Scaling-up production of endophytic bioactive metabolites

Available online freely at www.isisn.org

Bioscience Research Print ISSN: 1811-9506 Online ISSN: 2218-3973

Journal by Innovative Scientific Information & Services Network

RESEARCH ARTICLE BIOSCIENCE RESEARCH, 201815(3):1867-1878 OPEN ACCESS

Scaling-up production of endophytic Aspergillus fumigatus bioactive metabolites as anti-phytopathogenic agent

Shahira H. EL-Moslamy* and Ahmed H. Rezk Bioprocess Development Dept., Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technology Applications, New Borg El-Arab city, Alexandria, Egypt *Correspondence: [email protected],[email protected] Accepted: 05july.2018 Published online:13Aug. 2018

The endophytic isolate that produced the highest CMCase, FPase, β-endoglucanase, and β-glucosidase activities was isolated from Egypt and identified as the endophytic Aspergillus fumigatus strain EAF102. By using, proficient tools for scaling-up biomass production like Plackett-Burman and Box-Bahnken designs the maximum fungal mass weight, and cellulases enzymes by using low-cost medium ingredients can be increased more than 6.98 times compared with the basal condition. In addition, a large-scale production was studied by calculating the microbial kinetics via batch fermentation mode. Since the maximum FMW, (Xmax) recorded as 153.34 g/l after 65hr that increased more than 20.45 times compared with cultivation in the shack flask. In addition, the maximum cellulases enzymes were calculated kinetically as CMCase (275.59 IU/ml) at 70hr, Fpase (279.04 IU/ml) at 48hr, β-endoglucanase (480.74 IU/ml) at 72hr, and β-glucosidase (373.24 IU/ml) at 54hr that increased more than 26.86, 22.21, 21.59, and 21.5 times respectively compared with the basal condition via shack flask. Finally, bio-application of the crud enzymes as antiphytopathogens agent was applied and the highest antagonistic effect recorded against A. pasteurianus, 52.6mm followed by F. solani 40.6mm. So EAF 102 was reported as a promising cellulolytic hydrolyzer strain via low-cost industrial batch fermentation system.

Keywords: Endophytic fungi, phytopathogens, experimental design, batch fermentation, cellulases enzymes.

INTRODUCTION

The phytopathogens caused significant damage in many economically crops and vegetables (Syed et al., 2013, Bhat, 2000, Luo et al., 1997, Bhat and Bhat, 1997). Therefore, the discovering of eco-friendly products are the only solution to control these pathogens without increasing the production cost (Abrão et al., 2017, Tasia and Melliawati, 2017, Naseeb et al., 2015). There are some publications studied the antagonistic strains such as Paenibacillus ehimensis, Trichoderma reesei, Aspergillus niger, and Aspergillus fumigatus that produced high chitinase, cellulase, glucanase, protease β-

glucosidase and xylanase against phytopathogens (Sarkar and Aikat, 2014, Robl et al., 2013, Srividya et al., 2012, Kluczek-Turpeinen, 2005). Endophytic fungi are defined as the fungal cells that inhabit inside the healthy plant tissues (EL-Moslamy, 2018, Bomtempo et al., 2017, EL-Moslamy et al., 2017). These fungal cells produced promising new bioactive metabolites (enzymes, antibiotics, and phytohormones) that applied in medicine and agriculture industries (Uday et al., 2017, Bagewadi1 and Ninnekar, 2015, Yehia and Saleh, 2012, Lee et al., 2011). Since these metabolites have a significant effect as antimicrobial and

http://www.isisn.org/

mailto:[email protected]

mailto:[email protected]

EL-Moslamy and Rezk Scaling-up production of endophytic Aspergillus fumigatus bioactive metabolites

Bioscience Research, 2018 volume 15(3):1867-1878 1868

fertilizer agents with negligible side effects to manage the plant diseases and to promote the plant growth (Hong et al., 2015, Suwannarangsee et al., 2014, Naing et al., 2014, de Almeida, 2011). Little bioactive metabolites that extracted from endophytic fungi were used as biocontrol agents by applying bioprocessing strategies in Egypt (Lin et al., 2017, Diogo et al., 2015, Zhang et al., 2012, Vimalashanmugam and Viruthagiri, 2012, Mishra et al., 2012). Therefore, in this work, the Plackett-Burman, Box-Bahnken designs were applied to the scale-up production of high rate, easy handle, and low-cost production of endophytic fungal biomass to extract the CMCase, Fpase, β-endoglucanase, and β-glucosidase that applied as antiphytopathogen agents.

