Method for improving the fermentable sugar yield from lignocellulosic

Abstract

The invention relates to processes for the conversion of biomass into carbohydrates, notable fermentable sugars. It provides means and methods for increasing the yield of enzymatic digestion of a biomass, in particular in those cases where cellulose is converted into sugars using a cellulose converting enzyme. More in particular, the invention relates to a method for producing a fermentable sugar from a lignocellulosic material wherein the lignocellulosic material is contacted with a laccase and an enzyme capable of degrading cellulose, either simultaneously or in a sequentially deferred fashion, wherein the laccase is the Bacillus spore coat protein CotA.

Claims

1. A method for producing a fermentable sugar from a lignocellulosic material, the method comprising: contacting the lignocellulosic material with a laccase and a mixture of cellulose-degrading enzymes, wherein the contacting is performed simultaneously or in a sequential fashion, and wherein the laccase is the Bacillus spore coat protein CotA; wherein the mixture of cellulose degrading enzymes comprises an exoglucanase; and wherein at least 31% of theoretical fermentable sugar yield is released from the lignocellulosic material.

2. The method according to claim 1, wherein the lignocellulosic material is selected from the group consisting of corn stovers, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, yard waste, wood and forestry waste, sugar cane, switchgrass, wheat straw, hay, barley, barley straw, rice straw, grasses, waste paper, sludge or byproducts from paper manufacture, corn grain, corn cobs, corn husks, grasses, wheat, wheat straw, hay, rice straw, sugar cane bagasse, sorghum, soy, trees, branches, wood chips, sawdust and any combination thereof.

3. The method according to claim 1, wherein the cellulose-degrading enzymes of the mixture of cellulose-degrading enzymes are selected from the group consisting of cellulase, hemi-cellulase, [beta] 1-4 endoglucanases (E.C. 3.2.1.4), [beta] 1-4 exoglucanases (E.C. 3.2.1.9.1), [beta]-glucosidases (E.C. 3.2.1.2.1), and endoxylanase.

4. The method according to claim 1, wherein the lignocellulosic material is pretreated before the material is contacted with the laccase and the mixture of cellulose-degrading enzymes.

5. The method according to claim 4, wherein the pretreatment consists of a steam explosion step.

6. The method according to claim 1, wherein the CotA laccase has a primary amino acid structure having at least 60% sequence identity to the sequence of COT1 (SEQ ID NO:1) or COT2 (SEQ ID NO:2).

7. The method according to claim 6, wherein CotA is COT1 (SEQ ID NO:1) or COT2 (SEQ ID NO:2).

8. The method according to claim 2, wherein the cellulose-degrading enzymes of the mixture of cellulose-degrading enzymes are selected from the group consisting of cellulase, hemi-cellulase, [beta] 1-4 endoglucanases (E.C. 3.2.1.4), [beta] 1-4 exoglucanases (E.C. 3.2.1.9.1), [beta]-glucosidases (E.C. 3.2.1.2.1), and endoxylanase.

9. The method according to claim 2, wherein the lignocellulosic material is pretreated before the material is contacted with the laccase and the mixture of cellulose-degrading enzymes.

10. The method according to claim 3, wherein the lignocellulosic material is pretreated before the material is contacted with the laccase and the mixture of cellulose-degrading enzymes.

Description

LEGEND TO THE FIGURE

(1) FIG. 1 Effect of laccases on reducing sugar yield from lignocellulosic biomass. Enzymatic hydrolysis of the lignocellulosic material (old corrugated cardboard) was performed using commercial cellulase cocktail for biofuel applications under recommended conditions, with or without laccases. Laccases were added (as indicated) at the concentration 1 ukat/g (60 units/g) of dry substrate, directly to the hydrolysis mixtures together with cellulases. Following laccases were used: “Fungal laccase 1” from Trametes versicolor, “fungal laccase 2” from Pleurotus ostreatus, and “CotA” spore coat protein from Bacillus subtilis. Reducing sugar yields were determined by DNS-method. Yield of the control reaction without laccase, (“cellulase”) was taken as 100% and the relative yields for the corresponding samples containing laccases were calculated.

