Process for the hydrolysis of biomass

Abstract

The present invention is directed to a process for the hydrolysis of biomass as well as the saccharide-containing permeate product and the protein-containing product produced by this process. In a further aspect, the present invention is directed to a process for the production of organic compounds from the saccharide-containing product. In an additional aspect the present invention is directed to the use of the protein-containing product for the production of a fermentation medium.

Claims

1. A process for the hydrolysis of biomass comprising: a) Contacting the biomass with an enzyme-composition containing at least one enzyme selected from the class of hydrolases in a vessel; b) Eliminating at least part of said biomass; c) Subjecting said at least part of said biomass to a filtration and removing the permeate; and d) Backfeeding of at least part of the filtrated biomass to the vessel; wherein step b) is carried out in an outer circuit; wherein step c) is carried out in an inner circuit; wherein said outer circuit and said inner circuit are different; wherein the outer circuit is in closer proximity to step a) than the inner circuit; wherein filtration is carried out by use of at least one filtration module comprising at least one ceramic membrane; wherein dry matter content of the biomass is from 5 to 40 wt.-%; wherein each circuit is equipped with a pump; and wherein at least two of steps a) to d) are carried out simultaneously; and wherein after step d), the filtrated biomass that is not backfed to the vessel is circulated back to the at least one filtration module.

2. The process according to claim 1, wherein the enzyme-composition contains cellulases, hemicellulases and/or pectinases.

3. The process according to claim 2, wherein the enzyme-composition further contains at least one enzyme selected from pectinmethylesterases, rhamnogalacturonases, 1,3-/1,6-beta-D-glucanases and xylanases.

4. The process according to claim 1, wherein the biomass is selected from cellulose, hemicellulose and/or lignin-containing biomass.

5. The process according to claim 4, wherein the biomass is selected from sugar-beet, sugar-cane, straw, corn, wood, oilseed and mixtures thereof.

6. The process according to claim 1, further comprising: e) adding an amount of liquid corresponding to the amount removed by filtration according to step c).

7. The process according to claim 6, further comprising: f) Adding an amount of fresh biomass.

8. The process according to claim 7, wherein steps b) to e) or b) to f) are repeated at least once.

9. The process according to claim 6, wherein each of steps a) to e) are carried out simultaneously.

10. The process according to claim 1, wherein dry matter content of the biomass is from 15 to 30 wt.-%.

11. The process according to claim 1, wherein dry matter content of the biomass is 22 wt.-%.

Description

EXAMPLES AND FIGURES

(1) The present invention is now described by the following examples and figures. All examples and figures are for illustrative purposes only and are not to be understood as limiting the invention.

(2) FIG. 1 shows how by the particular combination of steps a) to e) of the inventive process for the hydrolysis of biomass the enzyme concentration increases over time within the vessel with ongoing filtration according to step c) of the process. Further, the reaction rate of the hydrolysis increases with ongoing filtration according to step c) and addition of liquid according to step e) of the process.

(3) FIG. 2 shows an exemplary process setup suitable for carrying out the process for the hydrolysis of biomass according to the invention implementing one filtration module and two circuits (inner and outer circuit).

(4) FIG. 3 shows an exemplary process setup suitable for carrying out the process for the hydrolysis of biomass according to the invention implementing two circuits (inner and outer circuit) and two filtration modules in parallel within the inner circuit.

(5) FIG. 4 shows an exemplary process setup suitable for carrying out the process for the hydrolysis of biomass according to the invention implementing two circuits (inner and outer circuit) and two filtration modules in series within the inner circuit.

(6) FIG. 5 shows an exemplary process setup suitable for carrying out the process for the hydrolysis of biomass according to the invention implementing one outer and two inner circuits in parallel.

(7) FIG. 1 Illustration of Temporal Courses for Volume, Enzyme Concentration and Saccharide Yield During the Process of the Invention

(8) The process is started by contacting the biomass with the enzyme-composition (step a). Within the period in which only step (a) is carried out, the volume and the enzyme concentration remain constant and a part of the biomass is hydrolyzed.