MATERIALS AND METHODS

Isolation and screening the cellulolytic hydrolyzer strains of endophytic fungi:

In this work, fresh healthy Zea mays tissues (roots, leaves, and stems) that collected El-Shahrqia government were used for endophytic fungi isolation step. Firstly, the plant surfaces were sterilized by immersion these parts sequentially in absolute ethyl alcohol for 3 min and 1.3M sodium hypochlorite for 2min then evenly washed with sterilized distilled water and dried (EL-Moslamy, 2018, EL-Moslamy et al., 2017a). The sterilized plant parts were mixed with sterilized distilled water then squeezed and grinded by using mortar. From this suspension, serial dilutions were prepared and evenly spread in sterile Czapex dox medium that amended with Streptomycin to eliminate other microbial growth. Czapex dox medium contained (g/l): 2x10 cm Whatman No.1 filter paper as the sole carbon, 3.0 NaNO3, 0.5 MgSO4.7H2O, 0.5 KCl, 0.01 FeSO4, and 1.0 KH2PO4 (Abdel-Azeem et al., 2016, Buyer et al., 2001, Bradner et al., 1999, Dahot and Noomrio, 1996). These plates were incubated at 300C for 4-8 weeks. The grown fungi were purified onto Potato dextrose agar (PDA) medium. The highly cellulolytic hydrolyzer fungal isolate was selected via CMCase plate assay by using CMC medium that contained (g/l): 6.5 NaNO3, 3.0 MgSO4.7H2O, 6.5 KCl, 6.5 K2HPO4, 0.3 yeast extract, 10.0 CMC and 20.0 agar. These plates were incubated at 300C for 3-5 days, and then re-incubated at 500C for 18hr. Then these plates were stained with 1% Congo red for 1hr and destained with 1M NaCl for 30 min (EL-Moslamy et al., 2017a&2016, Setewart and Parry, 1981).

Finally, the total CMCase, FPase, β-endoglucanase and β-glucosidase activities were observed and calculated according to the international union of pure and applied chemistry commission of biotechnology (Bradner et al., 1999, Ghose 1987, Setewart and Parry, 1981). In this study, antimicrobial activity was screened against phytopathogens by using well diffusion method (EL-Moslamy et al., 2017a&2016). The tested phytopathogenic fungi (Fusarium solani, Fusarium moniliforme, Fusarium semitectum, Fusarium oxysporum, Helminthosporium sp., Botrytis sp., and Phytophthora arenaria) were collected from Zagazig University, Faculty of Agriculture, and El-Sharkia, Egypt. The tested phytopathogenic bacteria (Clavibacter michiganensis, Erwinia carotovora, Xanthomonas oryzae and Acetobacter pasteurianus) that collected from Bioprocess development, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technology Applications, Universities and research center district New Borg El-Arab City, Alexandria, Egypt.

Molecular identification of the endophytic cellulase producer:

By using ITS ribosomal gene sequence, the phylogenetic analysis was carried out to identify the highly cellulosic hydrolyzer isolate. Genomic DNA was prepared and the ITS regions were amplified by using universal ITS primers via PCR (EL-Moslamy et al, 2017a). The purified PCR product was sequenced via DNA sequencer (Model 3130, Genetic Analyzer, Applied Biosystems, Hitachi, Japan), and analyzed by using NCBI BLAST website. The resulted sequence was aligned by using ClustalW via BioEdit software (Hall, 1999), and a phylogenetic tree was constructed by using MEGA6 software (EL-Moslamy et al., 2018&2017a).

Bioprocessing strategies for optimizing the endophytic fungal biomass production:

Firstly, the identified strain (5 agar blocks) was pre-cultured in Potato dextrose broth for 48hr at 300C with shaking at 150rpm. The extracellular cellulases enzymes were produced by inoculating the endophytic fungal cells into 250ml conical flasks containing 50ml of the Modified Mandels and Sternburg’s basal medium. This medium consisted of (g/l): 1.4 (NH4)2SO4, 2.0 KH2PO4, 0.3 Urea, 0.3g CaCl2, 0.3 MgSO4.7H2O, 0.005 FeSO4, 0.002 MnSO4, 0.014 ZnSO4.7H2O, 0.005 CoCl2, 10ml (10% v/v) of Tween 80, 0.75 Peptone and



5.0 carbon source (crystalline cellulose, L-arabinose, D-Xylose, Dextrose, milled sugarcane bagasse, corn straw or rice straw) that used a separately. Then these flasks incubated for 72hr at 300C under shaking condition (150 rpm). The culture aliquots were centrifuged at 10,000rpm at 40C for 20min to prepare the crude enzymes (CMCase, FPase, β-endoglucanase, and β-glucosidase) and the dried biomass then measured the enzyme activities and fungal mass weight. In this work, experimental design approaches (Plackett-Burman and Box-Bahnken designs) were applied to determine the optimal condition for fungal biomass. Firstly, all factors that affected the biomass production were screened by using Plackett-Burman design (2-level factorial design). Fifteen independent factors were tested at low (-1) and high (+) levels at 16 different experimental conditions that generated via Minitab® 15 Statistical Software (Minitab Inc., State College, PA, USA). Secondly, the most significant factors were selected for the next optimization step to determine the optimal conditions and to study the interactions among factors by using the response methodology approach. The Box-Behnken design was applied by using three factors at three levels (high, middle, and low). Fungal biomass dry weight was served as the response for the multiple regression analysis that fitted to a second-order polynomial equation (EL-Moslamy et al., 2018, Uday et al., 2017, Zhang et al., 2012, Vimalashanmugam and Viruthagiri, 2012). Then the final data were generated via 3D plot and the predicted conditions for maximum biomass production were verified.

