Surfactant-Improved Simultaneous Saccharification and Co-Fermentation Method for Lignocellulose

Abstract

A surfactant-improved simultaneous saccharification and co-fermentation method for lignocelluloses in which a pretreated lignocellulose substrate is directly subjected to surfactant-improved simultaneous saccharification and co-fermentation without any detoxification by adding a surfactant, and the surfactant may be selectively added before or after pre-enzymatic hydrolysis, having economic, effective and feasible application prospects in reducing the loss of glucose, simplifying the production process, reducing equipment investment, reducing water consumption, improving ethanol production and reducing the total cost of the ethanol production process.

Claims

1. A surfactant-improved simultaneous saccharification and co-fermentation method for lignocellulose, wherein a pretreated lignocellulose substrate is directly subjected to simultaneous saccharification and co-fermentation without any detoxification treatment by adding a surfactant, wherein the surfactant is optionally added before or after pre-enzymolysis.

2. The method, as recited in claim 1, wherein the specific steps of adding the surfactant before the pre-enzymolysis are as follows: utilizing the pre-treated lignocellulose as a substrate, firstly adding a cellulase, the surfactant, a pH buffer solution to incubate for a period of time under a high temperature, and then lowering the temperature and adding Saccharomyces cerevisiae for simultaneous saccharification and ethanol fermentation.

3. The method, as recited in claim 1, wherein the specific steps of adding the surfactant after the pre-enzymolysis are as follows: utilizing the pre-treated lignocellulose as a substrate, firstly adding a cellulase, a pH buffer solution to incubate for a period of time under a high temperature for a period of time, and then lowering the temperature and adding the surfactant and Saccharomyces cerevisiae for simultaneous saccharification and ethanol fermentation.

4. The method, as recited in claim 2, wherein the solid-liquid ratio of the substrate to buffer is 0.1-0.3 g/mL; the concentration of the surfactant is 0-0.4 g/mL; an loading of the cellulase is 10-30FPU/g substrate; the cell density of the Saccharomyces cerevisiae is 0.5*10.sup.8-1.8*10.sup.8 cells /mL; the pre-enzymolysis temperature is 50° C. and the pre-enzymolysis is conducted for 2 to 24 hours; the temperature of the simultaneous saccharification and ethanol fermentation is 30 to 39° C.; the simultaneous saccharification and ethanol fermentation is conducted for 16-96 hours, 150 to 300 rpm.

5. The method, as recited in claim 1, wherein the surfactant is selected from the group consisting of polyethylene glycol (PEG), polyethylene glycol monomethyl Ether (MPEG), polyethylene glycol dimethyl ether (NHD), polydimethylsiloxane (PDMS), and combinations thereof.

6. The method, as recited in claim 1, wherein the surfactant is polyethylene glycol having a molecular weight of 200˜8000.

7. The method, as recited in claim 2, wherein the pH buffer solution comprises at least one selected from the group consisting of acetic acid-sodium acetate, citric acid-sodium citrate, phosphoric acid—sodium phosphate buffer and sulfuric acid solution, wherein the pH buffer solution has a pH value of 4.0˜5.5.

8. (canceled) .

9. The method, as recited in claim 1, characterized in that wherein the surfactant has a preferable concentration of 0.125 g˜0.18 g/mL.

10. The method, as recited in claim 2, wherein the solid-liquid ratio of the substrate and the buffer is 0.125 g/mL.

11. The method, as recited in claim 2, wherein the loading of the cellulase is 30 FPU/g substrate.

12. The method, as recited in claim 2, wherein the cell density of Saccharomyces cerevisiae is 0.8*10.sup.8˜0.96*10.sup.8 cells /mL.

13. The method, as recited in claim 1, wherein the pre-enzymolysis is conducted for 8 to 12 hours; the simultaneous saccharification and ethanol fermentation are conducted at 33° C., the simultaneous saccharification and the ethanol fermentation are conducted for 72 hours.

