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Research Article
 

Cellulase Production by Fusarium graminearum and its Application in Ruminant’s Diets Degradation



H.H. Azzaz, H.A. Murad, A.A. Aboamer, Hoda Alzahar and M. Fahmy
 
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ABSTRACT

Background and Objective: Cellulase as a fibrolytic enzyme is a highly effective tool for agricultural waste treatments. Production of cellulase enzyme on medium of agricultural wastes by Fusarium graminearum to be used in ruminant feeding was the main objective of this study. Materials and Methods: Impact of initial pH of growth medium, different nitrogen sources and variety of agriculture by products as a carbon sources on cellulase production have been studied. Electron microscope was used for investigate the impact of the resultant cellulase on corn stover degradation, while batch culture technique was used for investigate impact of different levels of the produced and commercial cellulases on total mixed ration digestibility by rumen microorganisms (in vitro). Results: Cellulase maximum production by F. graminearum was obtained at 20% corn stover, initial pH of growth medium 5.0 and peptone as a nitrogen source. All addition levels of the produced cellulase increased dry matter (DM), neutral detergent fiber (NDF), acid detergent fiber (ADF), cellulose and hemicellulose degradability of the treated diets, but the maximum produced cellulase efficiency% for dry matter degradability was obtained at 1200 IU kg1 DM reached 23.19% over the control. Conclusion: Utilization of the produced cellulase in enrichment of the feeding value of the agricultural by-products may help in overcome of the feed gap with good impact on environment and public health.

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H.H. Azzaz, H.A. Murad, A.A. Aboamer, Hoda Alzahar and M. Fahmy, 2020. Cellulase Production by Fusarium graminearum and its Application in Ruminant’s Diets Degradation. Pakistan Journal of Biological Sciences, 23: 27-34.

DOI: 10.3923/pjbs.2020.27.34

URL: https://scialert.net/abstract/?doi=pjbs.2020.27.34
 
Copyright: © 2020. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

The continuous elevation of prices of feed ingredient and the wide gap between animal’s requirements and available feeds forced the nutritionists for searching for alternative feed resources1. The Egyptian agricultural sector is trying to cover the shortage in feedstuffs by importation which representing a burden on the Egyptian economy2. However, the agricultural by-products can play an important role for minimizing this feed gap3. The annual production of agricultural by-products estimated to be around 30 million t of dry material/year4. These abundant wastes are mostly left in the road sides or burnt in the fields which lead to environmental pollution and health hazards5. The main problem facing in the utilization of agricultural by-products as feed resources is their high content of crude fiber which limits their nutritive value6. Thus, to improve the nutritive value of these residues, it is important to breakdown the lignocellulosic bonds in it7. Cellulase as a fibrinolytic enzyme plays a vital role in saccharification of agricultural by-products to glucose, cellobiose or cellooligosaccharides8. Also, microbial cellulase represents a valuable tool for improving ruminant’s ability for fibrous feed digestion9,10. Many of in vitro studies showed that supplemented ruminal bacterial cultures with cellulase led to improve ruminal fermentation through increase diet’s dry matter (DM), organic matter (OM), neutral detergent fiber (NDF) and acid detergent fiber (ADF) digestibility, alter ruminal pH with increase microbial protein synthesis and volatile fatty acid production11-13.

Recent advances in biotechnology have allowed for large production of microbial active cellulases, but its production cost is still high14. Production of extracellular cellulase from anaerobic bacteria (Acetivibrio cellulolyticus, Ruminococcus albus and Fibrobacter succinogenes) and aerobic fungi (Fusarium, Aspergillus, Trichoderma and Penicillium) have been reported by Murad and Azzaz7. Bacterial cellulases are constitutively produced, whereas fungal cellulase is produced only in the presence of cellulose. Therefore, agro-industrial residues may play an important role in commercialization of new sources of fungal cellulases. Inclusion of agricultural by-products as carbon sources in fungal growth medium may reduces the cost of cellulose production7,15.

