Bioethanol production by Enzymatic Hydrolysis of Cellulose using immobilized Saccharomyces cerevisiae in fedbatch Fermentation: A Kinetic study.
Ahmad Mohammed Gumel,
Biotechnology Unit, Institute of Biological Sciences, University of Malaya, 50603, Kuala Lumpur, Malaysia
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 substrate concentrations were used; parameters such as enzymatic velocity, substrate concentration, glucose concentration, 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 and Michaelis-Menten models were used to plot and validate the experimental data respectively.
Biofuels such as bioethanol, biohydrogen and biodiesel are anticipated to be one of the feature alternatives to fossil fuels. The current world consumption of 474 exajoules (5×1020 J) with 80 to 90 percent derived from the combustion of fossil fuels1, which are associated with production instability, insecurity, sky rocketed prices as well as green house gasses emissions; are among the reasons that lead policy makers to seek for energy alternatives that are sustainable and environmental friendly. Today bioethanol is among the most widely employed biofuel in the world with current production of about 19 billion gallons world wide, in United State alone it provides about $12.3 billion capital market, 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 post harvest 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 most abundant organic substance in nature and considered to be promising and economically feasible feedstock for biofuel production 3. Enzymatic hydrolysis by cellulolytic enzymes that naturally degrade these cellulosic materials to monomeric sugars that are fermentable by Microbes are often required4. 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 acid hydrolysis. A number of bioethanol production processes that used microorganisms have been studied to produce ethanol from cellulosic materials. Among them, the stirred tank fedbatch fermentation process was cited by many researchers5-10.
2 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 be among the most effective lignocellulosic biomass degrading microorganisms for hexose sugars such as 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 stirred tank reactors utilizing cellulosic biomass have been reported, alas! There are limited citations on kinetic studies of the process; which we believed will help in understanding the overall process for maximum optimization.
2. Materials and Methods 2.1 Fermentation media and Culture
The substrate used for this study is carboxymethylcellulose (CMC) from laboratory stock to serve as the cellulose substrate, S. cerevisiae that was used was also obtained from laboratory stock. While chemicals 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 Immobilization The strain was subcultured on basic media containing yeast extract 3gl -1, (NH4)2SO4 2.7 gl-1, KH2PO4 2.3 gl-1 , MgSO4.7H2O 0.7 gl-1, CaCl2.2H2O 0.1 gl-1 , which was incubated at 30oC for 24 hours. 5ml hypodermic syringe was used to pour 100ml of (1% v/v) sodium alginate containing the cultured 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 and was incubated at 4oC 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 gl1 ,0.4 gl-1,0.5 gl-1), were made; following standard DNS glucose assay protocols by Miller 13, the spectrophotometric absorbance (OD575nm)of each sample was recorded and plotted against each concentration, which formed the glucose standard curve.
2.4 Enzymatic Hydrolysis
3 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 stock Distilled water Total mixture volume (ml) volume(ml) volume (ml) 6 75 152.5 247.5 8 100 147.5 247.5 10 125 122.5 247.5 the mixture was homogenized by stirring at 200rpm for 2 minutes. 3ml of each sample was withdrawn 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 was done every 3 minutes for 15minutes and analyzed for glucose assay13 and pH. The spectrophotometric absorbance of each sample was recorded and the concentration was determined using the glucose standard curve. The final glucose sampling was taken at 24hours of fermentation, was diluted to specified concentrations and analyzed for glucose assay and pH. Base on the data collected the below tables were formulated: Table1: Glucose standard solution Absorbance base on concentration Concentratio Absorbance n (gl-1) OD (575nm) 0.1 0.25 0.2 0.4915 0.3 0.736 0.4 0.893 0.5 1.1855
Fig1: Glucose Standard curve
Result and Discussion:
The production of bioethanol under different CMC concentrations is shown in figure 6. the bioethanol concentration increased with increased fermentation time, however it was found in all concentration experimental run to decrease at the end of fermentation time. Each experimental sampling was run in duplicate to carried out the enzymatic hydrolysis of cellulose for ethanol production using Saccharomyces cerevisiae in fedbatch fermentation using stirred tank reactor. At CMC concentration of 6g/l the highest glucose concentration produced by hydrolysis is found to be 0.085g/l at 9 minutes of experimental time, while in 8g/l and 10g/l CMC concentration, the highest glucose produced is 0.085g/l and 0.167g/l at 15 minutes of fermentation time respectively. The concentration of total glucose produced by enzymatic hydrolysis of the CMC as shown in figure 2-4; was found to fluctuate throughout the fermentation period, however this also indicates a significant reduction in glucose concentration within the fermentation media towards the end of fermentation time, a more clear picture of the glucose concentration by enzymatic hydrolysis can be seen when compared on a single chart as depicted in figure 5.