14. The method, as recited in claim 3, wherein the solid-liquid ratio of the substrate to buffer is 0.1-0.3 g/mL; the concentration of the surfactant is 0-0.4 g/mL; an loading of the cellulase is 10-30 FPU/g substrate; the cell density of the Saccharomyces cerevisiae is 0.5*10.sup.8-1.8*10.sup.8 cells /mL; the pre-enzymolysis temperature is 50° C. and the pre-enzymolysis is conducted for 2 to 24 hours; the temperature of the simultaneous saccharification and ethanol fermentation is 30 to 39° C.; the simultaneous saccharification and ethanol fermentation time is 16-96 hours, 150 to 300 rpm.

15. The method, as recited in claim 3, wherein the pH buffer solution comprises at least one selected from the group consisting of acetic acid-sodium acetate, citric acid-sodium citrate, phosphoric acid—sodium phosphate buffer and sulfuric acid solution, wherein the pH buffer solution has a pH value of 4.0˜5.5.

16. The method, as recited in claim 3, wherein the solid-liquid ratio of the substrate and the buffer is 0.125 g/mL.

17. The method, as recited in claim 3, wherein the loading of the cellulase is 30 FPU/g dry lignocellulose substrate.

18. The method, as recited in claim 3, wherein the cell density of Saccharomyces cerevisiae is 0.8*10.sup.8˜0.96*10.sup.8 cells /mL.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 illustrates an operation of SSCF in pure water.

[0023] FIG. 2 illustrates an operation 1 of a surfactant-improved SSCF.

[0024] FIG. 3 illustrates an operation 2 of a surfactant-improved SSCF.

[0025] FIG. 4 illustrates effects of different concentrations of PEG-1000 on enzymatic hydrolysis efficiency of non-detoxification steam exploded fast-growing poplar SSCF process.

[0026] FIG. 5 illustrates effects of different concentrations of PEG-1000 on the ethanol yield of non-detoxification steam exploded fast-growing poplar SSCF process.

[0027] FIG. 6 illustrates effects of different concentrations of PEG-1000 on the ethanol concentration of non-detoxification steam exploded fast-growing poplar SSCF process.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0028] The present invention will be described in further detail with reference to specific embodiments, but the scope of the invention is not to be limited by the scope of the present invention, and the following description of the invention and the description are merely illustrative of the principles of the present invention without departing from the spirit and scope of the present invention It will be understood that various changes and modifications may be made therein without departing from the scope of the present invention as claimed. It is intended that the scope of the present invention be defined by the appended claims and their equivalents.

[0029] In addition, it is worth mentioning that the content of each component in the fermentation in each of the following examples is determined by a high performance liquid chromatograph (Agilent 1260), and the conversion rate and ethanol yield are calculated based on the amount of the dropped lignocellulosic substrate, and the ethanol concentration is calculated according to the mass of ethanol in the fermentation broth, the volume of activated water and the pH value thereof

[0030] The conditions of chromatographic are as follows: ion exchange column, column temperature 65° C., parallax refractive index detector, detector temperature 50° C.; mobile phase: 5 Mm H.sub.2SO.sub.4, flow rate 0.6 mL/min, injection volume 25 uL.

[0031] The pretreatment method described in the examples of the present invention is the steam explosion method.

EXAMPLE 1

[0032] The pretreated fast-growing poplar powder of 1.5 g is used as a substrate, and firstly adding 10.5 mL buffer solution, 0.2 mL cellulase, wherein the buffer solution has a pH value of 4.86, incubating for 4 hours at a temperature of 50° C., and then lowering the temperature to 33° C., then adding Saccharomyces cerevisiae, wherein the cell density is maintained 0.8*10.sup.8 /mL, SSCF fermentation for 72 hours. The enzymatic hydrolysis rate, ethanol yield and concentration data of the pretreated fast-growing poplar are shown in Table 1.