Scaling-up strategy for fungal biomass production via batch fermentation mode:

From the statistical optimization step, the final optimized conditions were used for scaling-up fungal biomass production. Firstly, the spore suspension was prepared to inoculate the pre-culture medium and incubated for 72hr at 280C under shaking condition (150 rpm), EL-Moslamy et al. (2018). In this experiment, A 7.0 L BioFlo 310 stirred bioreactor (New Brunswick Scientific Co., USA), that equipped with automatic control of temperature, agitation, aeration, and pH was inoculated with the prepared pre-Inoculum. During this batch fermentation mode, samples (10 ml) were removed periodically to determine the time course of biomass and enzymes production as described previously. The fungal growth kinetic relationship was affected by biomass yield coefficient (YX/S), maximum fungal biomass weight

(Xmax), maximum enzyme activities (Pmax), and maximum specific growth rate (μmax) so the behaviour of fungal growth can be described kinetically via this cultivation mode by using different equations (EL-Moslamy et al., 2018, Uday et al., 2017, and Zhang et al., 2012). Finally, the antimicrobial activities were determined also against some phytopathogens (F. solani, F. moniliforme, F. oxysporum, P. arenaria, C. michiganensis, E. carotovora and A. pasteurianus, and X. oryzae) via well diffusion method (EL-Moslamy et al., 2018, Uday et al., 2017, Vimalashanmugam and Viruthagiri, 2012).

Statistical analysis: The significant differences (P>0.05) between

replicates were studied by using analysis of variance (ANOVA). Since the least significant difference teast (LSD) was used to determine where the differences occurred. RESULTS AND DISCUSSION

Screening of the endophytic cellulolytic hydrolyzer isolates as antimicrobial agent:

In this work, a total 6 isolates of endophytic fungi were isolated from fresh healthy Zea mays tissues (roots, leaves, and stems). Then these endophytic isolates were tested as cellulolytic hydrolyzer candidates by calculating the total CMCase, FPase, β-endoglucanase and β-glucosidase activities. The endophytic isolate that coded as EF5 produced the highest CMCase (10.26 IU/ml), FPase (12.56 IU/ml), β-endoglucanase (22.36 IU/ml) and β-glucosidase (17.36 IU/ml) activities as shown in Figure 1.

In addition, the antimicrobial activities were observed and calculated by using some phytopathogens as recorded in Table 1. EF5 endophytic isolate showed the highest inhibition zones against all tested pathogens especially against Acetobacter pasteurianus (4.05±0.09 cm) and Erwinia carotovora (3.95±0.04 cm) followed by Helminthosporium sp. (3.85±0.97 cm) and Fusarium solani (3.25±0.05 cm). ITS primers used to identify this promising endophytic isolate and the nucleotide sequence was recorded around 707pbs. The multiple sequence alignment revealed 97-99 % identity to the sequences of Aspergillus fumigatus. So endophytic Aspergillus fumigatus strain EAF102 has been deposited in NCBI gene bank (Accession Number no. MF429776). Finally, the phylogenetic tree was constructed by using the neighbor-joining method as shown in Figure 2.



Figure (1): The comparative study among isolated endophytic fungi hat coded as (EF1, EF2, EF3,

EF4, EF5, and EF6) by calculating the maximum cellulolytic enzymes.

Table (1): Antimicrobial activities of the crude cellulases enzymes extracted from isolated endophytic fungi against some phytopathogens measured by calculating the inhibition zone (cm).

Antimicrobial activities EF1 EF2 EF3 EF4 EF5 EF6

Ph

yto

pa

tho

ge

nic

fun

gi

Fusarium solani 1.5±0.36 0.5±0.03 0.56±0.04 0.89±0.03 3.25±0.05 1.02±0.65

Fusarium moniliforme 0.36±0.04 1.23±0.96 1.29±0.36 0.96±0.05 2.36±1.3 1.09±0.69

Fusarium semitectum 1.36±0.58 0.96±0.04 1.35±0.06 1.85±0.65 2.85±0.58 0.89±0.02

Fusarium oxysporum 0.98±0.34 1.06±0.08 1.25±0.59 1.47±0.58 3.09±1.36 0.98±0.04

Helminthosporium sp. 1.48±0.78 1.25±0.59 1.04±0.58 2.03±1.09 3.85±0.97 0.94±0.46

Botrytis sp. 0.25±0.04 0.48±0.3 1.26±0.05 1.09±0.35 2.14±0.57 1.06±0.81

Phytophthora arenaria 1.23±0.56 1.15±0.36 1.26±0.0 0.36±0.0 2.52±0.36 0.49±0.00

Ph

yto

pa

tho

ge

nic

ba

cte

ria

Clavibacter michiganensis

2.36±1.36 2.45±0.21 1.25±0.01 1.85±0.06 3.08±0.0 0.79±0.01

Erwinia carotovora 2.31±0.04 1.84±0.04 1.45±0.08 1.89±0.01 3.95±0.04 2.09±0.04

Acetobacter pasteurianus

1.75±0.08 2.56±1.0 2.89±0.03 1.69±0.05 4.05±0.09 3.08±0.05

Xanthomonas oryzae 2.09±1.2 1.78±0.05 2.12±1.09 1.72±0.04 3.78±0.06 0.75±0.01

There are little publications reported the

endophytic Aspergillus fumigatus as cellulolytic hydrolyzer candidate by using so wide a substrate range and applied as anti phytopathogens agent (Lin et al., 2017, Diogo et al., 2015, Zhang et al., 2012, Mishra et al., 2012). Since these secreted enzymes like CMCase, FPase, β-endoglucanase, and β-glucosidase were digested, the pathogenic cell wall components and inhibited its growth (Abdel-Azeem et al., 2016, Buyer et al., 2001, Bradner et al., 1999, Dahot and Noomrio, 1996). These results were supported by the previous

