IMPROVEMENT OF BIOETHANOL PRODUCTION FROM ENZYMATIC DEGRADATION OF SOFTWOOD BIOMASS BY STANDARDIZING FERMENTATION PROCESSES
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Date
2015
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Abstract
ABSTRACT
The present investigation was carried out to isolate, screen and identify the most efficient cellulolytic,
xylanolytic and lignolytic microorganisms from diverse sources. Enzyme production, optimization, partial
purification, bioconversion of cellulosic waste into bioethanol and scale up studies were performed with selected
strains to recommend their use for industries. In total 23 microorganisms including 20 bacteria and 3 fungi were
isolated. Among bacteria R2, R4 were found to be potent cellulase producer and SD9 were screened as hyper
xylanase producer while a fungal strain WF3 was screened as best hydrolytic enzymes producer so selected for
further studies. These microbial isolates were identified as B. licheniformis R2, B. mojavensis R4 B. atropheaus
SD and Amylomyces rouxii WF3 and registered with NCBI under accession no. |KJ588781|, |KJ588787| and
|KJ588788|. Cellulase and xylanase enzymes were optimized through classical approach one factor at a time
(OFAT) as well as response surface methodology under submerged fermentation varying medium, pH,
temperature, incubation time, substrate concentration and inoculums size. The percent increase in enzyme
activity obtained after optimization of different process parameters was 106.25%, 105.76% for cellulase of B.
licheniformis R2 and B. mojavensis R4 while 26.46% increase was noticed for xylanase of B. atropheaus SD9.
To reduce the production cost of enzymes, various softwood biomass were used as a substrate under SmF by B.
licheniformis R2 and B. mojavensis R4 and B. atropheaus SD9 and SSF by A.rouxii WF3and among them P.
roxburghii wood was found the best for hypercellulase and xylanase production under SmF as well as SSF. The
partial purification of hydrolytic enzymes was done by ammonium sulphate precipitation. Selected biomass was
subjected to various pretreatment for enhanced saccharification. Among all the pretreatments, NaOH+urea was
found as the best pretreatment and was selected for further studies. Bioconversion of pretreated P. roxburghii
wood to ethanol was studied under two different fermentation processes i.e separate hydrolysis and fermentation
(SHF) and simultaneous saccharification and fermentation (SSF). Different protocols had been designed to
delimit the constraints of fermentation process. SHF was evaluated by modifying four different sub-processes of
detoxification and non-detoxification as well as pooling and non pooling of pretreated liquor. Maximum ethanol
was achieved in protocol–II of SHF i.e. 11.08g/l by co-culture of S. cerevisiae I and P. stipitis with the
fermentation efficiency of 41.09%. Among all the three processes of fermentation evaluated in the present
study, SSF was found to be the best and in case of strains used for fermentation, co-culture of S. cerevisiae I and
P. stipitis was observed the best combination for highest bioethanol production. Scale up of ethanol production
was performed in 7.5 L bioreactor under different modes of fermentation. Simultaneous saccharification
produced maximum ethanol with efficiency of 70.12%. Mathematical model based on experimental results for
ethanol production was proposed. The production of ethanol was found as mixed growth associated. The model
consists of a set of ordinary differential equations taking into account the growth, substrate utilization and
ethanol production with time.
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Keywords
enzymes, productivity, bacteria, cellulose, fermentation, fungi, wood, biomass, polysaccharides, alcohols, cellulolytic, xylanolytic and lignolytic microorganisms