This study was conducted for production of cellulase by Fusarium graminearum under the optimum fermentation conditions and investigated the effect of the resultant cellulase on ruminant’s diet’s degradation (in vitro). This study would advance a new knowledge through, optimization of cellulase production using unfamiliar fungal strain and unfamiliar agricultural residues as a carbon sources in the growth medium with new techniques act for decreasing the cost of production by 50%.

MATERIALS AND METHODS

This study was conducted in the Laboratories of Dairy Department-National Research Centre, Egypt. Fusarium graminearum was obtained from laboratory of dairy microbiology and the agricultural by-products were obtained from suburbans of Giza province. This study has been extended for 4 months from 10 January, 2018 till 10 April, 2018.

Sample collection: For cellulase production, around 100 samples were taken. For the in vitro study around 250 samples were taken. Fusarium graminearum was obtained from laboratory of dairy microbiology, the agricultural by-products (substrate) were obtained from suburbans of Giza province, scanning electron micrographs were taken to observe the effect of the produced cellulase on corn stover fibers and the rumen contents were collected from the rumen of slaughtered rams , then moved directly to the laboratory in separate warmed oxygen-free plastic jars to observe the effect of the produced cellulase on ruminant's diets degradability.

Media and inoculum preparation: Spores of Fusarium graminearum was transferred from surface of the actively growing slants of potato dextrose agar medium to 250 mL conical flasks each containing 50 mL of malt medium (malt extract (30 g L1), yeast extract (5 g L1)). After incubation on a rotary shaker (120 rpm) at 29±1°C for 48 h, the grown culture has been employed as inocula (5% v/v) for 1000 mL conical flasks each containing 100 mL of cellulose powder medium (CPM) which composed of (g L1) NaCl; 6.0, (NH4)2So4; 1.0, K2HPO4; 1.0, MgSO4. 7H2O; 0.05, CaCl2; 0.1, Yeast extract; 0.5, Peptone; 0.5, Glucose; 4.0, Cellulose powder; 2.0 and adjusted to pH 6.0 as reported by Khattab et al.16.

Cellulase production culture conditions: Effect of initial pH of growth medium in a range between 3 and 8, nitrogen sources included NH4CL, (NH4)2SO4, urea, yeast and peptone at level of 0.33 g N L1 and different agriculture by products (corn stover, bean straw, pea pods peel, palm fronds, wheat straw and rice straw) as a carbon sources at level of 10% on cellulase production have been studied.

Cellulase assay: One cellulase unit was defined as the amount of enzyme that liberates reducing sugar at the rate of 1 μmole min1 under the conditions of the assay. The carboxymethyl-cellulase activity (CMC) for resultant enzyme was determined according to Mandels et al.17. The reducing sugar liberated was determined by modified dinitrosalicylic acid method (DNS) of Miller18.

Scanning electron micrographs for corn stover fibers: Corn stover fibers were dried and then treated with the produced cellulase (50 U cellulase/1 g DM/100 mL buffer acetate at pH 6.5) for 24 h at 40°C and 110 rpm in a rotary shaker. The enzymaticly untreated corn stover (control) was kept in flask at the same condition. Finally, scanning electron micrographs of both treated and untreated corn stover fibers were taken to observe the effect of enzyme treatment.

In vitro study: In vitro dry matter, neutral detergent fiber (NDF), acid detergent fiber (ADF), cellulose and hemicellulose degradability were determined for the experimental diet. A 400 mg sample of the control diet powder was weighed into 120 mL serum bottles. The control diet was consisted of 50% concentrate feed mixture (CFM), 25% Berseem hay and 25% corn stover. The bottles (3 replicates) were separately supplemented with rumen liquor, buffer solution and Pan-Zyme and the produced cellulase solutions at different levels (0, 600, 1200, 1800 and 2400 IU kg1 DM of the diet). Rumen contents were collected from the rumen of slaughtered rams fed berseem hay ration, then moved directly to the laboratory in separate warmed oxygen-free plastic jars. Rumen liquor contents were strained through 4 layers of cheese-cloth and the obtained liquor was mixed with the buffer solution at 39°C under continuous flushing19 of CO2. The bottles were sealed and maintained at 39°C in a shaking water bath (20 oscillations min1) for 24 h. After 24 h of incubation the pH value, total gas production (TGP) volume, NH3 and total volatile fatty acids (TVFA) concentrations were determined according to the method of Azzaz et al.20.

