الفهرس 
1. IntroductionOnly 14 pages are availabe for public view
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Abstract
Twenty-six isolates of Trichoderma spp. collected from various Egyptian locations were screened for their Cellulase activities via qualitative and quantitative tests. The main objective was to choose the best isolates that can degrade lignocellulosic biomass.
First, we carried out qualitative screening of Cellulases in Congo red dye medium. Six isolates showed clear zones larger than other isolates namely, MNF-MAS-Tricho1, 6, 23, and ASI-MAS-Tricho26. In addition, another qualitative test was done using 1% PASC media. Full plate radial growth was shown in isolates MNF-MAS-Tricho 2 ,3 ,5 ,6 ,12 ,15 ,18 ,21 , and 23 after 48 h incubation and the sporulation type was detected.
Second, quantitively enzymes activities (filter paper activity (FPase) assay, carboxymethyl cellulase assay for endo- β - 1,4-gluconase and cellobiose assay for β-glucosidase) were carried out for 2,3,4,6 and 7 days in Mandel’s Media with CMC 0.5% as a substrate of Cellulases. We detected four isolates that showed highly significant FPase activities after seven days of incubation namely, MNF-MAS-Tricho 2, MNF-MAS-Tricho 3 MNF-MAS-Tricho 15 and MNF-MAS-Tricho 17(0.49, 0.47, 0.47 and 0.45 IU mL-1, respectively).
Furthermore, Cellulase enzyme activities (FPase, CMCase and β-glucosidase) were measured in Mandel’s Media with Avicel 1% in SmF cultures for all isolates. Three isolates demonstrated highly significant FPase activities namely, MNF-MAS-Tricho1, MNF-MAS-Tricho 2 and MNF-MAS-Tricho 3 (0.50, 0.39 and 0.49 IU mL-1, respectively). While MNF-MAS-Tricho 1 showed the highest significant CMCase activity (0.80 IU mL-1 ). Concerning β-glucosidase activity, MNF-MAS-Tricho 1 was the highest (0.78 IU mL-1 ), while MNF-MSH-Tricho 11 and MNF-MAS-Tricho15 were the lowest (0.36 IU mL-1 ).
Cellulase enzyme activities (FPase, CMCase and β-glucosidase) were measured in SSF (Rice straw) for all isolates. Generally, all isolates indicated outstanding endoglucanase activity, when compared to FPase and β-glucosidase. Five isolates demonstrated highly significant FPase activities namely, MNF-MAS-Tricho2, 6,12,18 and 23 (0.114 ,0.102, 0.103, 0.105 and 0.103 IU g-1 , respectively). MNF-MAS-Tricho 13 showed the lowest FPase activity (0.06 IU g-1). While, MNF-MAS-Tricho 2,12,22 and 23 showed the highest significant CMCase activity (0.156, 0.159, 0.176 and 0.168 IU g-1 , respectively) and the lowest were MNF-MAS-Tricho 8,11,14, and 16 (0.112 IU g-1 ). As for β-glucosidase MNF-MAS-Tricho 2,6,12,18 and 23 were the highest in the same level without significant (0.15 IU g-1 ), while MNF-MSH-Tricho1 was the lowest (0.076 IU g-1 ).
In SSF of wheat straw, two isolates demonstrated highly significant FPase activities namely, MNF-MAS-Tricho2, and 18 (0.076 and 0.070 IU g-1 , respectively). MNF-MAS-Tricho 13 showed the lowest FPase activity (0.04 IU g-1 ). While MNF-MAS-Tricho 22 and 23 showed the highest significant CMCase activity (0.112 and 0.117 IU g-1 , respectively) and the lowest were MNF-MAS-Tricho 8,11,14, and 16 (0.075 IU g-1 ). As for β-glucosidase, MNF-MAS-Tricho 2 and 23 were the highest in the same level without significant (0.103 IU g-1 ) while MNF-MSH-Tricho 20 was the lowest (0.070 IU g-1 ).