publications that described the ability of endophytic fungi to produce cellulases enzymes as antimicrobial agents (Lin et al., 2017, Diogo et al., 2015, Zhang et al., 2012, Buyer et al., 2001, Bradner et al., 1999, Dahot and Noomrio, 1996).

Bioprocessing strategies for optimizing the biomass production of the endophytic Aspergillus fumigatus strain EAF102:

The endophytic microorganism’s especially endophytic fungi have a promising capacity to extract a large variety of different bioactive



metabolites (Bomtempo et al., 2017, EL-Moslamy et al., 2017a). In addition, these metabolic activities were extremely sensitive to the culturing conditions and the nutrient ingredients via fermentation process (Abrão et al., 2017, Tasia and Melliawati, 2017, Uday et al., 2017, Naseeb et al., 2015, Bagewadi1 and Ninnekar, 2015, Yehia and Saleh, 2012, Lee et al., 2011). So, the statistical optimization strategy was considered as an essential step for the overproduction of biomass and bioactive metabolites (EL-Moslamy, 2018, Bomtempo et al., 2017, EL-Moslamy et al., 2017a). The production cost of bioactive metabolites could be using low-cost carbon and nitrogen sources or growth reducers (Hong et al., 2015, Suwannarangsee et al., 2014, Naing et al., 2014, de Almeida, 2011). There are some carbons and nitrogen sources were published as a proficient an energy source to induce the cellulases enzymes production (Lin et al., 2017, Diogo et al., 2015, Zhang et al, 2012, Vimalashanmugam and Viruthagiri, 2012, Mishra et al., 2012). Therefore, the primary screening was studied in this work by using different nitrogen/carbon sources such as L-arabinose, D-Xylose, Dextrose, Crystalline cellulose, Sugarcane bagasse, Corn straw, and Rice straw. Figure 3 showed the crystalline cellulose as the excellent carbon source that produced the maximum fungal biomass weight (7.5 g/l), CMCase (15.56 IU/ml), FPase (14.26 IU/ml), β-endoglucanase (25.89 IU/ml), and β-glucosidase (19.75 IU/ml). In this stage, the advanced statistical medium optimization strategies were applied to overproduce the endophytic Aspergillus fumigatus strain EAF102 biomass production by using low-cost medium.

Since, application of the statistical experimental designs published recently as proficient tools for scaling-up biomass production by using low-cost medium ingredients. Firstly, the fires-order model Plackett-Burman design was used to select the significant variables. Then, the second-order Box-Bahnken design was applied to measure the optimal level and to study the interactions effects among the tested factors. Table 2 showed the tested factors that represented at two levels (low and high). The maximum FMW (22.9 g/l) was recorded in the trail no. 3, while the minimum value (5.0 g/l) was achieved in the trail no.1, as shown in Table 3. The relationship among independent factors and FMW was determined via multiple-regression

model. The main effects of the tested factors were calculated and represented in Figure 4A. In addition, the p-value and confidence levels (%) were determined by using ANOVA analysis as shown in Figure 4B

These results indicated three factors from the fifteen independent factors were recorded as the significant factors that named crystalline cellulose (F1), KH2PO4 (F2), and Tween 80 (F3). These factors were used for the further optimization step via the Box-Behnken design. Table 4 showed the total fifteen experimental Box-Behnken designs matrix, the factors levels, and the final FMW. ANOVA analysis was used to check the model adequate via fisher’s statistical analysis. The model F-value of 25.69 (P model > F 0.00115) implies the model significance. In addition, the analyzed data revealed that the tested factors and the interaction effects increased the final biomass production as shown in Table 5. The 3D-dimensional response surface curves of the raw data were illustrated to show the effect of all factors for maximizing the biomass production (Figure 5). Finally, to evaluate the relationship between the factors and response a second-order polynomial model was set-up to calculate the optimum levels that produced the maximum FMW as follows:

Y = 43.6 -8.65*F1 -2.517*F2 -1.68*F3-7.162*F1F2+ 10.55*F1F3-1.049*F2F3+ 2.087*F1F1-3.06*F2F2+1.720*F3F3

The final results indicated the maximum FMW (52.4 g/l), CMCase (75.236 IU/ml), Fpase (82.016 IU/ml), β-endoglucanase (92.296 IU/ml), and β-glucosidase (59.296 IU/ml) can be increased more than 6.98, 7.3, 6.5, 4.13, and 3.41 times respectively compared with the basal condition. Finally the optimized medium consisted of (g/L): 1.4 (NH4)2SO4, 3.25 KH2PO4, 0.3 Urea, 0.3g CaCl2, 0.3 MgSO4.7H2O, 0.005 FeSO4, 0.002 MnSO4, 0.014 ZnSO4.7H2O, 0.005 CoCl2, 4.45ml (10% v/v) of Tween 80, 0.75 Peptone and 7.5 Crystalline cellulose.

