Optimization and Characterization of Extracellular Cellulase Produced by Native Egyptian Fungal Strain

Since accumulation of agricultural wastes represents a huge problem, it was important to explore the available methods to help eliminate agricultural wastes safely, and simultaneously produce functional enzyme like cellulase. Six native Egyptian fungal strains were isolated, morphologically identified and screened for cellulose biodegradation potential, which was determined as endoglucanase or as carboximethylcellulase (CMCase). The most promising isolate ( Aspergillus terreus ) was selected for molecular characterizations based on sequencing of internal transcribed spacer (ITS). Further optimization experiments were accomplished on the selected strain. The strain with cellulolytic activity, 2.26 IU mL -1 was identified using ITS nucleotides (genes) sequences and the result confirmed that the strain is 99.8% homology with A. terreus. Then, it was submitted to GeneBank and given an accession number. Further optimization experiments revealed that 35ºC is the optimum temperature for cellulase production and raised the enzyme activity (EA) up to 3.19 IU mL -1 . Out of two organic nitrogen sources; peptone at concentration 6 gL -1 was found to be the optimum nitrogen source for cellulase production with the highest activity 4 IU mL -1 . Whereas, the different four carbon sources: microcellulose, corn stalks, wheat straw and rice straw showed significant differences in EA with values 11.07, 9.68, 7.87 and 3.71 IU mL -1 , respectively at pH 3. The maximum EA was recorded to be within 5-7 days of incubation, dependent on type of carbon sources. The optimization of different incubation conditions raised cellulolytic activity from 2.26 IU mL -1 up to 11.18 IU mL -1 .


Introduction
Agricultural waste biomass represents the most abundant renewable carbon source on earth, which could be used for production of bioenergy (bioethanol, biogas and biodiesel), chemicals, enzymes and other valuable products.During the procedure of the plant food processing, large quantities of by products are generated, that creates several environmental problems like water contamination, odor and greenhouse gases (GHGs) emission due to anaerobic mineralization process of the massive volumes of organic matters (McKendry, 2002).
In Egypt, according to the Egyptian Ministry of State for Environmental Affairs (EEAA, 2016), the amount of annual Egyptian agricultural wastes (agro-wastes) is about 31 million tons (MT).Nearly 4 MT of corn wastes, 6 MT of wheat wastes and 3 MT of rice straw are produced annually (FAO, 2001), a massive amount of these agricultural wastes are burned every year which cause a black smog and health problems such as respiratory diseases and allergies (Abdulrahman and Huisingh, 2018).
Cellulose is the most important component of agriculture and food wastes which represents most of the accumulated organic plant matter (about 60 % of agrowastes) (Clarke, 1996), and it is a branched polymer, consists of glucose units linked by a β (1→4) D-glycosidic straw and rice straw were collected from local farm in Giza, Egypt.They were air dried, milled with electric mill (model, CO.) and sieved into 2mm sieve.All reagents and chemicals were obtained from commercial sources and were of analytical grade.

Morphological identification of isolates
Six fungal strains were isolated from soil sample collected from the National Research Centre NRC farm, Dokki, Cairo, Egypt.These strains were identified in the Fungal Identification units in the Regional Center of Mycology and Biotechnology (RCMB, Al-Azhar University, Cairo, Egypt).