Statistical analysis: Data obtained from this study were statistically analyzed by IBM SPSS Statistics for Windows21 using the following general model procedure:

where, Yij is the parameter under analysis ij, μ is the overall mean, Ti is the effect due to treatment on the parameter under analysis, eij is the experimental error for ij on the observation, the Duncan's multiple range tests was used to test the significance among means using probability level less than 0.05 (p<0.05) for significance expression22.

RESULTS

Effect of different agricultural by products (carbon sources) on cellulase production: As shown in Fig. 1, corn stover as cellulolytic substrate gave the highest cellulase activity with Fusarium graminearum (1.01 μmole mL1 min1), while rice straw gave the lowest cellulases production (0.31 μmole mL1 min1). Therefore, corn stover selected for conducting further studies on cellulase production by Fusarium graminearum.

Effect of carbon source concentration on cellulase production: Data illustrated in Fig. 2 Showed effect of different concentration of corn stover powder ranged from 2.5-20% (w/v) on cellulase production by Fusarium graminearum.

Fig. 1:
Effect of different carbon sources on cellulase production

Fig. 2:
Effect of carbon source concentration (20 w/v) on cellulase production

Maximum cellulase activity reached (2.34 μmole mL1 min1) was obtained at 20% (w/v) corn stover concentration, while the minimum activity (0.71 μmole mL1 min1) was obtained at 2.5% of corn stover concentration of modified cellulose powder medium (CPM). Based on these data, corn stover concentration at 20 (w/v) was chosen for further studies on the modified CPM.

Effect of initial pH of growth medium on cellulase production: The cellulase production by Fusarium graminearum in varying initial pH of the growth medium showed that pH 5.0 was the optimum (Fig. 3). Moreover, when pH level increased, the enzyme production was decreased.

Fig. 3:
Effect of initial pH of growth medium on cellulase production

Fig. 4:
Effect of nitrogen sources on cellulase production

Accordingly, the initial pH for the modified CP medium was adjusted to pH 5.0 in the subsequent experiments.

Effect of nitrogen sources on cellulase production: Data of Fig. 4 showed that among 5 nitrogen sources tested for screening their effect on cellulase activity, peptone was found to be the best nitrogen source producing the highest level of cellulase activity reached 3.79 (μmole mL1 min1) by Fusarium graminearum. These data indicating that the source of nitrogen should to be organic for better cellulase production.

Scanning electron micrographs for corn stover: Electron micrographs showed that the fibers of corn stover were separated after cellulase treatment as a result of cellulose hydrolysis (Fig. 5a and b).

In vitro rumen fermentation: The obtained results showed that all levels of the produced cellulase and Pan-Zyme increased DM, NDF, ADF, cellulose and hemicellulose degradability of the treated diets compared with the control one, which gave the lowest values of diet degradability parameters (Table 1). The maximum produced cellulase efficiency percentage for dry matter degradability was obtained at 1200 IU kg1 DM reached 23.19% over the control:

Also, the Pan-Zyme takes the same trend and its efficiency percentage for dry matter degradability reached 25.15% over the control. There are no marked changes in the ruminal parameters pH and total gas production (TGP) due to treatment with the cellulases (Table 2). While marked changes have been found in total volatile fatty acids (VFA) and NH3 concentrations due to cellulases supplementation (Table 2).

Table 1:
Cellulases effects on degradability parameters of experimental diets
DM: Dry matter, NDF: Neutral detergent fiber, ADF: Acid detergent fiber, SE: Standard error, a-eSignificantly different (p<0.05)

Fig. 5(a-b):
Electron micrographs, (a) Before and (b) After treatment of corn stover fibers with the produced cellulase

Table 2:
Cellulases effects on ruminal parameters (in vitro)
TGP: Total gas production, TVFA: Total volatile fatty acids, SE: Standard error, a-csignificantly different (p<0.05)

The maximum increase in TVFA and NH3 concentrations has been noticed after diet treatment with the cellulases at 1200 IU kg1 DM.