(9) After a certain time period, steps (b), (c) and (d) are started additionally to step (a). Due to the removal of permeate the volume decreases and saccharides are obtained within the permeate. Therefore, the saccharide yield increases. Since the enzymes are retained by the membrane, the enzyme concentration increases, which improves the hydrolysis.

(10) When the volume reaches are certain minimum value, step (e) is additionally started by continuously adding the same amount of water as permeate is removed, Therefore, the volume remains constant as well as the enzyme concentration. The saccharides are washed out and so the saccharide yield increases further.

(11) When the economic optimum between addition of water and increase of saccharide yield is reached, the process is stopped.

(12) FIG. 2 Exemplary Process Setup Implementing One Filtration Module and Two Circuits (Inner and Outer Circuit)

(13) Carrying out the process for the hydrolysis of biomass according to the present invention by use of a process set-up according to FIG. 2, biomass is fed into the vessel (2) which is equipped with a stirrer (1). After (at least) partially carrying out step a) according to the process within the vessel (2), a part of biomass is eliminated according to step b) of the process and conveyed to the outer circuit (5) by use of a first pipeline (5a) and pumped by a first pump (4) to the inner circuit (8).

(14) Within the inner circuit (8) the eliminated part of biomass is conveyed to a second pump (6) by use of a second pipeline (8a). The second pump (6) is operated separately from the first pump (4) thereby selecting a pump-rate in order to achieve the required cross-flow velocity. The second pump (6) is pumping the eliminated part of biomass to the filtration module (7) in order to subject the biomass to a filtration according to step c) of the process. The permeate is then removed from the filtration module (7) through a pipeline (10) wherein the output volume is controlled by a valve (9).

(15) The filtrated biomass is then further transported within the inner circuit (8) by a third pipeline (8b) to the outer circuit (5) wherein the at least part of the volume of the filtrated biomass which is fed back to the vessel according to step d) of the process through a fourth pipeline (5b) is regulated by a valve (3). In case only a part of the filtrated biomass is fed back to the vessel (2) the rest of the filtrated biomass is circulated within the inner circuit (8) by use of a pipeline (8.1e). An amount of liquid corresponding to the amount removed during filtration is added to the vessel (2) from a reservoir (11) through a pipeline (12).

(16) FIG. 3 Exemplary Process Setup Implementing Two Circuits (Inner and Outer Circuit) and Two Filtration Modules in Parallel within the Inner Circuit

(17) Carrying out the process for the hydrolysis of biomass according to the present invention by use of a process set-up according to FIG. 3 biomass is fed into the vessel (2) which is equipped with a stirrer (1). After (at least) partially carrying out step a) according to the process within the vessel (2), a part of biomass is eliminated according to step b) of the process and conveyed to the outer circuit (5) by use of a first pipeline (5a) and pumped by a first pump (4) to the inner circuit (8).

(18) Within the inner circuit (8) the eliminated part of biomass is conveyed to a second pump (6) by use of a second pipeline (8.1a). The second pump (6) is operated separately from the first pump (4) thereby selecting a pump-rate in order to achieve the required cross-flow velocity. The second pump (6) is pumping the eliminated part of biomass to the first filtration module (7.1) by use of a pipeline (8.1c) in order to subject the biomass to a filtration according to step c) of the process but also, concurrently, by use of a pipeline (8.2a) to the second filtration module (7.2). Thus, according to the exemplary process setup according to FIG. 3 the first and second filtration module (7.1) and (7.2) can be operated parallel. The permeate is then removed from the first filtration module (7.1) and the second filtration module (7.2) through a pipeline (10.1) and a pipeline (10.2) wherein the output volume is controlled independently by a valve (9.1) and a valve (9.2).

