Improved Cellulose to Cellobiose Conversion Process

Abstract

A process to hydrolyze cellulose into cellobiose comprising the following steps: providing a reaction vessel; providing a Cellulomonas uda (ATCC 491) inoculum; exposing said Cellulomonas uda (ATCC 491) bacterium to a source of cellulose having a kappa number of less than 10 in an aqueous medium of pH of about 8 at a temperature ranging from 30° C. to 35° C. for a period of time ranging from 14 to 42 days; exposing the cellobiose to a bacterium or fungi or yeast, or combination which converts cellobiose to glucose or ethanol.

Claims

1. A process to hydrolyze cellulose into cellobiose, said process comprising the following steps: providing a reaction vessel; providing a source of cellulose into said reaction vessel; wherein said source of cellulose has a kappa number of less than 10; providing a Cellulomonas uda (ATCC 491) inoculum into said reaction vessel; exposing said Cellulomonas uda (ATCC 491) to said source of cellulose in an aqueous medium of pH of about 8 at a temperature ranging from 30° C. to 35° C. for a period of time ranging from 14 to 42 days; and optionally, recovering the supernatant comprising cellobiose to be converted to glucose and or ethanol using a bacterium, fungi, yeast or combination.

2. Process according to claim 1, wherein said source of cellulose has a particle size ranging between 30-50 μm.

3. The process according to claim 1 further comprising a step of exposing the cell supernatant to a bacterium or fungi or yeast, or combination to convert cellobiose to glucose or ethanol.

4. The process according to claim 1, wherein the source of cellulose is a lignocellulosic biomass delignified by exposure to a modified Caro's acid composition selected from the group consisting of: composition A; composition B and Composition C; wherein said composition A comprises: sulfuric acid in an amount ranging from 20 to 70 wt % of the total weight of the composition; a compound comprising an amine moiety and a sulfonic acid moiety selected from the group consisting of: taurine; taurine derivatives; and taurine-related compounds; and a peroxide; wherein said composition B comprises: an alkylsulfonic acid; and a peroxide; wherein the acid is present in an amount ranging from 40 to 80 wt % of the total weight of the composition and where the peroxide is present in an amount ranging from 10 to 40 wt % of the total weight of the composition; wherein said composition C comprises: sulfuric acid; a compound comprising an amine moiety; a compound comprising a sulfonic acid moiety; and a peroxide; for a period of time sufficient to remove substantially all of the lignin present on said biomass material.

5. The process according to claim 4, wherein said sulfuric acid, said compound comprising an amine moiety and a sulfonic acid moiety and said peroxide are present in a molar ratio of no more than 15:1:1.

6. The process according to claim 4, wherein said sulfuric acid and said compound comprising an amine moiety and a sulfonic acid moiety are present in a molar ratio of no less than 3:1.

7. The process according to claim 4, wherein compound comprising an amine moiety and a sulfonic acid moiety is selected from the group consisting of: taurine; taurine derivatives; and taurine-related compounds.

8. The process according to claim 4, wherein taurine derivative or taurine-related compound is selected from the group consisting of: taurolidine; taurocholic acid; tauroselcholic acid; tauromustine; 5-taurinomethyluridine and 5-taurinomethyl-2-thiouridine; homotaurine (tramiprosate); acamprosate; and taurates; as well as aminoalkylsulfonic acids where the alkyl is selected from the group consisting of C.sub.1-C.sub.5 linear alkyl and C.sub.1-C.sub.5 branched alkyl.

9. The process according to claim 8, wherein linear alkylaminosulfonic acid is selected form the group consisting of: methyl; ethyl (taurine); propyl; and butyl.

10. The process according to claim 4, wherein said compound comprising an amine moiety and a sulfonic acid moiety is taurine.