DISCUSSION

Production of cellulase by Fusarium graminearum requires optimal condition for their growth. The superiority of corn stover over the other agricultural wastes for cellulase production may be due to that corn stover contain growth factors (ex, minerals) or may be it act as a carbon and nitrogen source at the same time. Therefore the carbohydrate part of the lignocellulosic agricultural residues has received considerable interest in cellulase production process. Production of cellulase under the optimum fermentation conditions using the agricultural by-products as the substrate (carbon source) may give us highly effective feed additive product with low cost. The utilization of the produced cellulase in enrichment of the feeding value of the agricultural by-products may help in overcome of the feed gap with good impact on environment and public health. The carbon source of production medium is critically affecting the cellulase activity7.

It has been reported that, the impact of the agricultural wastes as carbon sources on cellulase production is vary due to their chemical nature and its effect on the growth of the cultivated fungi15. Cellulase production by different fungal strains on variety of agricultural residues has been reported with different yields8,13,15. It is will known that, fungal cellulase synthesis and secretion under submerged fermentation is mainly depending on moisture content of growth medium13. In the current study, the higher concentration of corn stover not negatively affects the active water of the growth medium and gave F. graminearum more ability for cellulase production. It seems that higher CPM moisture content (2.5% corn stover) decreases cellulase production yield due to suppression of F. graminearum growth through reduction in interparticle spaces and impaired oxygen transfer23. These findings are agree with those obtained by Aboul-Fotouh et al.13, who found that minimum cellulase production was obtained by A. niger at 2.5% rice straw concentration of growth medium.

Initial pH of the microbial growth medium has profound effect on cellulase production. It’s well known that the optimal pH for cellulase production varies with different fungal strains. In the current study, pH 5.0 was the optimum for cellulase production by Fusarium graminearum. Also, Sethi and Gupta24 reported that maximum cellulase activity by Penicillium chrysogenum and Aspergillus niger was observed in medium of pH 5.0. Similar observation was reported for cellulase production by A. tereus QTC 828 by Ali et al.25 and Trichoderma resei by Doppelbauer et al.26.

In current study, peptone was found to be the best nitrogen source producing the highest level of cellulase activity by Fusarium graminearum. Many researchers have noticed that organic nitrogen sources gave better cellulase activity than inorganic nitrogen sources8,13,15. In accordance, peptone was found to be the most promising and effective nitrogen source for cellulase production27 by Penicillium waksmanii F10-2, A. hortai 28 and A. niger13. It seems that the presence of the peptone at certain level in fungal growth medium was essential for high levels of cellulase production.

The produced cellulase in the current study was able to degrade the corn stover fibers as shown in electron micrographs. In accordance, Murad and Azzaz8 noticed the same impact of cellulase produced by A. flavus NRRL 5521on the rice straw fibers. The electron micrographs may give evidence for occur of fiber saccharification but the degree of saccharification should be assayed on the basis of release of reducing group.