(19) The filtrated biomass is then further transported within the inner circuit (8) by a pipeline (8.1b), (8.1d) and by a pipeline (8.2b) to the outer circuit (5) wherein the volume of the filtrated biomass which is fed back to the reactor according to step d) of the process through a pipeline (5b) is regulated by a valve (3).). In case only a part of the filtrated biomass is fed back to the vessel (2) the rest of the filtrated biomass is circulated within the inner circuit (8) by use of a pipeline (8.1e). An amount of liquid corresponding to the amount removed during filtration is added to the vessel from a reservoir (11) through a pipeline (12).

(20) FIG. 4 Exemplary Process Setup Implementing Two Circuits (Inner and Outer Circuit) and Two Filtration Modules in Series within the Inner Circuit

(21) Carrying out the process for the hydrolysis of biomass according to the present invention by use of a process set-up according to FIG. 4 biomass is fed into the vessel (2) which is equipped with a stirrer (1). After (at least) partially carrying out step a) according to the process within the vessel (2), a part of biomass is eliminated according to step b) of the process and conveyed to the outer circuit (5) by use of a first pipeline (5a) and pumped by a first pump (4) to the inner circuit (8).

(22) Within the inner circuit (8) the eliminated part of biomass is conveyed to a second pump (6.1) by use of a second pipeline (8a). The second pump (6.1) is operated separately from the first pump (4) thereby selecting a pump-rate in order to achieve the required cross-flow velocity. The second pump (6.1) is pumping the eliminated part of biomass to the first filtration module (7.1) in order to subject the biomass to a filtration according to step c) of the process. The permeate is then removed from the first filtration module (7.1)) through a pipeline (10.1) wherein the output volume is controlled independently by a valve (9.1).

(23) The filtrated biomass is then further transported within the inner circuit (8) by a pipeline (8c) and pumped by a third pump (6.2) to the second filtration module (7.2) thereby selecting a pump-rate in order to achieve the required cross-flow velocity. The third pump (6.2) is also operated separately from the first pump (4) and the second pump (6.1). The permeate is then removed from the second filtration module (7.2) through a pipeline (10.2) wherein the output volume is controlled independently by a valve (9.2). Thus, according to the exemplary process setup according to FIG. 4 the first and second filtration module (7.1) and (7.2) can be operated in series.

(24) The filtrated biomass is then further transported within the inner circuit (8) by a pipeline (8b) to the outer circuit (5) wherein the volume of the filtrated biomass which is fed back to the reactor according to step d) of the process through a pipeline (5b) is regulated by a valve (3). In case only a part of the filtrated biomass is fed back to the vessel (2) the rest of the filtrated biomass is circulated within the inner circuit (8) by use of a pipeline (8.1e). An amount of liquid corresponding to the amount removed during filtration is added to the vessel from a reservoir (11) through a pipeline (12).

(25) FIG. 5 Exemplary Process Setup Implementing One Outer and Two Inner Circuits in Parallel

(26) Carrying out the process for the hydrolysis of biomass according to the present invention by use of a process set-up according to FIG. 5 biomass is fed into the vessel (2) which is equipped with a stirrer (1). After (at least) partially carrying out step a) according to the process within the vessel (2), a part of biomass is eliminated according to step b) of the process and conveyed to the outer circuit (5) by use of a first pipeline (5a) and pumped by a first pump (4) to the first inner circuit (8.1) and concurrently to the second inner circuit (8.2).

(27) Within the first inner circuit (8.1) the eliminated part of biomass is conveyed to a second pump (6.1) by use of a pipeline (8.1a). The second pump (6.1) is operated separately from the first pump (4) thereby selecting a pump-rate in order to achieve the required cross-flow velocity. The second pump (6.1) is pumping the eliminated part of biomass to the first filtration module (7.1) in order to subject the biomass to a filtration according to step c) of the process.

(28) Within the second inner circuit (8.2) the eliminated part of biomass is conveyed to a third pump (6.2) by use of a pipeline (8.2a). The third pump (6.2) is operated separately from the first pump (4) and the second pump (6.1) thereby selecting a pump-rate in order to achieve the required cross-flow velocity. The third pump (6.2) is pumping the eliminated part of biomass to the second filtration module (7.2) in order to subject the biomass to a filtration according to step c) of the process. Thus, according to the exemplary process setup according to FIG. 5 the first and second inner circuit (8.1) and (8.2) can be operated parallel.