EXAMPLE 2

[0033] The pretreated fast-growing poplar powder of 1.5 g is used as a substrate, and firstly adding 10.5 mL buffer solution, 0.2 mL cellulase and 2.0 g PEG-1000, wherein the buffer solution has a pH value of 4.86, incubating for 4 hours at a temperature of 50° C., and then lowering the temperature to 33° C., then adding Saccharomyces cerevisiae, wherein the cell density is maintained 0.8*10.sup.8 /mL, SSCF fermentation for 72 hours. The enzymatic hydrolysis rate, ethanol yield and concentration data of the pretreated fast-growing poplar are shown in Table 1. It can be seen from the data in the table that the hydrolysis rate has almost no change after adding PEG-1000, while the ethanol yield and concentration is increased by nearly 1 times.

EXAMPLE 3

[0034] The experimental procedure is the same as in Example 1 except that the yeast cell density is 0.96*10.sup.8/mL. The enzymatic hydrolysis rate, ethanol yield and concentration data of the pretreated fast-growing poplar are shown in Table 1. It can be seen from the data in the table that the enzymatic efficiency, ethanol concentration and yield have almost no change after increasing the concentration of yeast cells in pure water system.

EXAMPLE 4

[0035] The experimental procedure is the same as in Example 2 except that the yeast cell density is 0.96*10.sup.8/mL. The enzymatic hydrolysis rate, ethanol yield and concentration data of the pretreated fast-growing poplar are shown in Table 1. It can be seen from the data in the table that in PEG-1000 system, when the concentration of yeast cells is increased, the enzymatic efficiency, ethanol concentration and yield have almost no change.

EXAMPLE 5

[0036] The experimental procedure is the same as in Example 1 except that the buffer solution is increased to 12.0 mL. The enzymatic hydrolysis rate, ethanol yield and concentration data of the pretreated fast-growing poplar are shown in Table 1. It can be seen from the data in the table that in pure water system, when the concentration of substrate is reduced, the enzymatic efficiency, ethanol concentration and yield have almost no change.

EXAMPLE 6

[0037] The experimental procedure is the same as in Example 2 except that the buffer solution is increased to 12.0 mL. The enzymatic hydrolysis rate, ethanol yield and concentration data of the pretreated fast-growing poplar are shown in Table 1. It can be seen from the data in the table that in PEG-1000 system, when the concentration of substrate is reduced, the enzymatic efficiency, ethanol concentration and yield are significantly improved.

EXAMPLE 7

[0038] The experimental procedure is the same as in Example 1 except that the buffer solution is reduced to 10.0 mL. The enzymatic hydrolysis rate, ethanol yield and concentration data of the pretreated fast-growing poplar are shown in Table 1. It can be seen from the data in the table that in pure water system, when the concentration of substrate is increased, the enzymatic efficiency, ethanol concentration and yield have almost no change.

EXAMPLE 8

[0039] The experimental procedure is the same as in Example 2 except that the buffer solution is reduced to 12.0 mL. The enzymatic hydrolysis rate, ethanol yield and concentration data of the pretreated fast-growing poplar are shown in Table 1. It can be seen from the data in the table that in PEG-1000 system, when the concentration of substrate is decreased, the enzymatic efficiency was decreased, ethanol concentration and yield are significantly increased.

TABLE-US-00001 TABLE 1 Simultaneous saccharification and co-fermentation of pretreated fast-growing poplar Saccharomyces Enzymatic Eth- Eth- Exam- cerevisiae PEG- Buffer/ hydrolysis anol anol/ ple cells/mL 1000 mL rate/% yield/% g/L 1 0.8 × 10.sup.8 0.0000 10.5 62.2 20.3 7.57 2 0.8 × 10.sup.8 2.0901 10.5 62.7 46.5 17.30 3 0.96*10.sup.8 0.0000 10.5 62.8 24.3 9.05 4 0.96*10.sup.8 2.0072 10.5 58.1 46.9 17.46 5  0.8*10.sup.8 0.0000 12.0 67.1 23.0 7.52 6  0.8*10.sup.8 2.0209 12.0 65.5 64.3 20.94 7 0.96*10.sup.8 0.0000 10.0 64.5 21.9 8.55 8 0.96*10.sup.8 2.0342 10.0 58.3 36.9 14.44