Production of cereal crop residues, especially corn stover has increased dramatically as a result of more corn production. Microbial treatments of such residues may improve its nutritive value and gave solution for feed gap in Egypt29,30. In current study, diets containing 25% corn stover have been treated with the produced fungal cellulase and commercial enzyme source. In fact, the increase DM, NDF, ADF, cellulose and hemicellulose degradability is reasonable as cellulases supplementation led to more hydrolysis and separation for fibers of feed particles (see electron micrographs). Positive effects of fibrolytic enzymes supplementation on the diets degradability have been observed in many of the in vitro studies by Azzaz et al.9, Aboul-Fotouh et al.13 and Azzaz et al.20. Aboul-Fotouh et al.13 suggested that cellulases supplementation might alter the carbohydrates structure of the treated diets during the interaction period, making it more amenable for rumen microorganisms. In current study, although ruminal TVFA and NH3 concentrations have been increased after cellulases addition, the ruminal pH and TGP are not affected. This might be due to increase extent of dietary amino acids deamination by rumen microorganisms and enhancement of microbial protein synthesis which may drive the pH toward alkalinity. In the same time, increase extent of dietary carbohydrate hydrolysis after cellulases addition let for more acids production especially TVFA which opposite action of amino acids deamination process. This may give an explanation for why the ruminal pH was not change. The current data supports the findings of Ribeiro et al.31, who noticed that in vitro DM, NDF and ADF disappearance tended to increase for barley straw treated with different types of fibrolytic enzymes with no effect of enzyme on the ruminal pH. Also, Addition of fibrolytic enzymes to a total mixed ration (TMR) in a RUSITEC did not affect pH32. On the other hand, the rapid fermentation of the concentrate portion the teasted diet may lead to higher propionate production with no effect on CH4 production. Propionate acts as alternative hydrogen sink in the rumen diverting hydrogen away from the reduction of CO2 to CH4, while the production of butyrate and acetate promotes methanogenesis33. This may give an explanation for why the ruminal TVFA production increased with no effect on TGP production in this study.

CONCLUSION

Finally, it could be concluded that production of cellulase under the optimum fermentation conditions using the agricultural by-products as the substrate (carbon source) may give us highly effective feed additive product with low cost. On the other hand, the utilization of the produced cellulase in enrichment of the feeding value of the agricultural by-products may help in overcome of the feed gap with good impact on environment and public health.

SIGNIFICANCE STATEMENT

This study discovered the potential production of cellulase by Fusarium graminearum under the optimum fermentation conditions using the agricultural by-products as the substrate. This may give feed factories highly effective feed additive product with low cost. The utilization of the produced cellulase in enrichment of the feeding value of the agricultural by-products can be beneficial for livestock breeders who suffering high prices of traditional feed stuff. This study will help the researchers to uncover the critical areas of using of biotechnology for enrichment of the feeding value of the agricultural residues and their impact on environment and public health.

ACKNOWLEDGMENTS

This research was funded by Science and Technology Development Fund in Egypt (STDF), in accordance with the Research Project ID Number: 15174. The authors are very grateful to the head of Science and Technology Development Fund in Egypt (STDF) and the head of the National Research Centre (NRC) who have allowed the implementation of this research.

REFERENCES
1:  Azzaz, H.H., H.A. Murad, A.M. Kholif, T.A. Morsy, A.M. Mansour and H.M. El-Sayed, 2013. Increasing nutrients bioavailability by using fibrolytic enzymes in dairy buffaloes feeding. J. Biol. Sci., 13: 234-241.
CrossRef  |  Direct Link  |  

2:  Khattab, M.S.A., E.A. El-Bltagy, A.M. Abd El Tawab, O.H. Matloup, T.A. Morsy, H.H. Azzaz and M.M. Abdou, 2019. Productive performance of lactating buffaloes fed ration containing date seed and fibrolytic enzymes. J. Applied Sci., 19: 241-246.
CrossRef  |  Direct Link  |  

3:  Azzaz, H.H., E.S.A. Farahat, T.A. Morsy, H.A. Aziz, F.I. Hadhoud and M.S. Abd-Alla, 2016. Moringa oleifera and Echinacea purpurea as supplements for rhamani lactating Ewe's diets and their effect on rumen characteristics, nutrients digestibility, blood parameters, milk production, composition and its fatty acid profile. Asian J. Anim. Vet. Adv., 11: 684-692.
CrossRef  |  Direct Link  |  

4:  Azzaz, H.H., H.A. Aziz, H. Alzahar and H.A. Murad, 2018. Yeast and Trichoderma viride don't synergistically work to improve olive trees by products digestibility and lactating Barki ewe's productivity. J. Biol. Sci., 18: 270-279.
CrossRef  |  Direct Link  |  

