Abstract: A process for enzymatic hydrolysis of non-edible lignocellulosic biomass including cellulose for bioethanol production is provided wherein mixing the ingredients for selective time, involving mixing for an initial period followed by no mixing for the rest of the reaction period, is surprisingly found to maximize the yields of sugars selected from glucose and reducing sugars from said cellulosic biomass while minimizing energy costs. Fig 1
DESC:FIELD OF THE INVENTION
The present invention relates to a process for enzymatic hydrolysis of non-edible lignocellulosic biomass including cellulose for bioethanol production, wherein mixing the ingredients for selective time, involving mixing for an initial period followed by no mixing for the rest of the reaction period, is surprisingly found to maximize the yields of sugars selected from glucose and reducing sugars from said cellulosic biomass while minimizing energy costs.
BACKGROUND ART
Enzymatic hydrolysis of cellulose to produce glucose and reducing sugar is a rate limiting step in the process of Bioethanol production. The yield and rate of glucose production is usually very low, thus posing a serious challenge to the commercial viability of the process. So, researchers are trying to solve the problem by adopting various approaches that involve drastic process modification, revamping equipments and finding new raw materials and enzymes.
Currently, all the technologies for enzymatic hydrolysis of cellulose use continuous mixing at sufficiently high mixing speeds (RPM) and current research is mostly directed to new enzyme strains involved in the hydrolysis process to obtain higher glucose yields.
Hence, it is thus apparent from the above-said that it is still a challenge in the art to attain higher yields of glucose and reducing sugar in the said enzymatic process of cellulose, and, therefore, there is a need in the art for a process relating to such enzymatic hydrolysis that would generate higher yields of glucose and reducing sugar while spending minimum energy.
OBJECTS OF THE INVENTION
It is thus a primary object of the present invention to provide for a process for enzymatic hydrolysis of cellulosic biomass for bioethanol production that would maximize glucose/ reducing sugar yield without the need of revamping the existing methodology of such enzymatic hydrolysis.
It is another object of the present invention to provide for the said process that would not require any drastic process modification or would require revamping the existing equipments for workability of the process to give improved yields of glucose/ reducing sugar.
It is still another object of the present invention to provide for said process that would neither need new raw materials and enzymes in the said enzymatic hydrolysis but would still minimize energy costs incurred from mixing.
SUMMARY OF THE INVENTION
Thus according to the basic aspect of the present invention there is provided a process for enzymatic hydrolysis of non-edible lignocellulosic biomass including cellulose for bioethanol production comprising:
carrying out the enzymatic hydrolysis of said lignocellulosic biomass including cellulose to produce glucose and reducing sugar under controlled mixing of the ingredients during said enzymatic hydrolysis involving a combination of enzymatic hydrolysis under mixing of ingredients and enzymatic hydrolysis under no-mixing of ingredients wherein essentially the enzymatic hydrolysis under mixing of ingredients is carried out at the initial period of said enzymatic hydrolysis.
According to another preferred aspect of the present invention there is provided a process wherein said combination of enzymatic hydrolysis under mixing of ingredients and enzymatic hydrolysis under no-mixing of ingredients is carried out such that early mixing reduces mass transfer resistances in the process and accelerates the reaction and wherein said no-mixing for the rest of the reaction time prevents slowing down the rate of cellulose hydrolysis by not allowing mass transfer-aided product inhibitors to inhibit the free enzymes.
According to yet another preferred aspect of the present invention there is provided a process wherein said enzymatic hydrolysis under mixing of ingredients carried out at the initial period of said enzymatic hydrolysis is carried out for upto 4 hours from initiation of the enzymatic hydrolysis followed by no-mixing for the rest of the reaction time to maximize the yield of bioethanol while minimizing energy costs.
Preferably, said process comprises the steps of
a. providing for cellulosic substrate;
b. providing for cellulase enzyme in a buffer solution;
c. early mixing for an optimal period of upto about 4 hrs followed by no-mixing for the rest of the reaction time of about 14-72 hrs and obtaining said bioethanol therefrom.