11. The process according to claim 4, wherein said alkylsulfonic acid is selected from the group consisting of: methanesulfonic acid; ethanesulfonic acid; propanesulfonic acid; 2-propanesulfonic acid; isobutylsulfonic acid; t-butylsulfonic acid; butane sulfonic acid; iso-pentylsulfonic acid; t-pentylsulfonic acid; pentanesulfonic acid; t-butylhexanesulfonic acid; and combinations thereof.

12. The process according to claim 4, where, in Composition C, said compound comprising an amine moiety is an alkanolamine selected from the group consisting of: monoethanolamine; diethanolamine; triethanolamine; and combinations thereof.

13. The process according to claim 4 where, in Composition C, said compound comprising an amine moiety is triethanolamine and said compound comprising a sulfonic acid moiety is methanesulfonic acid.

14. The process according to claim 4, wherein the delignification lasts from 2 to 20 hours.

15. The process according to claim 4, wherein the delignification is carried out at temperatures below 50° C.

16. The process according to claim 4, wherein said cellulose is characterized by an absence of prior exposure to bleaching chemicals selected from the group consisting of: sodium hydrosulphite (Na.sub.2S.sub.2O.sub.4); hydrogen peroxide; pentasodium salt diethylenetriaminepentaacetic acid; amine borane (CH.sub.3).sub.3CNH.sub.2-BH.sub.3; borane ammonia complex BH.sub.3—NH3; sodium percarbonate; formamidine sulphinic acid; sodium perborate; and chlorine dioxide.

17. A process to convert cellulose to glucose or ethanol, said process comprising the steps of: providing a reaction vessel; providing a source of cellulose into said reaction vessel; wherein said source of cellulose having a particle size ranging between 30-50 μm and a kappa number of less than 10; providing a Cellulomonas uda (ATCC 491) inoculum into said reaction vessel; exposing said Cellulomonas uda (ATCC 491) to said source of cellulose in an aqueous medium of pH of about 8 at a temperature ranging from 30° C. to 35° C. for a period of time ranging from 14 to 42 days, allowing sufficient exposure time to convert at least 90% of the cellulose into cellobiose and/or glucose; and optionally, recovering the supernatant comprising cellobiose and glucose.

18. The process according to claim 17, where the temperature inside the reaction vessel during said exposure time does not exceed 60° C.

19. The process according to claim 17, wherein said cellulose has an aspect ratio of 7.5.

20. A process to obtain a bacterium having a high cellulase activity for a low kappa number cellulose, wherein said a low kappa number cellulose has the following characteristics: particle size ranging from 30-50 microns and a kappa number of less than 10, more preferably less than 5 and even more preferably, less than 2, wherein said process comprising the steps of: providing a reaction vessel; providing a Cellulomonas uda (ATCC491) inoculum which has a cellulase enzyme activity when exposed to said low kappa number cellulose of less than 5 units/ml after said bacterium has been exposed to said low kappa number cellulose for 21 days; exposing said Cellulomonas uda (ATCC491) bacterium to said low kappa number cellulose in an aqueous medium of pH of about 8 at a temperature ranging from 30° C. to 35° C. for a period of time ranging from 14 to 42 days; recovering the bacterium and repeating the step of exposing said bacterium to said low kappa number cellulose for a number of cycles of exposure and recovery until the cellulase activity of the enzyme reaches at least 8 units/ml when said bacterium is exposed to said low kappa number cellulose for at least 18 days, wherein said bacterium has been optimized for cellulose hydrolysis; and optionally, recovering the optimized bacterium. optionally, removing the products and refeeding the culture for a continuous batch culture.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0076] Features and advantages of embodiments of the present application will become apparent from the following detailed description and the appended figures, in which:

[0077] FIG. 1 is a depiction of glucose monomers present in a starch polymer and their α-1,4-linkages compared to glucose monomers present in a cellulose polymer and their β-1,4-linkages;

[0078] FIG. 2 illustrates a comparative graphical representation of the enzymatic activity of an adapted strain of Cellulomonas uda (ATCC 491), after a few generations of exposure to the Modified Caro's Acid (MCA) cellulose, upon exposure to fine grade and coarse grade delignified cellulose.