5:  Azzaz, H.H., H.A. Murad and T.A. Morsy, 2015. Utility of Ionophores for ruminant animals: A review. Asian J. Anim. Sci., 9: 254-265.
CrossRef  |  Direct Link  |  

6:  Azzaz, H.H., E.S.A. Farahat and H.M. Ebeid, 2017. Effect of partial replacement of corn grains by date seeds on rahmani ram’s nutrients digestibility and Nubian goat’s milk production. Int. J. Dairy Sci., 12: 266-274.
CrossRef  |  Direct Link  |  

7:  Murad, H.A. and H.H. Azzaz, 2010. Cellulase and dairy animal feeding. Biotechnology, 9: 238-256.
CrossRef  |  Direct Link  |  

8:  Murad, H.A. and H.H. Azzaz, 2013. Cellulase production from rice straw by Aspergillus flavus NRRL 5521. Sci. Int., 1: 103-107.
CrossRef  |  Direct Link  |  

9:  Azzaz, H.H., A.M. Kholif, H.A. Murad, M.A. Hanfy and M.H. Abdel Gawad, 2012. Utilization of cellulolytic enzymes to improve the nutritive value of banana wastes and performance of lactating goats. Asian J. Anim. Vet. Adv., 7: 664-673.
CrossRef  |  Direct Link  |  

10:  Azzaz, H.H., T.A. Morsy and H.A. Murad, 2016. Microbial feed supplements for ruminant's performance enhancement. Asian J. Agric. Res., 10: 1-14.
CrossRef  |  Direct Link  |  

11:  Colombatto, D., F.L. Mould, M.K. Bhat and E. Owen, 2007. Influence of exogenous fibrolytic enzyme level and incubation pH on the in vitro ruminal fermentation of alfalfa stems. Anim. Feed Sci. Technol., 137: 150-162.
CrossRef  |  Direct Link  |  

12:  Giraldo, L.A., M.L. Tejido, M.J. Ranilla and M.D. Carro, 2008. Effects of exogenous fibrolytic enzymes on in vitro ruminal fermentation of substrates with different forage: Concentrate ratios. Anim. Feed Sci. Technol., 141: 306-325.
CrossRef  |  Direct Link  |  

13:  Aboul-Fotouh, G.E., G.M. El-Garhy, H.H. Azzaz, A.M. Abd El-Mola and G.A. Mousa, 2016. Fungal cellulase production optimization and its utilization in goat’s rations degradation. Asian J. Anim. Vet. Adv., 11: 824-831.
CrossRef  |  Direct Link  |  

14:  Kholif, A.E., A.Y. Kassab, H.H. Azzaz, O.H. Matloup, H.A. Hamdon, O.A. Olafadehan and T.A. Morsy, 2018. Essential oils blend with a newly developed enzyme cocktail works synergistically to enhance feed utilization and milk production of Farafra ewes in the subtropics. Small Rumin. Res., 161: 43-50.
CrossRef  |  Direct Link  |  

15:  Azzaz, H.H., H.A. Murad, A.M. Kholif, M.A. Hanfy and M.H. Abdel Gawad, 2012. Optimization of culture conditions affecting fungal cellulase production. Res. J. Microbiol., 7: 23-31.
CrossRef  |  Direct Link  |  

16:  Khattab, M.S.A., H.H. Azzaz, A.M. Abd El Tawab and H.A. Murad, 2019. Production optimization of fungal cellulase and its impact on ruminal degradability and fermentation of diet. Int. J. Dairy Sci., 14: 61-68.
CrossRef  |  Direct Link  |  

17:  Mandels, M., L. Hontz and J. Nystrom, 1974. Enzymatic hydrolysis of waste cellulose. Biotechnol. Bioeng., 16: 1471-1493.
CrossRef  |  Direct Link  |  

18:  Miller, G.L., 1972. Use of dinitrosalicyclic acid reagent for determination of reducing sugar. Biotechnol. Bioeng. Symp., 5: 193-219.