Figure 2: Glucose concentration by hydrolysis of 6g/l carboxymethylcellulose (CMC) over different experimental time in the course of the fedbatch fermentation
Figure 3: Glucose concentration by hydrolysis of 8g/l carboxymethylcellulose (CMC) over different experimental time in the course of the fedbatch fermentation
glucose conc (g/l
Figure 4: Glucose concentration by hydrolysis of 10g/l carboxymethylcellulose (CMC) over different experimental time in the course of the fedbatch fermentation
The kinetic parameters such as the initial enzymatic velocity (v) given by δP / δt was found to varied within the experimental runs of different concentrations; it was found to be 0.0192g[P]/min in 6g/l CMC concentration (figure2),while 0.0205g[P]/min, 0.0231g[P]/min in 8g/l and 10g/l CMC concentrations respectively (figure3 and 4). We noticed that increased in enzymatic concentration leads to an increased in velocity and conversion rate, however, this increase tends to stabilize when the maximum velocity is approach, so to optimized the production process the maximum velocity range in relation to substrate concentration has to be maintained.
[S ] Km [S ] = + , [V ] V max V max
Therefore, using Langmuir Transformation, : By plotting
[S ] 1 against [S] in Langmuir plot the slope denoted by , Vm ax v
Km V max
From the experimental data plotted on Langmuir model; X-intercept was used to determine the negative value of Km, therefore Km = 4.8, hence this was used in Y-intercept equation as gave the value of Vmax to be 0.034g[P]/min. while individually base on the data collected and computed the enzymatic maximum velocity (Vmax) within CMC concentrations of 6g/l, 8g/l and 10 g/l was found to be ranging from 0.03-0.034g[P]/min. these values were further confirmed theoretically using Michaelis-Menten equation of V= similar with difference of 0.17 in Km value. When the reaction stoicheometric equation is considered, following the fedbatch fermentation of 15 minutes period using CMC concentrations of 6g/l, 8g/l and 10g/l shown in figure 6, bioethanol concentrations were found to be 7.56E-3 g[P]/min, 6.9E-3g[P]/min and 5.6E-3g[P]/min respectively. The productivity value (P = amount of product produced per unit time) in g[P] was found to be 5.04E-4, 4.6E-4 and 3.73E-4 respectively. It is worth noting that the highest ethanol productivity shown in figure 7, is at CMC concentration of 10g/l with about 42% (w/v) productivity. When the initial glucose concentration of 38.90g/l is considered, the final glucose concentration after 24 hours fermentation was found to be 6.30 g/l, the total glucose consumed is 32.6g/l. hence total bioethanol concentration was found to be 16.66g/l therefore the total bioethanol productivity of the operational mode P =
( E max − Eo ) within 24 hours fermentation period was 0.69gL-1h-1. t
Vm ax[ S ] and were found to be almost [S ] + K m
Ethanol production concentration
ethanol concentration g[P]/min 8.00E-03 6.00E-03 4.00E-03 2.00E-03 0.00E+00 0 2 4 6 8 10 12 CMC concentration in g/l
Figure 6: Ethanol production concentration in g[P]/min using different CMC concentration as fermentation substrate
Bioethanol productivity from fedbatch fermentation using the following CMC concentration 6g/l,8g/l,10g/l at different experimental runs
10 42% 6 25%
Figure 7: Ethanol productivity in g[P] based on fermentation substrate (CMC concentration) used within 15 minutes fermentation time.
Bioethanol production by Fedbatch Fermentation of Hydrolyzed Carboxymethylcellulose (CMC) using Saccharomyces cerevisiae was achieved using different CMC concentrations, with highest production of 42% (w/v) in experimental running of CMC 10g/l concentration in 15 minutes, this process is an attractive process for producing sustainable cost effective and environmental friendly energy source. From the result discussed so far, leads to the conclusion that fermentation process can be optimized when kinetic parameters such as substrate concentration [S], enzymatic velocity (v) are carefully designed and monitored, for instance the enzymatic velocity has to be carefully designed and controlled to be within the range of maximum value (Vmax), in order to achieve maximum yield and maintain production cost as well as enzymatic speed, because once the maximum enzymatic velocity is reach, increased in substrate [S] concentration will do little or no effect at long last, resulting in none efficient production process.