[0079] FIG. 3 illustrates a graphical representation of the enzymatic activity of the original strain of Cellulomonas uda (ATCC 491) in a continuous feed model.

[0080] FIG. 4 illustrates a graphical representation of the beta-glucanase enzymatic activity for various substrates obtained either by the kraft process or by exposure to a modified Caro's acid;

[0081] FIG. 5 illustrates a graphical representation of the beta-glucosidase enzymatic activity for various substrates obtained either by the kraft process or by exposure to a modified Caro's acid;

[0082] FIG. 6 illustrates a graphical representation of the ethanol yield for various substrates obtained either by the kraft process or by exposure to a modified Caro's acid;

DETAILED DESCRIPTION OF THE INVENTION

[0083] The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles of the present invention. These examples are provided for the purposes of explanation, and not limitation, of those principles and of the invention.

[0084] According to a preferred embodiment of the present invention, C. uda is more efficient at hydrolyzing the modified Caro's acid delignified cellulose (MCA cellulose) which has a kappa number of β-2 than a softwood cellulose with a kappa number of 27.

[0085] U.S. Pat. No. 4,496,656A describes a medium used to grow the bacteria used in the present invention. Several other media were tested, but this turned out to be the most suitable for the methods according to preferred embodiments of the present invention.

[0086] According to a preferred embodiment of the present invention, the process to hydrolyze cellulose into cellobiose comprises the following steps: [0087] providing a reaction vessel; [0088] providing an enzyme adapted to convert a source of cellulose having a particle size of 30-50 microns and a kappa number of less than 10, more preferably less than 5 and even more preferably, less than 2, in an aqueous medium of pH of about 8 at a temperature ranging from 30° C. to 35° C. for a period of time ranging from 14 to 42 days, into cellobiose; [0089] exposing the cell supernatant to a bacterium or fungi or yeast, or combination, which converts cellobiose to glucose or ethanol.

[0090] According to a preferred embodiment of the present invention, the process to hydrolyze cellulose into cellobiose comprises the following steps: [0091] providing a reaction vessel; [0092] providing a Cellulomonas uda (ATCC 491) inoculum; [0093] exposing said Cellulomonas uda (ATCC 491) to a source of cellulose having a particle size of 30-50 microns and a kappa number of less than 10, more preferably less than 5 and even more preferably, less than 2, in an aqueous medium of pH of about 8 at a temperature ranging from 30° C. to 35° C. for a period of time ranging from 14 to 42 days; [0094] exposing the cell supernatant to a bacterium or fungi or yeast, or combination, which converts cellobiose to glucose or ethanol.

[0095] Cellulomonas uda (ATCC 491) is an aerobic bacterium isolated and characterized from compost. It is a gram-positive rod-shaped bacterium of the phylum Actinobacteria and is known for its ability to metabolize cellulose.

[0096] In referring to FIG. 4, one can notice that the MCA cellulose with a lower kappa number have generated higher enzyme activity in the first exposure to a bacterium. This first exposure is meant to hydrolyze the β-1,4-glucosidic bonds of the cellulose to generate smaller cellulose molecules and cellobiose and therefore generate the bulk of the glucose precursor material (in this case, cellobiose). It is understood that the second step of the process according to a preferred embodiment of the present invention is straightforward as it does not deviate from the common approach of cellobiose conversion to glucose. It is established that the first step is more determinant of the extent of biomass conversion to glucose since it will generate as main primary product, the cellobiose, which is subsequently employed in conversion to glucose. It is imperative that the cellulose degradation to cellobiose be maximized at this step, otherwise it will prevent the second step of the process from having a significant impact.