(29) The permeate is then removed from the first filtration module (7.1) and the second filtration module (7.2) through a pipeline (10.1) and a pipeline (10.2) wherein the output volume is controlled independently by a valve (9.1) and a valve (9.2). The filtrated biomass is then further transported within the first inner circuit (8.1) by a pipeline (8.1b) and within the second inner circuit (8.2) by a pipeline (8.2b) to the outer circuit (5) wherein the volume of the filtrated biomass which is fed back to the reactor according to step d) of the process through a pipeline (5b) is regulated by a valve (3). In case only a part of the filtrated biomass is fed back to the vessel (2) the rest of the filtrated biomass is circulated within the inner circuit (8.1) and/or the inner circuit (8.2) by use of a pipeline (8.1e). An amount of liquid corresponding to the amount removed during filtration is added to the vessel from a reservoir (11) through a pipeline (12).

EXAMPLE 1

(30) Whole sugar beet material was prepared from fresh sugar beet roots sampled in Bedburg, Germany. Beet roots were washed to remove remaining soil and cut. The material was then treated by a high shear mixer in order to allow pumping. The sugar beet material on average had a d.m. content of 22%.

(31) The following enzymes were used: 43.4%(w/w) Celluclast, 6.3%(w/w) Novo 188 and 50.3% (w/w) Pectinex Ultra SP-L. These products were mixed in 50 mM NaAc buffer (pH 5).

(32) This enzyme mixture was mixed with 200 kg fresh sugar beet material at 0.2% wt.-% E/S. The final reaction mixture contained 18% d.m. of sugar beet material. The mixture was incubated with slight stirring at 50 C. After incubation for 4 hours, the ultrafiltration step was started using two circuits equipped each with a pump (one for increasing the transmembrane pressure and one for the transportation of the biomass through the membrane unit). The membrane used was a ceramic membrane with 10 kDa cut-off (GEA Filtration, GEA Wiegand GmbH). The transmembrane pressure applied was 0.5-3 bar and the cross-flow velocity was 3-4 m/s. The resulting permeate flux was 15-20 L/h. After 9 h of ultrafiltration, deionized water was added into the reactor at 15-20 L/h, while the filtration was continued at the same permeate flow rate. After 2 h the process was stopped. The final permeate mass and retentate mass recovered were 180 kg and 16 kg, respectively. The C6 sugar yield was >98% and the liquefaction reached >85 wt.-%.

(33) Samples were subsequently applied to HPLC analysis. The resulting hydrolysis mixture was analyzed by HPLC (Agilent, Germany) with an Aminex HPX 87 (BioRad Labs, Hercules, USA) ion exchange column (Eluent: 100% water, T: 85 C., Flow: 0.6 ml/min, RI detection).

(34) The results are shown in table 1.

(35) TABLE-US-00001 Permeate Permeate obtained obtained Filtrated during step during step biomass (final b) to d) b) to e) composition) Glucose [g/l] 74 34 12 Fructose [g/l] 77 36 11 Arabinose [g/l] 7 4 1 Cellobiose [g/l] 6 4 1

(36) Table 1 shows the obtained sugar concentrations in permeate obtained during step b) to d), permeate obtained during step b) to e) and filtrated biomass (the error is estimated to +/10%)

EXAMPLE 2

(37) Whole sugar beet material was prepared from fresh sugar beet roots sampled in Bedburg, Germany. Beet roots were washed to remove remaining soil and. The material was then treated by a high shear mixer in order to allow pumping. The sugar beet material on average had a d.m. content of 22%.

(38) The following enzymes were used: 43.4% (w/w) Celluclast, 6.3%(w/w) Novo 188 and 50.3% (w/w) Pectinex Ultra SP-L. These products were mixed in 50 mM NaAc buffer (pH 5).