Molecular identification
The internal transcribed spacer region (ITS) was used to identify the superior cellulase producer isolate based on molecular genetic analysis.Partial sequences of the isolates 18S rDNA were obtained.The universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG -3') and (ITS4) (5'-TCC TCC GCT TAT TGA TAT GC -3') (Operon Technologies Company, Netherlands) were used.For the amplification of the ITS region, 12 ng of the used primer and 40 ng of the purified DNA sample were added to each polymerase chain reaction (PCR) vial.The total volume of the amplification reaction was completed to 25 µL using sterile distilled water.Then, the amplification was carried out under the following thermal conditions: 95 °C for 5 min, 35 cycles of (95 °C for 1 min, 50 °C for 1 min and 72 °C for 2 min).A final extension for 5min at 72 °C followed by an infinite hold at 4 °C finished the run.The amplified DNA products from ISSR analysis was electrophoresed against plus DNA ladder and photographed using Gel Documentation System with UV Trans eliminator after staining with red safe dye.The PCR product was cleaned up using GeneJET™ PCR purification Kit (Thermo K0701).The DNA sequencing of the purified PCR products was directly sequenced by using ABI 3730xl DNA sequencer (GATC Company, Germany) and forward primer.The DNA sequences were compared with the sequences available by the Basic Local Alignment Search Tool (BLAST) in the program of National Center Biotechnology Information (NCBI), GenBank database (http://www.ncbi.nlm.nih.gov).The sequences were aligned together with those of reference taxa retrieved from public databases.The evolutionary distances were generated based on parameter model (Jukes and Cantor, 1969) and bond (Gielkens et al., 1999).The degradation of cellulosic biomass demonstrates a critical part of the carbon cycle within the biosphere (Béguin and Aubert, 1994).
Cellulose is degraded by cellulases which cleave the β-1,4 bond (Béguin and Aubert, 1994).Recently, cellulase has shown potential application in pulp and paper industrial, biofuel production, food, feed and agriculture industrial.Furthermore, there has been an increasing interest in the biodegradation of cellulose to glucose using cellulases, which produced by a diversity of microorganisms including both fungi and bacteria during their growth on cellulosic materials ( Kulkarni et al., 2018;Islam and Roy 2018;Srivastava et al., 2018).
However, the mechanism of cellulose degradation by aerobic fungi is similar to that of aerobic bacteria.The exoglucanase (Exg) acts on the ends of the cellulose chain to produce ß-cellobiose unites as the end product, endoglucanase (EG) randomLy attacks the internal Oglycosidic bonds, resulting in glucan chains of different lengths and the ß-glycosidases act specifically on the ßcellobiose disaccharides and produce glucose (Bayer et al., 1991).
Recently, several researchers investigated the ability of fungi to use agricultural wastes as carbon sources rich in cellulosic material, of which A. terreus species found to be a promising source of cellulose (Olanbiwoninu and Odunfa 2016;Lee et al., 2017;Ranjith et al., 2018).An isolated species of A. terreus from soil showed high levels of cellulases (Sohail et al., 2016).The efficiency of the isolated A. terreus cellulase was increased through fermentation with delignified rice straw (Narra et al., 2014), however it still important to study the stimulation and optimization of cellulase production, using fermentation of A. terreus with different agro wastes as carbon sources.Besides, the application of cellulases enzymes to hydrolyze agro waste cellulose under mild conditions, is an attractive process as the enzymatic depolymerization of agro waste is environmentally friendly (Kuhad et al., 2011).
Therefore, in order to avoid agro wastes problems in Egypt, this study were aimed to isolate some native Egyptian fungi strains and study there ability to produce cellulases, and identify the superior cellulase producer isolate by sequencing of the ribosomal ITS region.Different cultivation media contained agricultural wastes and physical conditions were compared in terms of cellulase activity.

Substrates, chemicals and media
Three agro waste as carbon sources corn stalks, wheat phylogenetic tree was constructed by using the neighborjoining method (Saitou and Nei, 1987).Alignment of ITS DNA sequences was done using Clustal_X program (Thompson et al., 1997).

Inoculums preparation
Fungal spores of each tested isolate fungus were collected from Potato Dextrose agar (PDA) slants which had been inoculated and incubated for 5 days at 37 ºC.Spores were collected by adding 0.01% (v/v) tween 80 with the aid of wire loop.Each of fermentation flasks was inoculated with 5 mL containing almost (7 ×106 spores mL -1 ) (Shahriarinour and Wahab, 2011).

Collection and storage of samples
After incubation, the cell-free culture supernatant was centrifuged at 1050 xg for 2 min and then the aliquots of supernatant were dispensed in sterile Eppendorf tubes and stored at -20 °C until used as a source of enzyme in further studies.

Determination of enzyme activity
Carboxymethyl cellulose (CMC, 1% W/V in citrate buffer, pH 4.6) was used as substrate to determine Endoglucanase or carboxymethylcellulase (CMCase) EA of tested fungal strains isolates.The reducing sugars released from CMC were spectrophotometrically measured as glucose at 540 nm using 3,5-Dinitrosalicylic acid (DNS) method.One unit of EA was defined as the amount of enzyme required to release 1 µmol of reducing sugar per min under assay conditions.The following equation: 1 IU = 1 μmol min -1 of glucose equivalent released = 0.18 mg min -1 of glucose was used to calculate EA of the tested fungi (Wood and Bhat, 1988).

Optimization of culture conditions
The most promising cellulolytic isolate fungus strain A. terreus was selected for further studies to optimize the culture conditions for increasing cellulase activity.

Nitrogen sources
Only two types of nitrogen source [peptone and yeast extract] at different concentrations (6, 9, and 12 gL -1 ) were used separately in the medium formulation as described by Shahriarinour and Wahab (2011).

Carbon source
Three agro wastes; corn stalk, wheat straw and rice straw as well as microcellulose at concentration 30 gL -1 were used as a carbon source.