After all these tests we can compare enzyme production by SmF and SSF. Firstly, Using SmF production for high yield and high quality, but silt it can’t be used in second generation biofuel. Moreover, SmF still very expensive. Secondly, SSF are producing low yield of enzymes and can be used in second generation biofuel. So, we need to improve the isolates that have high yield of enzyme and releasing high amount of glucose in SSF.
Moreover, the genetic diversity among the tested isolates was tested using rep-PCR marker. The polymorphism percentage ranged from 46.15 to 83.33%. (GTG)5 marker produced the maximum number of polymorphic loci (13 loci out of 18 loci) with about 83.33% polymorphism, followed by rep-10 with 69.2% polymorphism. Furthermore, the polymorphism information content (PIC) values ranged between 0.285 for Rep-10 and 0.340 for (GTG)5 with an average of 0.306. The tested primers exhibited high discriminating and resolving power.
Moreover, we extract total genomic DNA to detect Cellulase genes by using Chb gene cellobiohydrolase (CBH), Bgl gene β-glucosidase (BGL) and Egl gene endoglucanase (EG).
In order to select the Trichoderma harzianum isolates to improve the cellulase production for second-generation biofuel, we selected the best isolates in SSF for FPase and β-glucosidase activities. Moreover, selection of the best isolates that have high concentrations of glucose level after the incubation periods that refers to didn’t have feedback inhibition. After this selection we select MNF-MAS-Tricho 2, 6,12 ,18, and 23 to improve them with the protoplast fusion and genome shuffling. FU7 showed the highest number of observed colonies (45) in plate. FU1, FU5 and FU6 showed the lowest number of colonies (12,11 and 14, respectively). We selected 11 fusions from stability test.
We can conclude that the protoplast fusion improves FPase enzyme activity in all fusants strains except (FU2.1 and FU2.2) in SmF, (FU1.1 and FU4.1) in SSF of untreated rice straw and (FU3.1 and FU3.2) in SSF of pretreated rice straw. CMCase enzyme activity was improved in all fusant strains except (FU2.2, FU3.1, FU6.1 and FU7.2) in SmF, and (FU4.1 and FU7.1) in SSF of untreated rice straw. β-glucosidase enzyme activity was improved in all fusant strains except (FU2.2) in SmF.
In general, the highest improvement of FPase activity was detected in SmF with about 153 % in FU1.1, SSF untreated rice straw, 283.8 % in FU4.2 and in SSF pretreated straw 431% in FU4.2. Moreover, the highest improving in CMCase was shown in SmF 200% in FU4.2, SSF untreated rice straw 350% (FU2.1 and FU2.2) and SSF pretreated straw 540% in FU7.1. Whereas β-glucosidase activity showed highest improvement in SmF 189% in FU5.1, SSF untreated rice straw, in 365.8% FU4.1 and SSF pretreated straw 406%.
The results of genome shuffled indicated higher improving in SH1 in SSF of pretreated straw in CMCase 560% (0.56 IU/g) higher than the lower parent, and 412.9 % FPase (0.266 IU/g) from lower parent. The improvement was detected in each SSF and SmF. Moreover, Improvement reached 310 % in β-glucosidase (0.62 IU/g) higher than the lowest parent. Finaly, it was clear that the glucose concentration in pretreated straw SSF was the highest (10.1 mg/ml).
The bioethanol production by fermentation process started by the addition of Saccharomyces cerevisiae cells 8x108 cfu /mL. The glucose was consumed rapidly during the first 4 h of fermentation, and then it was highly consumed after 72 h this increased ethanol production which was significantly higher after 7 days. whereas the bioethanol was the lowest showed in SmF (2.23 ± 0.05g/l, 0.02 g/g) and shown maximum ethanol production by the saccharification of SSF (4.20 ± 0.13 g/l , 0.04 g/g).