According to another preferred aspect of the present invention there is provided said process wherein said cellulosic substrate includes non-edible lignocellulosic biomass, said cellulase enzyme includes endoglucanase, exoglucanase and ß-glucosidase, said buffer solution involves 0.1 M Sodium acetate buffer (pH 5.0).
According to yet another preferred aspect of the present invention there is provided said process for enzymatic hydrolysis of cellulosic biomass to bioethanol wherein said bioethanol obtained involves sugars, reducing sugars including glucose, cellobiose.
According to another preferred aspect of the present invention there is provided said process comprising terminating the mixing after 4 hours to maximise the yield of glucose and other reducing sugars, and wherein at the end of 14 hour of hydrolysis the concentration of glucose for 4 hour mixing achieved is 15% higher than no-mixing and 14.76% higher than continuous mixing, while the concentration of total reducing sugar achieved is 11% higher than no-mixing and 19% higher than continuous mixing.
The nature of the invention, its objects and advantages are explained hereunder in greater detail in relation to the following non-limiting exemplary illustrations as per the following accompanying figures and examples:
BRIEF DESCRIPTION OF FIGURES
Figure 1(a). Effect of mixing time on glucose concentration at the end of 14 hour of hydrolysis reaction;
Figure 1(b). Effect of mixing time on reducing sugar concentration at the end of 14 hour of hydrolysis reaction;
Figure 2(a). Plot of increase in yield (%) of glucose versus hydrolysis reaction time;
Figure 2(b). Plot of increase in yield (%) of reducing sugar versus hydrolysis reaction time.
DETAILED DESCRIPTION OF THE INVENTION
As discussed hereinbefore, the present invention provides for enzymatic hydrolysis of cellulosic biomass for bioethanol production, wherein by mixing of the ingredients for selective time period involving mixing for an initial period followed by no mixing for the rest of the reaction period maximizes glucose and reducing sugar yields while minimizing energy costs.
The interlinked dynamics between chemical kinetics (including product inhibition) and mass transfer in the process of the present invention is selectively and surprisingly found to be based on the time period of mixing of the ingredients, whereby mixing for an initial period followed by no mixing for the rest of the reaction period maximizes glucose and reducing sugar yields while minimizing energy costs.
Therefore, by way of the process of the present invention of cellulose hydrolysis – the rate-limiting step in the production of cellulosic ethanol, glucose and reducing sugar yields were maximized. In the said process of enzymatic hydrolysis of cellulose, the products (glucose and cellobiose), which are considered as inhibitors of the process, should be at minimum in the reaction mixture to prevent them from binding with the enzymes and inhibiting the reactions significantly. Therefore, it was selectively found that a complete mixing for the certain period initially helped in easy mass transport of the enzymes to the cellulose molecules leading to faster hydrolysis of the cellulose. However, followed by complete mixing it was after the reaction has significantly occurred and the concentration of products (glucose and cellobiose) has reached a sufficiently high level at which it can inhibit the reaction significantly, it was surprisingly found that further mixing induces mass transport of the inhibitors (products) to the free enzymes, which in turn, slows down the rate of cellulose hydrolysis. Therefore, it was thus found by way of the selective process of the present invention that there exists an optimal period of early mixing which reduces mass transfer resistances in the system and accelerates the reaction, followed by no mixing for the rest of the reaction time in order to prevent the mass transfer-aided product inhibition, wherein an optimal initial mixing time (of upto about 4 hours) maximizes glucose and reducing sugar yield while minimizing energy costs incurred due to mixing in the reactor.
The process of the present invention, thus, in involving cellulose selected from microcrystalline cellulose (Avicel PH101) as the cellulosic substrate advantageously needs no additional equipments or any revamping of the plant, wherein existing bioethanol processes can easily be used without any capital investment. Hence, the energy costs could be thus effectively minimized by replacing continuous mixing by only 4 hours of initial mixing.
