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Bio Ethanol Production by Enzymatic Hydrolysis of Cellulose

Bio Ethanol Production by Enzymatic Hydrolysis of Cellulose

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Published by: Ahmad Mohammed Gumel on Mar 22, 2010
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11/19/2012

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Bioethanol production by Enzymatic Hydrolysis of Cellulose usingimmobilized
Saccharomyces cerevisiae
in fedbatch Fermentation:A Kinetic study.
Ahmad Mohammed Gumel,
 Biotechnology Unit, Institute of Biological Sciences, University of Malaya, 50603, Kuala Lumpur, Malaysia
Abstract:
The purpose of this study was to highlight the enzymatic kinetics in the hydrolysis of cellulose for Bioethanol production. Immobilized
Saccharomyces
 
cerevisiae
was used as fermentative organism,while Carboxymethylcellulose (CMC) was used as fermentation substrate. Different substrateconcentrations were used; parameters such as enzymatic velocity, substrate concentration, glucoseconcentration, bioethanol yield and productivity were studied under 15 minutes fermentation time.Under optimized condition of 10g/l CMC with glucose hydrolysis concentration of 0.166g/l bioethanol production was found to be 42% (w/v) at 15 minutes of fermentation time. Langmuir andMichaelis-Menten models were used to plot and validate the experimental data respectively.
1. Introduction:
Biofuels such as bioethanol, biohydrogen and biodiesel are anticipated to be one of the featurealternatives to fossil fuels. The current world consumption of 474 exajoules (5×10
20
J) with 80 to 90 percent derived from the combustion of fossil fuels
1
, which are associated with productioninstability, insecurity, sky rocketed prices as well as green house gasses emissions; are among thereasons that lead policy makers to seek for energy alternatives that are sustainable and environmentalfriendly.Today bioethanol is among the most widely employed biofuel in the world with current productionof about 19 billion gallons world wide, in United State alone it provides about $12.3 billion capitalmarket, and creates 238,541 jobs as at 2007
2
, bioethanol production is expected to pass over 20 billion gallons by 2012.Although, food-crops are known to be the main feedstock in bioethanol production, the use of postharvest agro-waste and non-food crops cellulosic materials are highly encouraged, this is because of the food security issue, though there are surplus amount of food in some countries, millions of  people in other countries most especially developing countries face food shortage scenario.Therefore extensive used of food crops such as corn, wheat, soybeans, palm oil as a feedstock in bioethanol production, may result in a serious food security problems.Cellulosic resources, such as agricultural residues, paper wastes and wood chips, are the mostabundant organic substance in nature and considered to be promising and economically feasiblefeedstock for biofuel production
3
. Enzymatic hydrolysis by cellulolytic enzymes that naturallydegrade these cellulosic materials to monomeric sugars that are fermentable by Microbes are oftenrequired
4
. Although cellulose may be hydrolised by non- enzymatic methods, i.e. by acid hydrolysis,the advantages and utility cost of enzymatic hydrolysis are better and lower compared to the acidhydrolysis. A number of bioethanol production processes that used microorganisms have beenstudied to produce ethanol from cellulosic materials. Among them, the stirred tank fedbatchfermentation process was cited by many researchers
5-10
.1
 
Although, several microorganisms were employed in bioethanol production process, but direct bioconversion of cellulosic materials for example by
Clostridium thermocellum
is not attractive because of low ethanol productivity and high energy requirements compared to ethanol production by
Saccharomyces cerevisiae
using molasses
11
.
S. cerevisiae
is a yeast, which is believed to beamong the most effective lignocellulosic biomass degrading microorganisms for hexose sugars suchas glucose, mannose and galactose, it has a capability of rapid rates of glycolysis and ethanol production under optimal conditions, producing over 50 mmol of ethanol per h per g of cellulose
12
.However, this high rate is maintained for only a brief period during batch fermentation and declines progressively as ethanol accumulates in the surrounding broth
12
.To date numerous research studies regarding the fermentative activities of 
S. cerevisiae
in stirredtank reactors utilizing cellulosic biomass have been reported, alas! There are limited citations onkinetic studies of the process; which we believed will help in understanding the overall process for maximum optimization.
2. Materials and Methods2.1 Fermentation media and Culture
The substrate used for this study is carboxymethylcellulose (CMC) from laboratory stock to serve asthe cellulose substrate, S. cerevisiae that was used was also obtained from laboratory stock. Whilechemicals and reagents such as sodium alginate, calcium chloride, yeast extract, ammonium sulfate, potassium phosphate, hydrated magnesium sulfate, hydrated calcium chloride, DNS reagents,Rochelle salt solution reagents were all obtained from Merck and are of pure quality.2.2 Microbial culture and ImmobilizationThe strain was subcultured on basic media containing yeast extract 3gl
-1
, (NH
4
)
2
SO
4
2.7 gl
-1
, KH
2
PO
4
2.3 gl
-1
, MgSO
4
.7H
2
O 0.7 gl
-1
, CaCl
2
.2H
2
O 0.1 gl
-1
, which was incubated at 30
o
C for 24 hours.5ml hypodermic syringe was used to pour 100ml of (1% v/v) sodium alginate containing thecultured yeast into a beaker containing 200ml of 0.75% w/v calcium chloride, in a drop wise fashion.The solution in the beaker was then discarded and replaced with 0.11% w/v calcium chloride andwas incubated at 4
o
C for an hour. And the beads were filtered for fermentation process.
2.3 Standard Glucose Assay
From the standard glucose stock solution, Different samples concentrations (0.1 gl
-1
,0.2 gl
-1
,0.3 gl
-1
,0.4 gl
-1
,0.5 gl
-1
), were made; following standard DNS glucose assay protocols by Miller 
13
, thespectrophotometric absorbance (OD
575nm
)of each sample was recorded and plotted against eachconcentration, which formed the glucose standard curve.
2.4 Enzymatic Hydrolysis
2
 
19ml of acetate buffer (100mM, 4.6pH) was measured into reaction vessel; the following volumes of the samples concentration were used and top up with distilled water, which formed final volume of 247.5ml as presented in the table below:Concentration (gl
-1
)CMC stocvolume (ml)Distilled watevolume(ml)Total mixturevolume (ml)675152.5247.58100147.5247.510125122.5247.5the mixture was homogenized by stirring at 200rpm for 2 minutes. 3ml of each sample waswithdrawn and glucose concentration was assayed as the initial glucose concentration.2.5ml of cellulase solution was added to each sample while stirring at 200rpm, 3ml sampling wasdone every 3 minutes for 15minutes and analyzed for glucose assay
13
and pH. Thespectrophotometric absorbance of each sample was recorded and the concentration was determinedusing the glucose standard curve.The final glucose sampling was taken at 24hours of fermentation, was diluted to specifiedconcentrations and analyzed for glucose assay and pH. Base on the data collected the below tableswere formulated:Table1: Glucose standard solution Absorbance base on concentration
Concentration (gl
-1
)AbsorbanceOD (575nm)
0.10.250.20.49150.30.7360.40.8930.51.18553
Fig1: Glucose Standard curve

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