).The residue was selected as a carbon source for enzyme suitable for production of cellulase and xylanase by A. melleus UPAG01.Under acidic conditions with pH 5.5 and 7 days of incubation, A. melleus UPAG01 produced 0.280.04,9.450.33,1.200.12and 12.580.08U/mg of FPase, endoglucanase, -glucosidase, and xylanase, respectively.The high yield might result from its lignocellulosic materials present in inducers and nutrients.Several reports indicated that cellulase and xylanase production were favored in the acidic range of pH 4 to 6 (Brijwani et al., 2010;Romero et al., 1999;Tishkov et al., 2013).Similarly, Pothiraj et al. (2006) and Mrudula and Murugammal (2011) reported that A. terreus and A. niger produced high value of cellulase activities in the sixth to eight day of incubation by Solid State Fermentation (SSF).For optimal pH and temperature, the overall second order polynomial equation for the activities as measured in term of Unit (U/ml) is shown in Table 2.The three dimensional plots of the combined parameters between   and   on the enzyme activities are shown in Figure 2. The negative significant coefficient of    and   values are expressed as a linear effect; this is meant for decreasing enzyme activities ().
For the other terms, two quadratic terms of 

Table 1 .
Variables and their level for 2 2 factorial central composite designs for cellulase and xylanase activities (U/ml).

Table 2 .
Equations analysis for optimal pH and temperature (coded values).