Examples-I: Materials
Commercially available microcrystalline cellulose, Avicel PH101, with average particle size of 50 µm and particle density of 0.600g/cm3, was used for conducting experiments. Dry solid purified cellulase enzyme with an activity of 1 U/mg of solid. The enzyme is derived from Trichoderma viride. The enzyme has a maximum activity at a pH range of 4.0-5.0 and a temperature of 40–50°C. The purified enzyme contains all the three components, i.e., endoglucanase, exoglucanase and ß-glucosidase.
For maintaining the pH of the reaction mixture, 0.1 M Sodium acetate buffer (pH 5.0) was used. The amount of glucose was estimated using the Glucose Oxidase-Peroxidase (GOD-POD) method. The reducing sugars were estimated by the Dinitrosalicylic acid (DNS) method.
Example-II: Procedure for Enzymatic hydrolysis
The effect of initial mixing for a specified time (followed by no mixing for the rest of the reaction period) on the glucose and reducing sugar concentration levels in the enzymatic hydrolysis of cellulose in a batch reactor lead to the surprising results as discussed herebelow.
A reactor (conical flask) of volume 100 ml was used. The Avicel substrate was directly added to the reaction volume of 10 ml. The composition of the reaction mixture was
• Cellulose (Avicel) = 20 mg/ml i.e. a total of 200 mg.
• 10 ml of Buffer to maintain a pH of 5.
• Cellulase enzyme = 1mg/ml i.e. a total of 10 mg.
The reaction was initiated by adding the enzyme after the reaction contents attained a temperature of 50° C in incubator under aseptic conditions and at an RPM of 150. The hydrolysis reaction was continued up to 72 hours and the product concentrations were measured at various intervals of time ranging from 1 to 72 hours. The samples (0.1ml) were taken out of the reaction mixtures at various time intervals and cooled immediately to stop the reaction. For initial mixing, the experiments were conducted following initial mixing durations: 0 hour (no mixing), 1 hour, 2 hour, 3 hour, 4 hour, 5 hour, 6 hour, 7 hour, 8 hour and continuous mixing. All experiments were repeated.
Surprisingly, it was found that by terminating the mixing after 4 hours even as the hydrolysis reaction is allowed to continue results in maximum yield of glucose and other reducing sugars. As shown in Figs. 1 a) and b), at the end of 14 hour of hydrolysis, the concentration of glucose for 4 hour mixing case is 15% higher than no mixing case (i.e., 0 hr of mixing) and 14.76% higher than continuous mixing case (14 hours), while the concentration of total reducing sugar for this case is 11% higher than no mixing case and 19% higher than continuous mixing case. The results were found to be consistent under repetition and persistent with increasing hydrolysis reaction time all the way up to 72 hours.
For example, as shown in Figs. 2 a) and b), after 36 hours of hydrolysis, the optimum (4 hour) mixing case gives 32% higher yield of reducing sugar and 11.75% higher glucose yield over no mixing case, and 34.3% and 12.7%, respectively, over the continuous mixing case. It is significantly found by way of the process of the present invention that while glucose and cellobiose – the two products of cellulose hydrolysis – inhibit the hydrolysis reaction, the initial 4 hours of mixing aids to depolymerize the cellulose by facilitating the contact between the enzyme and the substrate molecules, whereby further mixing decelerates the reaction by bringing the product molecules (glucose and cellobiose, which are inhibitors) in contact with the reactant molecules. It may be noted that the latter phenomenon of mixing-aided inhibition becomes dominant only after enough products have been generated that could inhibit the reaction, which, in this case, was found to be about 4 hours. Therefore, the process of the present invention significantly provides for an optimal period of early mixing which accelerates the reaction followed by no mixing to prevent the mixing-aided product inhibition.
It is thus possible by way of the present invention to provide for a process for enzymatic hydrolysis of non-edible lignocellulosic biomass including cellulose for bioethanol production, wherein by mixing of the ingredients for selective time involving mixing for an initial period followed by no mixing for the rest of the reaction period, maximizes the yields of sugars selected from glucose and reducing sugars from said non-edible lignocellulosic biomass while advantageously minimizing energy costs.