[0097] According to a preferred embodiment of the present invention, it is observed that, in the last data point, that the enzymatic activity is higher with the MCA cellulose than the enzymatic activity when such is exposed to a a kraft processed hardwood cellulose (kappa number=15.82) or raw corn stover biomass (kappa number=47.74). The MCA cellulose is obtained according to a process (or approach) to obtain low lignin cellulose (also referred to as a low kappa number cellulose, also referred to as modified Caro's acid delignified cellulose) as prepared according to a preferred process described hereinbelow and has a kappa number=β-2.

Process to Obtain a Low Kappa Number Delignified Cellulose

[0098] According to a preferred embodiment of the present invention, the method of delignification of biomass material which yields a Modified Caro's Acid (MCA) cellulose used in the cellulose to cellobiose (and ultimately, glucose) conversion experiments comprise: [0099] providing a biomass material comprising cellulose fibers and lignin; [0100] exposing said biomass material requiring delignification to a modified Caro's acid composition selected from the group consisting of: composition A; composition B and Composition C;

[0101] wherein said composition A comprises: [0102] sulfuric acid in an amount ranging from 20 to 70 wt % of the total weight of the composition; [0103] a compound comprising an amine moiety and a sulfonic acid moiety selected from the group consisting of: taurine; taurine derivatives; and taurine-related compounds; and [0104] a peroxide;

[0105] wherein said composition B comprises: [0106] an alkylsulfonic acid; and [0107] a peroxide; wherein the acid is present in an amount ranging from 40 to 80 wt % of the total weight of the composition and where the peroxide is present in an amount ranging from 10 to 40 wt % of the total weight of the composition;

[0108] wherein said composition C comprises: [0109] sulfuric acid; [0110] a compound comprising an amine moiety; [0111] a compound comprising a sulfonic acid moiety; and [0112] a peroxide;
for a period of time sufficient to remove substantially all of the lignin present on said biomass material. The process can be carried out for a varying duration of time depending on the particle size of the biomass being fed into the process. The process can last from 2 to 20 hours depending on that characteristic. Moreover, the temperature of the resulting mixture also has an impact on the duration of the process as the reaction is highly exothermic, precautions are taken to prevent a runaway degradation of the cellulose. This would result in a carbon black resulting product with no value. The process is preferably run at temperatures below 50° C., more preferably at temperatures below 40° C. the process of delignification is preferably performed with a cooling means adapted to control the heat generated by the chemical reaction of delignification and maintain the temperature to avoid an undesirable ‘runaway’ reaction.

[0113] Preferably, said sulfuric acid, said compound comprising an amine moiety and a sulfonic acid moiety and said peroxide are present in a molar ratio of no more than 15:1:1.

[0114] According to a preferred embodiment of the approach to obtain low lignin cellulose, said sulfuric acid and said compound comprising an amine moiety and a sulfonic acid moiety are present in a molar ratio of no less than 3:1.

[0115] Preferably, said compound comprising an amine moiety and a sulfonic acid moiety is selected from the group consisting of: taurine; taurine derivatives; and taurine-related compounds.

[0116] According to a preferred embodiment of the approach to obtain low lignin cellulose, said taurine derivative or taurine-related compound is selected from the group consisting of: taurolidine; taurocholic acid; tauroselcholic acid; tauromustine; 5-taurinomethyluridine and 5-taurinomethyl-2-thiouridine; homotaurine (tramiprosate); acamprosate; and taurates; as well as aminoalkylsulfonic acids where the alkyl is selected from the group consisting of C.sub.1-C.sub.5 linear alkyl and C.sub.1-C.sub.5 branched alkyl.

[0117] Preferably, said linear alkylaminosulfonic acid is selected form the group consisting of: methyl; ethyl (taurine); propyl; and butyl.

[0118] Preferably, branched aminoalkylsulfonic acid is selected from the group consisting of: isopropyl; isobutyl; and isopentyl.

[0119] According to a preferred embodiment of the approach to obtain low lignin cellulose, said compound comprising an amine moiety and a sulfonic acid moiety is taurine.