(39) This enzyme mixture was mixed with 150 kg fresh sugar beet material at 0.1% wt.-% EIS. The final reaction mixture contained 18% dm. of sugar beet material. The mixture was incubated with slight stirring at 50 C. After incubation for 5 hours, the ultrafiltration step was started using two circuits equipped each with a pump (one for increasing the transmembrane pressure and one for the transportation of the biomass through the membrane unit). The membrane used was a ceramic membrane with 10 kDa cut-off (GEA Filtration, GEA Wiegand GmbH). The transmembrane pressure applied was 0.5-3 bar and the cross-flow velocity was 3-4 m/s. The resulting permeate flux was 10-20 L/h. After 9 h of ultrafiltration, deionized water was added into the reactor at 10-20 L/h, while the filtration was continued at the same permeate flow rate. After 1.5 h the process was stopped. The final permeate mass and retentate mass recovered were 126 kg and 16 kg, respectively. The C6 sugar yield was >90% and the liquefaction reached >80 wt.-%.

(40) Samples were subsequently applied to HPLC analysis. The resulting hydrolysis mixture was analyzed by HPLC (Agilent, Germany) with an Aminex HPX 87 (BioRad Labs, Hercules, USA) ion exchange column (Eluent: 100% water, T: 85 C., Flow: 0.6 ml/min, RI detection).

(41) The results are shown in table 2.

(42) TABLE-US-00002 Permeate Permeate Filtrated obtained obtained biomass during step during step (final b) to d) b) to e) composition) Glucose [g/l] 75 45 23 Fructose [g/l] 72 44 22 Arabinose [g/l] 7 5 2 Cellobiose [g/l] 7 6 3

(43) Table 2 shows the obtained sugar concentrations in permeate obtained during step h) to d), permeate obtained during step b) to e) and filtrated biomass (the error is estimated to +1-10%)

COMPARATIVE EXAMPLE 2

(44) Whole sugar beet material was prepared according to example 2.

(45) The following enzymes were used: 43.4%(w/w) Celluclast, 6.3%(w/w) Novo 188 and 50.3% (w/w) Pectinex Ultra These products were mixed in 50 mM NaAc buffer (pH 5).

(46) This enzyme mixture was mixed with 150 kg fresh sugar beet material at 0.1 wt.-% E/S. The final reaction mixture contained 18% dm. of sugar beet material in 50 mM sodium acetate buffer (pH5).

(47) The reaction mixture was incubated for 30 min to 5 hours at 50 C. After liquefaction and hydrolysis the reaction mixture was centrifuged for 30 min at 3200 g and the liquid supernatant was separated and weighted. 1 ml of the supernatant was heat inactivated at 95 C. for 10 min and the amount of sugar released was analyzed by HPLC (Agilent, Germany) with an Aminex HPX 87 (BioRad Labs, Hercules, USA) ion exchange column (Eluent: 100% water, T: 85 C., Flow: 0.6 ml/min, RI detection).

(48) The results are shown in table 3

(49) TABLE-US-00003 Supernatant Glucose [g/l] 54 Fructose [g/l] 53 Arabinose [g/l] 9 Cellobiose [g/l] 5

(50) Table 3 shows the obtained sugar concentrations in the supernatant.

(51) Since the supernatant of comparative example 2 has a lower sugar concentration compared to the permeate of example 2 the process according to the invention was more efficient.

EXAMPLE 3

Ethanol Production Using Saccharide-Containing Permeate-Product

(52) 750 mL of saccharide-containing permeate-product was inoculated with 50 mL inoculate of Saccharomyces cerivisiae resulting in a start optical density of 1.8. The fermentation medium was stirred with 400 rpm at pH 4.5 and 32 C. using a Multifors lab fermenter (Infors, Switzerland). After 70.5 hours the fermentation was stopped. Samples were subsequently applied to HPLC and GC analysis. The results show that glucose as well as fructose were completely consumed and 65 g/L Ethanol produced.