EXAMPLE 9

[0040] The experimental procedure is the same as in Example 5 except that the cellulase is 0.3 mL. The enzymatic hydrolysis rate, ethanol yield and concentration data of the pretreated fast-growing poplar are shown in Table 2. It can be seen from the data in the table that in pure water system, when the concentration of cellulase is improved, the enzymatic efficiency is increased a little, and ethanol concentration and yield have almost no change.

EXAMPLE 10

[0041] The experimental procedure is the same as in Example 6 except that the cellulase is 0.3 mL. The enzymatic hydrolysis rate, ethanol yield and concentration data of the pretreated fast-growing poplar are shown in Table 2. It can be seen from the data in the table that in PEG-1000 system, when the concentration of cellulase is improved, the enzymatic efficiency, ethanol concentration and yield are improved.

EXAMPLE 11

[0042] The experimental procedure is the same as in Example 9 except that the pretreated fast-growing poplar is subjected to pre-enzymatic hydrolysis at 50° C. for 8 hours. The enzymatic hydrolysis rate, ethanol yield and concentration data of the pretreated fast-growing poplar are shown in Table 2. It can be seen from the data in the table that in pure water system, when the co-fermentation time is prolonged in pure water system, the enzymatic efficiency is increased, and ethanol concentration and yield have almost no change.

EXAMPLE 12

[0043] The experimental procedure is the same as in Example 10 except that the pretreated fast-growing poplar is subjected to pre-enzymatic hydrolysis at 50° C. for 8 hours. The enzymatic hydrolysis rate, ethanol yield and concentration data of the pretreated fast-growing poplar are shown in Table 2. It can be seen from the data in the table that in pure water system, when the co-fermentation time is prolonged in the PEG-1000 system, the enzymatic efficiency, ethanol concentration and yield are improved.

EXAMPLE 13

[0044] The experimental procedure is the same as in Example 12 except that PEG-1000 is added after the pretreated fast-growing poplar is subjected to pre-enzymatic hydrolysis. The enzymatic hydrolysis rate, ethanol yield and concentration data of the pretreated fast-growing poplar are shown in Table 2. It can be seen from the data in the table that the order of addition of the surfactant has no significant effect on the fermentation efficiency.

EXAMPLE 14

[0045] The experimental procedure is the same as in Example 10 except that PEG-1000 is 3.0 g. The enzymatic hydrolysis rate, ethanol yield and concentration data of the pretreated fast-growing poplar are shown in Table 2. It can be seen from the data in the table that the excess of PEG-1000 can lead to the decrease of SSCF process efficiency, the enzymolysis efficiency, ethanol concentration and yield are reduced.

TABLE-US-00002 TABLE 2 Simultaneous saccharification and co-fermentation of pretreated fast-growing poplar Enzymolysis Ethanol Example PEG-1000 Time/h efficiency/% yield/% Ethanol/g/L 9 0.0000 4 + 72 71.13 23.28 7.57 10 2.0039 4 + 72 72.12 71.20 23.17 11 0.0000 8 + 72 75.53 22.52 7.32 12 2.0085 8 + 72 76.51 75.63 24.61 13 2.0303 8 + 72 74.41 73.44 23.89 14 3.0300 4 + 72 62.16 60.89 19.81

EXAMPLE 15

[0046] The experimental procedure is the same as in Example 11 except that the pretreated fast-growing poplar is subjected to pre-enzymatic hydrolysis for 24 hours. The enzymatic hydrolysis rate, ethanol yield and concentration data of the pretreated fast-growing poplar are shown in Table 3. It can be seen from the data in the table that in pure water system, the prolonged reaction time of SSCF process does not improve the ethanol concentration and yield.