[0120] According to a preferred embodiment of the approach to obtain low lignin cellulose, said sulfuric acid and compound comprising an amine moiety and a sulfonic acid moiety are present in a molar ratio of no less than 3:1.

[0121] According to a preferred embodiment of the approach to obtain low lignin cellulose, said compound comprising an amine moiety is an alkanolamine is selected from the group consisting of: monoethanolamine; diethanolamine; triethanolamine; and combinations thereof.

[0122] According to a preferred embodiment of the approach to obtain low lignin cellulose, said compound comprising a sulfonic acid moiety is selected from the group consisting of: alkylsulfonic acids; arylsulfonic acids; and combinations thereof.

[0123] Preferably, said alkylsulfonic acid is selected from the group consisting of: alkylsulfonic acids where the alkyl groups range from C.sub.1-C.sub.6 and are linear or branched; and combinations thereof. More preferably, said alkylsulfonic acid is selected from the group consisting of: methanesulfonic acid; ethanesulfonic acid; propanesulfonic acid; 2-propanesulfonic acid; isobutylsulfonic acid; t-butylsulfonic acid; butanesulfonic acid; iso-pentylsulfonic acid; t-pentylsulfonic acid; pentanesulfonic acid; t-butylhexanesulfonic acid; and combinations thereof.

[0124] Preferably, said arylsulfonic acid is selected from the group consisting of: toluenesulfonic acid; benzesulfonic acid; and combinations thereof.

[0125] According to a preferred embodiment of the approach to obtain low lignin cellulose, said alkylsulfonic acid; and said peroxide are present in a molar ratio of no less than 1:1.

[0126] Preferably, said compound comprising a sulfonic acid moiety is methanesulfonic acid.

[0127] According to a preferred embodiment of the approach to obtain low lignin cellulose, said Composition C may further comprise a compound comprising an amine moiety. Preferably, the compound comprising an amine moiety has a molecular weight below 300 g/mol. Preferably also, the compound comprising an amine moiety is a primary amine. More preferably, the compound comprising an amine moiety is an alkanolamine. Preferably, the compound comprising an amine moiety is a tertiary amine. According to a preferred embodiment of the approach to obtain low lignin cellulose, the alkanolamine is selected from the group consisting of: monoethanolamine; diethanolamine; triethanolamine; and combinations thereof. Preferably, the alkanolamine is triethanolamine.

[0128] According to a preferred embodiment of the approach to obtain low lignin cellulose, said in Composition C, said sulfuric acid and said a compound comprising an amine moiety and said compound comprising a sulfonic acid moiety are present in a molar ratio of no less than 1:1:1.

[0129] Preferably, in Composition C, said sulfuric acid, said compound comprising an amine moiety and said compound comprising a sulfonic acid moiety are present in a molar ratio ranging from 28:1:1 to 2:1:1.

[0130] Preferably, in Composition C, said compound comprising an amine moiety is triethanolamine and said compound comprising a sulfonic acid moiety is methanesulfonic acid.

[0131] In referring to FIG. 2, which illustrates a comparative graphical representation of the enzymatic activity of an adapted strain of Cellulomonas uda (ATCC491) between fine grade MCA cellulose (30-50 μm) and coarse grade MCA cellulose (1-2 mm) according to a preferred embodiment of the present invention, both of which are obtained from the process described hereinabove. It is worth noting that the particle size of the cellulose feedstock has a non-negligible impact on the enzymatic activity of the bacterium used in this series of experiments.

[0132] In referring to FIG. 3, which illustrates a graphical representation of the enzymatic activity of the original strain of Cellulomonas Uda (ATCC491) in a continuous feed model according to a preferred embodiment of the present invention, it is noted that after re-feeding of the culture the lag phase is minimal and it takes 5-7 days for the organism to return to the enzymatic activity seen in the first culture. This demonstrates that a continuous feed culture model will allow for enzyme activity to remain high and reduce lag periods that are seen with batch or fed-batch culture systems.