Batch fermentation strategy for scaling-up of the endophytic Aspergillus fumigatus strain EAF102 biomass production:

In this experiment, a large-scale production of the biomass and hence the cellulases enzymes was studied by calculating the microbial kinetics via batch fermentation mode.



Figure 2. Phylogeentic tree of the isolated endophytic fungi based on combined Internal Transcribed Spacer (ITS). The scale bar indicates nucleotide substitutions per position.

Figure (3): Different carbon/nitrogen sources that used to produced the maximum biomass production and its cellulolytic enzymes activities by using the endophytic Aspergillus fumigatus strain EAF102.

Table 2. Factors that tested in Plackett-Burman design and their levels for optimizing the biomass

production of the endophytic Aspergillus fumigatus strain EAF102.

Codes Factors Unit Low value High value

(-1) (+1)

X1 (NH4)2SO4 g/l 1 4

X2 * KH2PO4 g/l 2 6

X3 * Crystalline cellulose g/l 4 10

X4 CaCl2 g/l 0.2 0.5

X5 * Tween 80 v/v 5ml 15ml

X6 FeSO4 g/l 0.001 0.005

X7 MnSO4 g/l 0.001 0.005

X8 ZnSO4.7H2O g/l 0.014 0.05

X9 CoCl2 g/l 0.005 0.01

X10 MgSO4.7H2O g/l 0.05 0.2

X11 Peptone g/l 0.5 2

X12 Urea g/l 0.5 2

X13 Agitation rpm 50 250

X14 Inoculum concentration Spore/ml 2x105 5x105

X15 Initial pH 4 7



Table (3): Plackett-Burman design matrix and their levels that used for optimizing the biomass production of the endophytic Aspergillus fumigatus strain EAF102.

Trail X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15 FMW (g/l)

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5

2 -1 1 -1 -1 1 1 1 1 -1 1 -1 1 -1 -1 -1 6.5

3 -1 -1 1 -1 -1 1 1 1 1 -1 1 -1 1 -1 -1 22.9

4 1 -1 -1 1 -1 -1 1 1 1 1 -1 1 -1 1 -1 11

5 1 1 -1 -1 1 -1 -1 1 1 1 1 -1 1 -1 1 8

6 -1 1 1 -1 -1 1 -1 -1 1 1 1 1 -1 1 -1 13.3

7 -1 -1 1 1 -1 -1 1 -1 -1 1 1 1 1 -1 1 21.58

8 -1 -1 -1 1 1 -1 -1 1 -1 -1 1 1 1 1 -1 10

9 -1 -1 -1 -1 1 1 -1 -1 1 -1 -1 1 1 1 1 11

10 1 -1 -1 -1 -1 1 1 -1 -1 1 -1 -1 1 1 1 15

11 -1 1 -1 -1 -1 -1 1 1 -1 -1 1 -1 -1 1 1 12

12 1 -1 1 -1 -1 -1 -1 1 1 -1 -1 1 -1 -1 1 20

13 -1 1 -1 1 -1 -1 -1 -1 1 1 -1 -1 1 -1 -1 13

14 1 -1 1 -1 1 -1 -1 -1 -1 1 1 -1 -1 1 -1 16

15 1 1 -1 1 -1 1 -1 -1 -1 -1 1 1 -1 -1 1 10

16 1 1 1 -1 1 -1 1 -1 -1 -1 -1 1 1 -1 -1 15

Significance F= 0.0012, R Square= 0.96, Adjusted R Square= 0.89, Standard Error= 1.55

Figure 4: (A) column chart sowed the calculated main effect. (B) The column chart represents the p-value and confidence level of the tested factors that affected on the biomass production of the endophytic Aspergillus fumigatus strain EAF102



Table (4): Box-Behnken experimental design matrix of tested three factors that named crystalline cellulose (F1), KH2PO4 (F2), and Tween 80 (F3) at three levels

Table (5): Regression statistical analysis for Box-Behnen design that used for optimizing the endophytic Aspergillus fumigatus strain EAF102 biomass production

Significance F= 0.0012, R Square= 0.98, Adjusted R Square= 0.94, Standard Error= 2.5

Figure 5: Three dimensional response surfaces representing the produced the endophytic Aspergillus fumigatus strain EAF102 biomass production via Box-Behnen design.