EXAMPLE 16

[0047] The experimental procedure is the same as in Example 12 except that the pretreated fast-growing poplar is subjected to pre-enzymatic hydrolysis for 24 hours. The enzymatic hydrolysis rate, ethanol yield and concentration data of the pretreated fast-growing poplar are shown in Table 3. It can be seen from the data in the table that in PEG-1000 system, the prolonged reaction time of SSCF process does not improve the ethanol concentration and yield.

EXAMPLE 17

[0048] The experimental procedure is the same as in Example 15 except that the buffer solution is reduced to 10mL. The enzymatic hydrolysis rate, ethanol yield and concentration data of the pretreated fast-growing poplar are shown in Table 3. It can be seen from the data in the table that in pure water system, when the concentration of substrate is increased in SSCF process, and the ethanol concentration is not improved.

EXAMPLE 18

[0049] The experimental procedure is the same as in Example 12 except that the buffer solution is reduced to 10mL. The enzymatic hydrolysis rate, ethanol yield and concentration data of the pretreated fast-growing poplar are shown in Table 3. It can be seen from the data in the table that in PEG-1000 system, when the concentration of substrate in SSCF process is increased, the ethanol yield and concentration are significantly reduced, but it is still higher than the pure water system.

TABLE-US-00003 TABLE 3 Simultaneous saccharification and co-fermentation of pretreated fast-growing poplar Enzymatic hydrolysis Ethanol Example PEG-1000 Buffer/mL rate/% yield/% Ethanol/g/L 15 0.0000 12 82.88 21.55 7.01 16 2.0059 12 76.62 75.74 24.66 17 0.0000 10 77.41 19.59 7.65 18 1.9966 10 76.12 28.42 11.10

EXAMPLE 19

[0050] The experimental procedure is the same as in Example 16 except that the fermentation temperature is 36oC. The enzymatic hydrolysis rate, ethanol yield and concentration data of the pretreated fast-growing poplar are shown in Table 4. It can be seen from the data in the table that in PEG-1000 system, during the SSCF process, the proper increase of temperature can effectively improve the ethanol yield and concentration.

EXAMPLE 20

[0051] The experimental procedure is the same as in Example 16 except that the fermentation temperature is 39oC. The enzymatic hydrolysis rate, ethanol yield and concentration data of the pretreated fast-growing poplar are shown in Table 4. It can be seen from the data in the table that in PEG-1000 system, during the SSCF process, the proper increase of temperature can effectively improve the ethanol yield and concentration.

EXAMPLE 21

[0052] The experimental procedure is the same as in Example 16 except that the added surfactant is 2.0 g PEG-200. The enzymatic hydrolysis rate and ethanol concentration data are shown in Table 4. It can be seen from the data in the table that PEG-200 can effectively improve the ethanol yield and concentration.

EXAMPLE 22

[0053] The experimental procedure is the same as in Example 16 except that the added surfactant is 2.0 g PEG-400. The enzymatic hydrolysis rate and ethanol concentration data are shown in Table 4. It can be seen from the data in the table that PEG-400 can effectively improve the ethanol yield and concentration.

EXAMPLE 23

[0054] The experimental procedure is the same as in Example 16 except that the added surfactant is 2.0 g PEG-4000. The enzymatic hydrolysis rate and ethanol concentration data are shown in Table 4. It can be seen from the data in the table that PEG-4000 can effectively improve the ethanol yield and concentration.

EXAMPLE 24

[0055] The experimental procedure is the same as in Example 16 except that the added surfactant is 2.0 g polyethylene glycol monomethyl ether (MPEG). The glucose conversion rate and ethanol concentration data are shown in Table 4. It can be seen from the data in the table that MPEG can effectively improve the ethanol yield and concentration.