19:  Ismail, S.A., A.M. Abdel-Fattah, M.A. Emran, H.H. Azzaz, M.S. El-Gamal and A.M. Hashem, 2018. Effect of partial substitution of ration's soybean meal by biologically treated feathers on rumen fermentation characteristics (in vitro). Pak. J. Biol. Sci., 21: 110-118.
CrossRef  |  Direct Link  |  

20:  Azzaz, H.H., A.A. Aboamer, H. Alzahar, M.M. Abdo and H.A. Murad, 2019. Effect of xylanase and phytase supplementation on goat's performance in early lactation. Pak. J. Biol. Sci., 22: 265-272.
CrossRef  |  Direct Link  |  

21:  IBM., 2011. IBM SPSS Statistics for Windows, Version 20.0. IBM Corporation, Armonk, New York, USA.

22:  Duncan, D.B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42.
CrossRef  |  Direct Link  |  

23:  Krishna, C. and M. Chandrasekaran, 1996. Banana waste as substrate for α-amylase production by Bacillus subtilis (CBTK 106) under solid-state fermentation. Applied Microbiol. Biotechnol., 46: 106-111.
CrossRef  |  Direct Link  |  

24:  Sethi, S. and S. Gupta, 2014. Optimization of cultural parameters for cellulase enzyme production from fungi. Biolife, 2: 989-996.
Direct Link  |  

25:  Ali, S., A. Sayed, R.T. Sarker and R. Alam, 1991. Factors affecting cellulase production by Aspergillus terreus and Aspergillus niger. Enzyme Microb. Technol., 11: 606-616.

26:  Doppelbauer, R., H. Esterbauer, W. Steiner, R.M. Lafferty and H. Steinmuller, 1987. The use of lignocellulosic wastes for production of cellulase by Trichoderma reesei. Applied Microbiol. Biotechnol., 26: 485-494.
CrossRef  |  Direct Link  |  

27:  Han, L., J. Feng, C. Zhu and X. Zhang, 2009. Optimizing cellulase production of Penicillium waksmanii F10-2 with response surface methodology. Afr. J. Biotechnol., 8: 3879-3886.
Direct Link  |  

28:  El-Hadi, A.A., S. Abu El-Nour, A. Hammad, Z. Kamel and M. Anwar, 2014. Optimization of cultural and nutritional conditionsfor carboxymethylcellulase productionby Aspergillus hortai. J. Radiat. Res. Applied Sci., 7: 23-28.
CrossRef  |  Direct Link  |  

29:  Azzaz, H.H., H.M. Ebeid, T.A. Morsy and S.M. Kholif, 2015. Impact of feeding yeast culture or yeast culture and propionibacteria 169 on the productive performance of lactating buffaloes. Int. J. Dairy Sci., 10: 107-116.
CrossRef  |  Direct Link  |  

30:  Abd El Tawab, A.M., H.A. Murad, M.S.A. Khattab and H.H. Azzaz, 2019. Optimizing production of tannase and in vitro evaluation on ruminal fermentation, degradability and gas production. Int. J. Dairy Sci., 14: 53-60.
CrossRef  |  Direct Link  |  

31:  Ribeiro, G.O., A. Badhan, J. Huang, K.A. Beauchemin and W. Yang et al., 2018. New recombinant fibrolytic enzymes for improved in vitro ruminal fiber degradability of barley straw. J. Anim. Sci., 96: 3928-3942.
CrossRef  |  Direct Link  |  

32:  Mohamed, M.A.E., C. Yangchun, B.M. Bodinga, Z. Lixin and Y. Zekun et al., 2017. Effect of exogenous fibrolytic enzymes on ruminal fermentation and gas production by RUSITEC, in vitro abomasum and ileum digestibility. Int. J. Pharmacol., 13: 1020-1028.
CrossRef  |  Direct Link  |  

33:  Aboamer, A.A., M.S.A. Khattab, S.A.H. Abo El-Nor, H.M. Saleh and A.M. Kholif et al., 2017. A study of nutrient digestibility, milk production and performance of lactating Barki ewes fed synchronous least cost ration. Int. J. Dairy Sci., 12: 114-121.
CrossRef  |  Direct Link  |  

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