Exp # F1 F2 F3 FMW (g/l)

1 0 0 0 43.2

2 -1 -1 0 58.2

3 0 1 1 50.5

4 0 -1 1 52

5 0 0 0 42.4

6 1 1 0 40

7 1 0 1 20.4

8 1 0 -1 29.5

9 -1 1 0 51.3

10 -1 0 1 47.4

11 0 1 -1 50

12 0 -1 -1 59.9

13 -1 0 -1 44.2

14 0 0 0 45.2

15 1 -1 0 40.2

Levels Unit (g/l)

-1 10 2 2

0 15 4 4

1 20 6 6

Coefficients Standard Error t Stat P-value Confidence level (%)

Intercept 43.6 1.428953 30.51186 7.10E-07

*F1 -8.6517765 0.887826 -9.7449 0.000193 99.98

F2 -2.5174235 0.887826 -2.83549 0.036439 96.36

F3 -1.6876 0.875051 -1.92857 0.111694 88.83

*F1F1 -7.1626235 1.296753 -5.5235 0.002665 99.73

*F2F2 10.550176 1.296753 8.135838 0.000455 99.95

F3F3 -1.0493765 1.296753 -0.80923 0.455149 54.49

F1 F2 2.0876471 1.273387 1.639445 0.162046 83.8

F1 F3 -3.06 1.237509 -2.47271 0.056343 94.37

F2 F3 1.7201882 1.20056 1.432821 0.211347 78.87



In batch fermentation system, the optimized medium inoculated with the endophytic Aspergillus fumigatus strain EAF102 spores into 7L stirred bioreactor, all culturing parameters controlled at the zero time then the final product was harvested at the end of this run (EL-Moslamy, 2018, Lin et al., 2017, Diogo et al., 2015, Mishra et al., 2012). The behaviour of this microbial cells was described kinetically via many parameters like biomass yield coefficient (YX/S), maximum fungal biomass weight (Xmax), maximum enzyme activities (Pmax), and maximum specific growth rate (μmax). Since the substrate was consumed completely after, 48hr and the maximum FMW, (Xmax) recorded as 153.356 g/l after 65hr that increased more than 20.447 times compared with cultivation in the shack flask. In addition, the maximum cellulases enzymes were calculated kinetically as CMCase (275.59 IU/ml) at 70hr, Fpase (279.04 IU/ml) at 48hr, β-endoglucanase (480.74 IU/ml) at 72hr, and β-glucosidase (373.24 IU/ml) at 54hr that increased more than 26.86, 22.21, 21.59, and 21.5 times respectively compared with the basal condition via shack flask. Due to the controlled conditions in stirred

bioreactor such as agitation, airflow and pH the reported fermentation data could be increased than the cultivation system by using shack flask.

Finally, Table 6 summarized the maximum biomass and cellulase enzymes during the small and large scale bioprocessing strategies. Antimicrobial activity of the diluted crude enzyme mixes (0.25X) against phytopathogens (F. solani, F. moniliforme, F. oxysporum, P. arenaria, C. michiganensis, E. carotovora, A. pasteurianus, and X. oryzae) was calculated via measuring the inhibition zones (mm). The highest inhibition zones were recorded against phytopathogenic bacteria (A. pasteurianus, 52.6mm and C. michiganensis 48.5mm) followed by phytopathogenic fungi (F. solani 40.6mm, P. arenaria 36.5mm, and F. oxysporum 35.6mm) as shown in Figure 6. The reported results are in accordance with previous results where the production of the endophytic Aspergillus fumigatus strain EAF102 biomass production and cellulase enzymes was improved by using experimental designs (Abrão et al., 2017, Tasia and Melliawati, 2017, Naseeb et al., 2015).

Table (6): Summary for the scaling-up production of the endophytic Aspergillus fumigatus strain EAF102 biomass, CMCase, Fpase, β-endoglucanase, and β-glucosidase during the application of

statistical experimental designs and batch fermentation mode compared with basal condition.

Parameters Basal

conditions Plackett-

Burman design Box-Behnken

design Batch

fermentation mode

Biomass (g/l) 7.5 12.7 52.4 193.356

CMCase (IU/ml) 10.26 20.007 88.236 220.59

Fpase (IU/ml) 12.56 24.492 108.016 270.04

β-endoglucanase (IU/ml) 22.36 43.602 192.296 480.74

β-glucosidase (IU/ml) 17.36 33.852 149.296 373.24

Figure (6): The chart shows the inhibition zones (mm) that produced against some

phytopathogens



Nevertheless, the highly efficient production in

this study was different to that previously concluded, where low levels of biomass and hence cellulase enzymes were recorded after optimization step (EL-Moslamy, 2018, and EL-Moslamy et al., 2017a). Finally, these results suggested the endophytic Aspergillus fumigatus strain EAF102 as a promising fungus to produce cellulases enzymes as antiphytopathogens by using industrial low-cost medium via submerged fermentation system.

CONCLUSION In this work, a total 6 isolates of endophytic

fungi were isolated from fresh healthy Zea mays tissues as cellulolytic hydrolyzer candidates. The endophytic isolate that produced the highest CMCase, FPase, β-endoglucanase, and β-glucosidase activities was identified as the endophytic Aspergillus fumigatus strain EAF102. By using, statistical, experimental designs the maximum FMW, and cellulases enzymes can be increased more than 6.98 times compared with the basal condition. Also, a large-scale production was studied by calculating the microbial kinetics via batch fermentation mode. Since the maximum FMW, (Xmax) recorded as 153.356 g/l after 65hr that increased more than 20.447 times compared with cultivation in the shack flask. In addition, the maximum cellulases enzymes were calculated kinetically as CMCase (275.59 IU/ml) at 70hr, Fpase (279.04 IU/ml) at 48hr, β-endoglucanase (480.74 IU/ml) at 72hr, and β-glucosidase (373.24 IU/ml) at 54hr that increased more than 26.86, 22.21, 21.59, and 21.5 times respectively compared with the basal condition via shack flask. The highest inhibition zones were recorded against phytopathogenic bacteria (A. pasteurianus, 52.6mm and C. michiganensis 48.5mm) followed by phytopathogenic fungi (F. solani 40.6mm, P. arenaria 36.5 mm, and F. oxysporum 35.6mm). Finally, these results suggested the endophytic Aspergillus fumigatus strain EAF102 as a promising fungus to produce cellulases enzymes as antiphytopathogens by using industrial low-cost medium via submerged fermentation system.