,CLAIMS:We Claim:
1. A process for enzymatic hydrolysis of non-edible lignocellulosic biomass including cellulose for bioethanol production comprising:
carrying out the enzymatic hydrolysis of said lignocellulosic biomass including cellulose to produce glucose and reducing sugar under controlled mixing of the ingredients during said enzymatic hydrolysis involving a combination of enzymatic hydrolysis under mixing of ingredients and enzymatic hydrolysis under no-mixing of ingredients wherein essentially the enzymatic hydrolysis under mixing of ingredients is carried out at the initial period of said enzymatic hydrolysis.
2. A process as claimed in claim 1 wherein said combination of enzymatic hydrolysis under mixing of ingredients and enzymatic hydrolysis under no-mixing of ingredients is carried out such that early mixing reduces mass transfer resistances in the process and accelerates the reaction and wherein said no-mixing for the rest of the reaction time prevents slowing down the rate of cellulose hydrolysis by not allowing mass transfer-aided product inhibitors to inhibit the free enzymes.
3. A process as claimed in anyone of claims 1 or 2 wherein said enzymatic hydrolysis under mixing of ingredients carried out at the initial period of said enzymatic hydrolysis is carried out for upto 4 hours from initiation of the enzymatic hydrolysis followed by no-mixing for the rest of the reaction time to maximize the yield of bioethanol while minimizing energy costs.
4. A process as claimed in anyone of claims 1 to 3 comprising the steps of
a. providing for cellulosic substrate;
b. providing for cellulase enzyme in a buffer solution;
c. early mixing for an optimal period of upto about 4 hrs followed by no-mixing for the rest of the reaction time of about 14-72 hrs and obtaining said bioethanol therefrom.
5. A process as claimed in claim 4 wherein said cellulosic substrate includes non-edible lignocellulosic biomass, said cellulase enzyme includes endoglucanase, exoglucanase and ß-glucosidase, said buffer solution involves 0.1 M Sodium acetate buffer (pH 5.0).
6. A process for enzymatic hydrolysis of cellulosic biomass to bioethanol as claimed in claim 4 wherein said bioethanol obtained involves sugars, reducing sugars including glucose, cellobiose.
7. A process as claimed in anyone of claims 1-6 comprising terminating the mixing after 4 hours to maximise the yield of glucose and other reducing sugars, and wherein at the end of 14 hour of hydrolysis the concentration of glucose for 4 hour mixing achieved is 15% higher than no-mixing and 14.76% higher than continuous mixing, while the concentration of total reducing sugar achieved is 11% higher than no-mixing and 19% higher than continuous mixing.
Dated this the 29th day of April, 2014 Anjan Sen
Of Anjan Sen and Associates
(Applicants Agent)
Application Number | Applicant | Section | Controller | Decision Date | |
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1 | 509/KOL/2013 | INDIAN INSTITUTE OF TECHNOLOGY,KHARAGPUR | Order us 15 NBA permission | Monika Yadav | 2022-08-31 |