EXAMPLE 25

[0056] The experimental procedure is the same as in Example 16 except that the added surfactant is 2.0 g polyethylene glycol dimethyl ether (NHD). The glucose conversion rate and ethanol concentration data are shown in Table 4. It can be seen from the data in the table that NHD can effectively improve the ethanol yield and concentration.

TABLE-US-00004 TABLE 4 Simultaneous saccharification and co-fermentation of pretreated fast-growing poplar Enzymatic Temperature/ hydrolysis rate/ Ethanol Ethanol/ Example Surfactant ° C. % yield/% g/L 19 PEG-1000 55 + 36 97.5 96.8 31.5 20 PEG-1000 55 + 39 89.1 89.0 29.0 21 PEG-200 55 + 33 77.4 60.8 19.8 22 PEG-400 55 + 33 76.1 66.8 22.8 23 PEG-4000 55 + 33 76.6 75.7 24.6 24 MPEG 55 + 33 77.2 28.4 11.1 25 NHD 55 + 33 74.5 26.5 10.0

EXAMPLE 26

[0057] The pretreated fast-growing poplar powder of 1.5 g is used as a substrate, and firstly adding 12.0 mL buffer solution, 0.3 mL cellulase, 0-2.0 g surfactant, wherein the buffer solution has a pH value of 4.86, incubating for 24 hours at a temperature of 50° C., and then lowering the temperature to 33° C., then adding Saccharomyces cerevisiae, wherein the cell density is maintained 0.8*10.sup.8 /mL, SSCF fermentation for 72 hours. The enzymatic hydrolysis rate, ethanol yield and concentration data of the pretreated fast-growing poplar are shown in FIGS. 4, 5 and 6. It can be seen from the data in the Figs that PEG-1000 has little effect on the enzymolysis rate, while the ethanol yield and concentration increase with the increase of PEG-1000 concentration.

EXAMPLE 27

[0058] The pretreated fast-growing poplar powder of 1.5 g is used as a substrate, and firstly adding 12.0 mL buffer solution, 0.3 mL cellulase, wherein the buffer solution has a pH value of 4.86, incubating for 4 hours at a temperature of 50° C., and then lowering the temperature to 33° C., then adding 1.5002 g surfactant of PEG-1000, wherein the cell density is maintained 0.8*108 /mL, SSCF fermentation for 72 hours. The enzymatic hydrolysis rate, ethanol yield and concentration data of the pretreated fast-growing poplar are shown in table 5. It can be seen from the data in the table that the addition order of PEG-1000 has no significant effect on the enzymolysis rate, the ethanol yield and concentration, as compared with Example 10 and Example 12.

EXAMPLE 28

[0059] The experimental procedure is the same as in Example 27 except that the added surfactant is 2.0039 g PEG-1000. The enzymatic hydrolysis rate and ethanol concentration data are shown in Table 5.

EXAMPLE 29

[0060] The experimental procedure is the same as in Example 27 except that the pretreated fast-growing poplar is subjected to pre-enzymatic hydrolysis for 8 hours, the enzymatic hydrolysis rate, ethanol yield and concentration data are shown in Table 5.

EXAMPLE 30

[0061] The experimental procedure is the same as in Example 30 except that the pretreated fast-growing poplar is subjected to pre-enzymatic hydrolysis for 8 hours, and the added surfactant is 2.0103 g PEG-1000. The enzymatic hydrolysis rate, ethanol yield and concentration data are shown in Table 5.

TABLE-US-00005 TABLE 5 Simultaneous saccharification and co-fermentation of pretreated fast-growing poplar Enzymatic hydrolysis Ethanol yield/ Ethanol/ Example PEG-1000 Time/h rate/% % g/L 27 1.5002 4 + 72 71.63 71.42 22.89 28 2.0039 4 + 72 71.82 70.90 22.17 29 1.5003 8 + 72 75.51 74.63 23.61 30 2.0103 8 + 72 74.93 73.84 24.01