CONFLICT OF INTEREST

The present study was performed in absence of any conflict of interest. ACKNOWLEGEMENT

The author acknowledges the Bioprocess development department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City of Scientific Research and Technological Applications (SRTA-City), New Borg El-Arab, Alexandria, Egypt for funding the activities presented in this study. They also recognize the valuable technical help of the Central Laboratory, Advanced Technology and New Materials Research Institute, City for Scientific Research and Technological Applications, Alexandria, Egypt. AUTHOR CONTRIBUTIONS

Shahira H. EL-Moslamy, proposed the research concept, designed the experiments, providing necessary tools for experiments, experimental instructions, conducted most of the experiments, analyzed, and interpreted the data and wrote the manuscript. Ahmed H. Rezk, designed some of the experiments, as well as contributed to reviewing process and had given final approval of the version to be published. All authors read and approved the manuscript.

Copyrights: © 2017 @ author (s). This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author(s) and source are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

REFERENCES Abdel-Azeem MA, YA Gherbawy, and AM Sabry

(2016). Enzyme profiles and genotyping of Chaetomium globosum isolates from various substrates. Journal Plant Biosystems, 150 (3): 420-428.

Abrão FO, RE Duarte, SM Pessoa, LdV Santos, FdL Freitas Ju ´nior, dOK Barros, AF dS Hughes, TD Silva, N M Rodriguez (2017) Notable fibrolytic enzyme production by Aspergillus spp. isolates from the gastrointestinal tract of beef cattle fed in lignified pastures. PLoS ONE 12(8): e0183628.

Bagewadi1 KZ, and HZ Ninnekar (2015). Production, Purification and Characterization of Endoglucanase from Aspergillus fumigatus

https://creativecommons.org/licenses/by/4.0/

https://creativecommons.org/licenses/by/4.0/



and Enzymatic Hydrolysis of Lignocellulosic Waste. International Journal of Biotechnology and Biomedical Sciences, 1(1): 25-32.

Bhat MK, and S Bhat (1997). Cellulose degrading enzymes and their potential industrial applications. Biotechnol. Adv., 15: 583-620.

Bhat, M.K, (2000). Cellulases and related enzymes in biotechnology. Biotechnol. Adv., 18: 355-383.

Bomtempo VSF, FMM Santin, RS Pimenta, DP de Oliveira, and EA Guarda (2017). Production of cellulases by Penicillium oxalicum through solid state fermentation using agroindustrial substrates. Acta Scientiarum. Biological Sciences Maringá, 39(3): 321-329.

Bradner JR, M Gillings and KMH Nevalainen (1999). Qualitative assessment of hydrolytic activities in antarctic microfungi grown at different temperatures on solid media. World J. Microbiol. Biotechnol., 15: 131-132.

Buyer JS, DP Roberts, P Millner, and E Russek-Cohen (2001). Analysis of fungal communities by sole carbon source utilization profiles. J. Microbiol. Methods, 45: 53-60.

Dahot UM, and Noomrio HM (1996). Microbial production of cellulases by Aspergillus fumigatus using wheat straw as a carbon source. Journal of Islamic Academy of Sciences 9:4, 119-124.

de Almeida NM, VM Guimarães, KM Bischoff, DL Falkoski, OL Pereira, DS O Gonçalves, and ST de Rezende (2011). Cellulases and Hemicellulases from Endophytic Acremonium Species and Its Application on Sugarcane Bagasse Hydrolysis. Appl Biochem Biotechnol,165:594–610.

Diogo R, PdS Delabona, PdS Costa, DJdS Lima, SC Rabelo, IC Pimentel, F Büchli, FM Squina, G Padilla and JGdC Pradella (2015). Xylanase production by endophytic Aspergillus niger using pentose-rich hydrothermal liquor from sugarcane bagasse. Journal Biocatalysis and Biotransformation, 33(3): 175-187.

EL-Moslamy HS (2018). Bioprocessing strategies for costefective large-scale biogenic synthesis of nano-MgO from endophytic Streptomyces coelicolor strain E72 as an anti-multidrugresistant pathogens agent. Sci. Rep. 8, 3820.

EL-Moslamy HS, Elkady FM, Rezk HA and Abdel-Fattah RY (2017a). Applying Taguchi design and large-scale strategy for mycosynthesis of

nano-silver from endophytic Trichoderma harzianum SYA.F4 and its application against phytopathogens. Sci. Rep. 45297.

EL-Moslamy HS, Kabeil SAS and Hafez EE (2016). Bioprocess development for Chlorella vulgaris cultivation and biosynthesis of antiphytopathogens silver nanoparticles. J Nanomater Mol Nanotechnol. 5(1).