[0133] In referring to FIG. 4, which illustrates a comparative graphical representation of the enzymatic activity of an adapted strain of Cellulomonas uda (ATCC491), after a few generations of exposure to different grades of cellulose, it is noted that the bacterium exhibits very high affinity for the MCA cellulose obtained from a process as described hereinabove. This is surprising knowing the fact that, as mentioned above, the enzymatic hydrolysis of a cellulase bacterium is typically hindered by the level of crystallinity of the cellulose it is exposed to. The conclusion upon reviewing the graph is that the kappa number of a source of cellulose has a direct and non-negligible impact on the ability of the adapted strain of Cellulomonas uda (ATCC491) to metabolize cellulose into cellobiose.

[0134] FIG. 5 shows the enzyme activity of beta-glucosidase (from Aspergillus brasiliensis) is much greater in the modified Caro's acid delignified cellulose samples compared to those made by the kraft process, increasing by approximately 20% in hardwood and 65% in corn stover. This enzymatic reaction is converting the cellobiose to glucose. This second reaction occurs in a semi-anoxic co-culture of Aspergillus brasiliensis and Saccharomyces cerevisiae (yeast), so that as A. brasiliensis converts the cellobiose to glucose, the yeast can then ferment the glucose to ethanol.

[0135] FIG. 6 shows the ethanol yields obtained per gram of cellulose. Ethanol yields from a process using a modified Caro's acid and kraft process are also listed in the table below. Using the modified Caro's acid delignification process, ethanol yields increased by 85% in hardwood (compared to the kraft) and 92% in corn stover (compared to raw biomass).

TABLE-US-00001 TABLE 1 Ethanol yield from cellulose obtained from various source material using either the Kraft delignification process or using a modified Caro’s acid g (EtOH)/ Sample g (cellulose) Hardwood (pref. emb.) 0.47 Hardwood (Kraft process) 0.25 Corn stover (pref. emb.) 0.44 Corn stover (Kraft process) 0.23

[0136] According to a preferred embodiment of the process of the present invention, the step of exposing the adapted strain of Cellulomonas uda, (ATCC491) to a source of cellulose generates over 40% more enzymatic activity than the same bacterium on a kraft pulp with a kappa number of 27. Moreover, in the same circumstance, the β-1,4-glucanase enzyme activity (in unit per ml) was approximately 38% more active when exposed to a cellulose having aspect ratio of 7.5, a particle size ranging from 30 to 50 μm and a kappa number ranging from β-2 than when it was exposed to a kraft pulp where the cellulose has a kappa number of 27.

[0137] According to a preferred embodiment of the process of the present invention, this process will enable a higher conversion of cellulose to glucose by overcoming the first step in the chain of reactions which is the conversion of cellulose to cellobiose.

[0138] Given this information, it is believed that idle ethanol plants located around the world could re-start operations of cellulose conversion to glucose (and subsequently, ethanol) if a biomass feedstock according to the following specifications was employed rather than using corn, sugar cane or conventional kraft pulp. Moreover, the implementation of a process according to a preferred embodiment of the present invention would essentially “dovetail” with the delignification process of a lignocellulosic biomass by using a modified Caro's acid, and the production of ethanol with the cellulose obtained from the delignification process. As mentioned previously, the person skilled in the art will recognize that by employing a cellulose obtained from a process using a modified Caro's acid, one will circumvent the need of any further or subsequent bleaching step following the delignification. It is to be understood that the bleaching refers to a separate and distinct step of pulp processing. Consequently, the pulp used obtained using a modified Caro's acid driven delignification process, is intended on being a pulp which has not undergone a separate bleaching step post-delignification. As is also understood by the person skilled in the art, such a treatment step (bleaching) is understood to not be economically viable when the ultimate goal of the cellulose is to be further converted in order to generate ethanol.

[0139] While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by those skilled in the relevant arts, once they have been made familiar with this disclosure that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims.