الفهرس 
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Abstract
6. Summary
Several fungal candidates were investigated for their
cellulytic activities including two strains Trichoderma viride
(NRRL1698) and Aspergillus terreus (NRRL260), isolates from soil
at Agricultural Research Center, mangrove soil and compost samples.
Cellulytic activity of isolates and strains
All fungal candidates found to possess cellulytic activities
using treated rice straw (RS) all over the 12 days of production and
mostly achieved their maximum cellulytic activity at the end, of
which strain NRRL1698 and isolates 516, 217, 504 and 517were the
most promising fungal candidates.
Optimizing cellulase production
Cellulase production by any of the fungal candidates was
subjected to successive optimization studies. The five fungal
candidates 217, NRRL1698, 504, 516 and 517 achieved their
maximum cellulytic production statistically after 5 days to be 7220U,
8124U), 7487U, 6203U and 7856U, at pH values of 5, 4, 4, 6 and 4,
respectively. Exceptionally, both fungal candidates 504 and 517
achieved their maximum cellulytic production not only at pH 4 but
also at pH 5, while 516 achieved its maximum cellulytic production
at both pH 6 and 7.
The optimization of N source included studying the impact of
two inorganic and two organic types on cellulase production. All
fungal candidates achieved their maximum cellulytic activities
statistically using organic nitrogen sources than inorganic ones, as
isolates 217 and 517 recorded their best cellulytic activities using
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beef extract to be 7065U and 6842), strain NRRL1698 and isolate
516 recorded their best cellulytic activities using yeast extract to be
6499U and 8495U, while isolate 504 recorded its best cellulytic
activities using either yeast or beef extracts to be 6293U and 6264U,
respectively.
Saccharification
As both isolates 217 and 516 proved their superiority among
other fungal candidates as efficient cellulase producers, their
cellulases were tested for their efficiency in saccharifying treated rice
straw (RS).
Optimizing saccharification in test tubes
Saccharification of RS was much better increasing by
incubation period progressing on. As emphasized by statistical
analysis the cellulases produced by isolate 217 maximum
saccharification was better with pH 6 and 7 than with pH 4 at 40°C,
while it was better with pH 5 than pH 6 and 7 at 50°C, proving that
the cellulases preferred acidic pH at higher incubation temperature
and neutral pH at lower temperature. On the other hand, the cellulase
produced by isolate 516 was superior in saccharification results
according to statistical analysis at pH 7 and temperature 50°C,
followed by pH 6 at both temperatures 40°C and 50°C. Depending on
those results, the cellulases produced isolate 516 proved to be more
superior than that produced by isolate 217 in saccharification of
treated RS.
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Saccharification in flasks
The saccharification % of RS at 40°C / pH=6 fluctuated
during incubation period achieving its highest result to be 72.19 %
after 4 days and increased by 4.7 times compared to that in the test
tube test. On the other hand, the saccharification % increased at the
end of incubation period recording 89.11% that leveled up
saccharification to 5.4 times compared to that achieved in previous
test tube test.
Fermentation efficiency in bottles as affected by initial pH
Fermentation efficiency % increased parallel to fermentation
period under each pH value, where it didn’t exceed 13% up till 45 hr
under both pH values 4 and 5, achieving their maximum after 48 hr to
be 79.4 and 98.1 %, respectively. Fermentation efficiency % at pH=6
exceeded 50% after 30 hr and reached after 48 hr to be 99.2% where
its ethanol yield was close to the ideal theoretical yield.
Rates of ethanol production in fermentation bottles
The production rate after 1 hr recorded 1.212, 1.013 and 3.001
g ethanol/bottle/hr declining afterwards it fluctuated to give
occasional losses in ethanol produced where negative production
rates appeared and finally achieving 2.358, 2.917 and 0.652 g
ethanol/bottle/hr at the end of fermentation period, under pH values
of 4, 5 and 6, respectively. It was an obvious increase in production
rates under pH 4 and 5 nearly by 200% and 290%, respectively,
comparing first hour and final 48 hr fermentation rates. Under pH 6
the first hour fermentation rate decreased by more than 78%,
regardless to the accumulation final results.
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Fermentation tests in fermenter
The fermentation efficiency in the first day achieved 64% and
then decreased down till 4 hr after which it continued increasing up to
84% at 8 hr. In the second day an obvious decrease down to 63%
followed by fluctuating increase achieving its maximum at 29 hr with
a final fermentation efficiency of 100% after 48 hr.
Rate of ethanol production (g/fermenter/hr)
The rate of ethanol production spotted on the apparent loss in
ethanol specifically after 2, 4, 24 27 30 and 48 hrs. Never the less, the
fluctuation prolonged the accumulation period needed to achieve the
100% fermentation efficiency after 48 hr.
The comparison of fermentation efficiencies of reducing
sugar by S. cerevisiae in both the 200ml fermentation bottles and 2L
fermenter was crucial to emphasize the effect of fermentation volume
and accordingly the effect of S. cerevisiae inoculum size on this
process. Fermentation efficiency in fermenter was higher than that in
fermentation bottle under the same fermentation conditions. This
made the ethanol harvesting during fermentation period more feasible
from fermenter than bottles, if it was intended to be used.
The genetic identification of isolate 516
Sequences of fungal isolate 516 designated as Am1 in this
study were assembled using DNASTAR computer package (version
5.05). Assembled sequence of isolate Am1 was uploaded to GenBank
as OM760501. The closely similar sequences to Aspergillus: section
Fumigati including sequences of type and ex-type species were
downloaded from GenBank. The isolate AM 1 occupied the same
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branch as Aspergillus neoellipticus ATCC 16903 (type strain) with
100% (562/562) similarity between both species. As a result, this
isolate was identified as Aspergillus neoellipticus.
GC mass results
Fermentation final sample was assessed qualitatively by GCMS
analysis, as the constituents in the mass spectrum fragmentation
pattern obtained by electron ionization (EI) were compared with
those stored in Wiley and NIST Mass Spectral Library data. Cation
fragments appearing at specific m/z segmented from the main parent
molecule by losing specific part. The comparative spectrum proved
that the final product was ethyl alcohol.