Initial culture pH
The optimum pH was determined by incubation of A. terreus with the different carbon sources at pH values ranging from two to seven.The culture initial pH was adjusted using 4M HCl or KOH.

Incubation time
To detected the optimum incubation time, the inoculated flasks were incubated at 35 ± 2 °C temperature and the EA was measured daily for 11 days.

Statistical analysis
All measurements were carried out in triplicate.Statistical analyses were performed using one way analysis of variance (ANOVA), and the equality of variance between means was determined by Bartlett's test, CoStat Software Computer Program.Differences at p < 0.05 or less were considered statistically significant.The results were presented as mean values ± SD, standard deviations (CoStat, 2004).

Morphological identification
Based on identification of the six isolated native fungi from Egyptian soil by the RCMB, the investigation data revealed that five of them belonged to genera Aspergillus: A. parasiticus, A. fumigatus var.ellipticus, A. japonicus, A. terreus and A. fumigatus.The sixth isolated fungus was T. harzianum.Fig. 1 demonstrates microscopic examination of the most cellulolytic promising strain A. terreus.Colonies of A. terreus on Czapek agar at 25 ºC attaining a diameter of 3.0-3.5 cm within 7 days gives buff to yellow brown color.

Screening of CMCase activity
According to the results of CMCase EA determined by DNS method out of six fungal isolates produces the enzyme by various degrees, the most promising CMCase producer was A. terreus with EA 2.26 ± 0.04 IU mL -1 .The rest of the five fungi strains could not be considered as extremely cellulolytic active strains.

Molecular identification of fungal strain associated with the highest cellulase production Fungal DNA isolation and amplification by ITS primers
The fungal DNA was isolated and determined using spectrophotometer, the ITS1 and ITS4 primers were used to amplify the region of the rDNA repeat unit that includes ITS1, 5.8S, ITS2 and 28S from the genomic DNA of the fungal strain.A band of approximately 500 to 550 bp was obtained after performing PCR (Fig. 2).DNA sequencing of the purified PCR products was done using ABI 3730xl DNA sequencer (GATC Company, Germany) by the use of forward primer, the obtained DNA sequence with the identified fungal strain was applied and conserved in the GenBank under the following accession number (MH997666.1).DNA sequencing of the ITS1, 5.8S, ITS2 and 28S regions was conducted for the differentiation of fungal strain in comparison with the reference strains from GeneBank.The amplified sequences showed that this strain had the highest homology with Aspergillus terreus strain.Furthermore, the phylogenetic tree of the fungal strain which isolated from soil was recorded in Fig. 3.

Optimization Effect of temperature on Endoglucanase enzyme activity
CMCase activity of A. terreus was tested during incubation at different temperatures (Fig. 4).The data revealed that the highest CMCase activity (3.19 ± 0.093 IU mL -1 ) was recorded at range 35 -40 °C, after 7 days of incubation.Also it was observed that the EA decreased to almost 84% at 30 °C and 70.5% at 25 °C while the EA significantly decreased to 35.7 and 33.5% at 20 and 45 °C.

Effect of nitrogen source
Various studies has investigated the effect of organic and inorganic nitrogen sources on cellulase production during fungal cultivation and demonstrated that using of organic nitrogen sources causes high efficiency (Shahriarinour and Wahab 2011;Narra et al., 2014;Yadav et al., 2016;Lee et , 2017).Based on these studies the effect of two organic nitrogen sources peptone and yeast extract was studied with three different concentrations (6, 9, 12 gL -1 ).The results highlighted that peptone concentrations 6 and 9 gL -1 had the greatest effect on cellulase production and increased the activity from 3 IU mL -1 to 4 IU mL -1 .While the EA decreased by 74.8% at peptone concentration 12 gL -1 .No significant difference was found among the different concentrations of yeast extract and the activity was significantly decreased compared to peptone at 6 gL -1 (Fig. 5).These results are analogous to the previous studies (Gautam et al., 2011;Narra et al., 2014;Yoon et al., 2014).