EL-Moslamy HS, Shokry HH, Rezk HA and Abdel-Fattah RY (2017b). Bioprocess Strategies and Characterization of Anti- Multidrug Resistant Human Pathogens Copper/Copper Oxide Nanoparticles from Citrus Peel Waste Extracts. JNanomater Mol Nanotechnol. 6(1).

Ghose K. T. (1987): measurement of cellulase activities. Pure & App. Chem., 59(2): 257-268.

Hong J-H, S Jang, YM Heo, M Min, H Lee, YM Lee, H Lee, and J-J Kim (2015). Investigation of marine-derived fungal diversity and their exploitable biological activities. Mar. Drugs, 13: 4137-4155.

Kluczek-Turpeinen B, P Maijala, M Tuomela, M Hofrichter, and A Hatakka (2005). Endoglucanase activity of compost-dwelling fungus Paecilomyces inflatus is stimulated by humic acids and other low molecular mass aromatics. World J. Microbiol. Biotechnol., 21: 1603-1609.

Lee C, I Darah, and C. O. Ibrahim (2011). Production and Optimization of Cellulase Enzyme Using Aspergillus niger USM AI 1 and Comparison with Trichoderma reesei via Solid State Fermentation System. Biotechnology Research International, Article ID 658493.

Lin J, X Zhang, B Song, W Xue, X Su, X Chen, and Z Dong (2017). Improving cellulase production in submerged fermentation by the expression of a Vitreoscilla hemoglobin in Trichoderma reesei. AMB Expr,7:203.

Luo J, L Xia, J Lin, and P Cen (1997). Kinetics of simultaneous saccharification and lactic acid fermentation processes. Biotechnol. Prog, 13: 762-767.

Mishra P, PR Kshirsagar, SS Nilegaonkar, and SK Singh (2012). Statistical optimization of medium components for production of extracellular chitinase by Basidiobolus ranarum: a novel biocontrol agent against plant pathogenic fungi. Journal of Basic Microbiology, 52: 539–548.

Naing WK, M Anees, SJ Kim, Y Nam, YC Kim, and KY Kim (2014). Characterization of antifungal activity of Paenibacillus ehimensis



KWN38 against soilborne phytopathogenic fungi belonging to various taxonomic groups. Ann Microbiol, 64:55–63.

Naseeb S, M Sohail, A Ahmed, and SA Khan (2015). Production of xylases and cellulases by Aspergillus fumigatus Ms16 using crude lignocellulosic substrates. Pak. J. Bot., 47(2): 779-784

Robl D, PdS Delabona, CM Mergel, JD Rojas, PdS Costa, IC Pimentel, VA Vicente, JGdC Pradella, and G Padilla (2013). The capability of endophytic fungi for production of hemicellulases and related enzymes. BMC Biotechnology, 13:94.

Sarkar N, and K Aikat (2014). Aspergillus fumigatus NITDGPKA3 provides for increased cellulase production. International Journal of Chemical Engineering, Article ID 959845.

Setewart CJ, and JB Parry (1981). Factors Influencing the Production of Cellulase by Aspergillus f umigatus (Freseniu s). Journal of General Microbiology, 125, 33-39.

Srividya S, A Thapa, DV Bhat, K Golmei ,and N Dey (2012): Streptomyces sp. sp as effective biocontrol against chilli soilborne fungal phytopathogens. European Journal of Experimental Biology, 2 (1):163-173.

Suwannarangsee S, J Arnthong, L Eurwilaichitr, and V Champreda (2014). Production and characterization of multi-polysaccharide degrading enzymes from Aspergillus aculeatus BCC199 for saccharification of agricultural residues. J. Microbiol. Biotechnol., 24(10):1427–1437.

Syed S, SRU Hassan, and S Johri (2013). A Novel Cellulase from an Endophyte, Penicillium Sp. NFCCI 2862. American Journal of Microbiological Research, 1(4): 84-91.

Tasia WY, and R Melliawati (2017). Cellulase and Xylanase Production from Three Isolates of Indigenous Endophytic Fungi. IOP Conf. Series: Earth and Environmental Science. 101: 12-35.

Uday SPU, TK Bandyopadhyay, S Goswami, and B Bhunia (2017). Optimization of physical and morphological regime for improved cellulase free xylanase production by fed batch fermentation using Aspergillus niger (KP874102.1) and its application in bio-bleaching. Bioengineered 8(2): 137-146.

Vimalashanmugam K, and T Viruthagiri (2012). Statistical Optimization of Media Components for Xylanase Production by

Aspergillus fumigatus using SSF. International Journal of Engineering Research and Applications, (2)5: 1320-1329.

Yehia SR, and AM Saleh ( 2012). Antifungal activity of rice straw extract on some phytopathogenic fungi. African Journal of Biotechnology 11(71): 13586-13590.

Zhang H, Q Sang and W Zhang (2012). Statistical optimization of cellulases production by Aspergillus niger HQ-1 in solid-state fermentation and partial enzymatic characterization of cellulases on hydrolyzing chitosan. Ann Microbiol, 62:629–645.

Documents

Scaling-up production of endophytic bioactive metabolites