2 | 509/KOL/2013 | INDIAN INSTITUTE OF TECHNOLOGY,KHARAGPUR | 15 | Monika Yadav | 2024-02-12 |
3 | 509/KOL/2013 | INDIAN INSTITUTE OF TECHNOLOGY,KHARAGPUR | 15 | Monika Yadav | 2024-02-12 |
4 | 509/KOL/2013 | INDIAN INSTITUTE OF TECHNOLOGY,KHARAGPUR | 15 | Monika Yadav | 2024-02-12 |
Name | Date | |
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1 | 509-KOL-2013-EDUCATIONAL INSTITUTION(S) [19-02-2024(online)].pdf | 2024-02-19 |
2 | 509-KOL-2013-EVIDENCE FOR REGISTRATION UNDER SSI [19-02-2024(online)].pdf | 2024-02-19 |
3 | 509-KOL-2013-FORM-8 [19-02-2024(online)].pdf | 2024-02-19 |
4 | 509-KOL-2013-Written submissions and relevant documents [07-12-2023(online)].pdf | 2023-12-07 |
5 | 509-KOL-2013-Correspondence to notify the Controller [27-11-2023(online)].pdf | 2023-11-27 |
6 | 509-KOL-2013-US(14)-ExtendedHearingNotice-(HearingDate-28-11-2023).pdf | 2023-10-23 |
7 | 509-KOL-2013-NBA INTIMATION TO APPLICANT COMPLY WITH REQUIREMENT-28-06-2023.pdf | 2023-06-28 |
8 | 509-KOL-2013-NBA INTIMATION TO APPLICANT COMPLY WITH REQUIREMENT-12-01-2023.pdf | 2023-01-12 |
9 | 509-KOL-2013-Written submissions and relevant documents [12-04-2022(online)].pdf | 2022-04-12 |
10 | 509-KOL-2013-Correspondence to notify the Controller [21-03-2022(online)].pdf | 2022-03-21 |
11 | 509-KOL-2013-US(14)-ExtendedHearingNotice-(HearingDate-28-03-2022).pdf | 2022-03-02 |
12 | 509-KOL-2013-Correspondence to notify the Controller [18-02-2022(online)].pdf | 2022-02-18 |
13 | 509-KOL-2013-FORM-26 [18-02-2022(online)].pdf | 2022-02-18 |
14 | 509-KOL-2013-Proof of Right [18-02-2022(online)].pdf | 2022-02-18 |
15 | 509-KOL-2013-US(14)-HearingNotice-(HearingDate-22-02-2022).pdf | 2022-02-08 |
16 | 509-KOL-2013-FER.pdf | 2021-10-03 |
17 | 509-KOL-2013-COMPLETE SPECIFICATION [28-04-2021(online)].pdf | 2021-04-28 |
18 | 509-KOL-2013-FER_SER_REPLY [28-04-2021(online)].pdf | 2021-04-28 |
19 | Form 18 [25-04-2017(online)].pdf | 2017-04-25 |
20 | 509-KOL-2013-(02-05-2014)-ABSTRACT.pdf | 2014-05-02 |
21 | 509-KOL-2013-(02-05-2014)-CLAIMS.pdf | 2014-05-02 |
22 | 509-KOL-2013-(02-05-2014)-CORRESPONDENCE.pdf | 2014-05-02 |
23 | 509-KOL-2013-(02-05-2014)-DESCRIPTION (COMPLETE).pdf | 2014-05-02 |
24 | 509-KOL-2013-(02-05-2014)-FORM-1.pdf | 2014-05-02 |
25 | 509-KOL-2013-(02-05-2014)-FORM-2.pdf | 2014-05-02 |
26 | 509-KOL-2013-(02-05-2014)-FORM-5.pdf | 2014-05-02 |
27 | ASA Complete specification for filing_29.04.2014.pdf | 2014-05-02 |
28 | Figure for Abstract-29.04.2014.pdf | 2014-05-02 |
29 | FORM 5.pdf | 2014-05-02 |
30 | 509-KOL-2013-(24-07-2013)-CORRESPONDENCE.pdf | 2013-07-24 |
31 | 509-KOL-2013-(24-07-2013)-PA.pdf | 2013-07-24 |
32 | 509-kol-2013-(03-05-2013)-CORRESPONDENCE.pdf | 2013-05-03 |
33 | 509-kol-2013-(03-05-2013)-DESCRIPTION (PROVISIONAL).pdf | 2013-05-03 |
34 | 509-kol-2013-(03-05-2013)-DRAWINGS.pdf | 2013-05-03 |
35 | 509-kol-2013-(03-05-2013)-FORM-1.pdf | 2013-05-03 |
36 | 509-kol-2013-(03-05-2013)-FORM-2.pdf | 2013-05-03 |
37 | 509-kol-2013-(03-05-2013)-FORM-3.pdf | 2013-05-03 |
1 | searchstrategyE_29-10-2020.pdf |