Effect of initial culture pH and different carbon sources
A range of pH (2-7) was used with corn stalk and wheat straw, and a range of pH (2-5) was used with rice straw, in case of rice straw there was no activity detected at pH 6 and 7.The production of CMCase using the three carbon sources was compared by microcellulose as carbon source during incubation.It was observed that optimum pH for A. terreus growing on all the four carbon sources was pH 3 and the highest EA with microcellulose, corn stalk, wheat straw and rice straw was 11.07 ± 0.322, 9.68 ± 0.388, 7.87 ± 0.017 and 3.71 ± 0.016 IU mL -1 respectively.The enzyme was active in the pH range of (3-6) during incubation with

Optimization of incubation time
On the way to detect the optimum incubation time for the highest EA for each one of the carbon sources, fermentation was carried out up to 11 days.The greatest CMCase activity was found to be 11.18 ± 0.625 IU mL -1 at day 5, in case of microcellulose as carbon source; corn stalk also gave the highest activity 9.97 ± 0.163 IU mL -1 after 5 days (Fig. 7) .Otherwise wheat straw and rice straw gave the maximum EA after 8 days of incubation with values of 8.15 ± 0.13, 3.96 ± 0.072 IU mL -1 , respectively.

Screening of CMCase activity
Several Aspergilli species were studied for cellulolytic activity, such as A. nidulans, A. niger, A. fumigatus and A. terreus, the last one was recorded with a high cellulolytic enzyme capacity and this is similar to the results of this study (Jahangeer et al., 2005;Gao et al., 2008;Liu et al., 2011;Shahriarinour and Wahab, 2011;Narra et al., 2014;Benoit et al., 2015).Cellulolytic microorganisms can exist in all biota where cellulosic waste assembles.They usually arise in mixed populations consist of cellulolytic and noncellulolytic species, which often cooperate synergistically.These cooperation lead to the complete utilization of cellulose, and eventually transform it into carbon dioxide and water (Béguin and Aubert, 1994).

Effect of temperature on Endoglucanase enzyme activity
Enzymes activity is proportional to the temperature because of the increasing of the collisions frequent between the substrate and the enzyme.After a specific temperature degree the thermal agitation affect the tertiary conformation of the enzyme, causing denaturation and an intense derease in solubility (Palmer and Bonner, 2007).The optimum growth at 35 ºC may be attributed to the optimal growth rate of A. terreus as it is a thermotolerant fungi (Mueller et al., 2011).The decline of EA may be because of the effect of temperature on enzymatic Km (Michaelis constant) and this directly affects the enzyme velocity and carbon utilization and consequently affect the fungal growth and enzyme production (Copeland, 2004).

Effect of nitrogen source
Peptone is a result of incomplete hydrolysis of protein which contains peptides and amino acids while yeast extract is the soluble part of digested yeast cell (Sumbali and Mehrotra, 2009).The peptone composition which is a source for additional carbon minerals and vitamins may be responsible for its enhancing effect, because these nutrients are important for fungal growth (Karim et al., 2015).This theory had been confirmed by Ilmén et al. (1997) who reported that enhancing effect of peptone is not attributed to cellulase gene induction but to intensive fungal growth.On the other hand, the decline of the enzyme production in case of yeast extract can be related to nitrogen availability and chemical component interaction (Panagiotou et al., 2003;Sarria-Alfonso et al., 2013).

Effect of initial culture pH and different carbon sources
The main structure of enzymes is proteins which make them very sensitive to pH, every enzyme has a specific pH range at which its activity can be detected and this range differs from fungi to another according to species.Cellulases activity is based on acid hydrolysis mechanism in which the enzyme break down the β-1,4-glucosidic bonds via a proton donor, so it was expected to see a different EA during incubation at different pH (Lynd et al., 2002).Optimal pH results consistent with previous studies at which suitable pH range for cellulolytic fungi is between 3-5 (Milala et al., 2005;Kadarmoidheen et al., 2012;Sivaramanan 2014).pH range may effect on the affinity between the enzyme and the substrate and the substrate ionization, and this might be a good reason for the different enzyme activity between pH ranges at each one of the carbon sources (Copeland, 2004;Sivaramanan, 2014).

Optimization of incubation time
EA at different incubation time was found to be similar to previous studies which showed the best incubation period to produce the highest CMCase activity was ranged between 3-7 incubation days (Ahmed et al., 2009;Narra et al., 2014;Sohail et al., 2016).Incubation period may be affected by different factors such as the microorganism species, heterogeneity of the substrate, changing in pH during fermentation, byproduct production and diminishment of nutrients in the medium (Narasimha et al., 2006;Karim et al., 2015).This difference in CMCase activity and fermentation period may be due to the difference in the carbon bioavailability of the four sources; it was found that corn stalk contains high cellulose content followed by wheat straw, followed by rice straw (Bakker et al., 2013;Daud et al., 2014).Lignin content of the substrates may be attributed, since lignin can affect the attachment between cellulose and cellulase via lignincellulase interaction (Saini et al., 2016), consequently more studies on pretreatment of agricultural wastes like delignification are required.

Table 1 .
CMCase activity of six fungal isolates