EXAMPLES

Example 1

Effect of Different Laccases on Sugar Yield from Enzymatic Hydrolysis of a Lignocellulosic Substrate

(2) Pieces of old corrugated cardboard were subjected to enzymatic hydrolysis. We carried out parallel experiments where hydrolysis of cellulose was performed in the absence of laccase or in the presence of one of the three laccases: (1) Spore coat protein from Bacillus subtilis CotA (COT2 (SEQ ID NO:2, recombinantly expressed in E. coli), (2) commercially available fungal laccase from white-rot-fungi Trametes versicolor (available from Sigma-Aldrich), and (3) laccase from white-rot-fungi Pleurotus ostreatus recombinantly expressed in Yeast Saccharomyces cerevisiae (Piscitelli, A., Giardina, P., Mazzoni, C., & Sannia, G. (2005). Recombinant expression of Pleurotus ostreatus laccases in Kluyveromyces lactis and Saccharomyces cerevisiae. Applied microbiology and biotechnology, 69(4), 428-39. doi:10.1007/s00253-005-0004-z).

(3) Pieces of old corrugated cardboard were pre-treated with 0.5% NaOH at 15% consistency (consistency means percentage of dry matter in the slurry, w/v) for 1 h at 90 degrees Celcius, then the material was washed with water, dried, and subjected to enzymatic hydrolysis at 5% consistency in 100 mM Succinic acid (pH 5.0).

(4) Enzymatic hydrolysis of cellulose was carried out using commercially available cellulase cocktail for biofuel applications, CMAX (Alternafuel) from Diadick using manufacturer recommended concentration of cellulases.

(5) All laccases were used at concentration 1 microkatal/g (60 units/g, of dry feedstock. One katal is defined as the amount of enzyme needed to convert 1 mole of substrate (ABTS) in 1 sec. A catalytic unit is defined as the amount of enzyme needed to convert 1 micromole of substrate (ABTS) in 1 min) and added directly to the hydrolysis reaction.

(6) Hydrolysis was carried out at 60 degrees Celsius for 72 h. After the hydrolysis, reducing sugar levels were determined by Dinitrosalicylic Acid Method (DNS method, Sadasivam S., Manickam A., “Carbohydrates” in Biochemical methods, New Age Internatioal Ltd Publishers, 2nd edition, 2005, p. 6).

(7) The results are shown in FIG. 1. We observed a large increase in yield when the lignocellulosic material was incubated simultaneously with CotA and the cellulase enzymes. An increase of approximately 250% was achieved in comparison to cellulases combined with white-rot-fungi Trametes versicolor or laccase from white-rot-fungi Pleurotus ostreatus. Very similar results were obtained when the laccase treatment was performed before the cellulase treatment.

Example 2

Enzymatic Hydrolysis of Various Lignocellulosic Feedstocks

(8) Enzymatic hydrolysis of various lignocellulosic feedstocks was carried out in order to evaluate the effect of CotA laccase treatment on sugar yield (Table 1).

(9) The following ligno-cellulosic substrates were used:

(10) Steam exploded wheet straw: steam explosion was performed in a steam explosion instrument at 200 degrees Celsius for 2.5 min, the slurry after steam explosion was washed with water and dried. Old corrugated cardboard was treated with 0.5% NaOH at 15% consistency for 1 h at 90 degrees Celsius, then the material was washed with water and dried. Eucalyptus pulp is pulp (wood fibers) obtained by chemical pulping (kraft pulping) and collected after bleaching step. It is practically pure cellulose fibers with small traces of lignin. Blow pulp Soft wood (Spruce) and Blow pulp, Hard wood (birch) are pulps (wood fibers) obtained by chemical pulping (kraft pulping) collected before bleaching step from the Blow tank which drys pulp after chemical cooking. These pulps contain about 3% lignin and 97% cellulose. Pine pulp is pulp obtained from pine by thermomechanical pulping process, it was collected before bleaching and contains about 25% lignin and 75% cellulose.

(11) Enzymatic hydrolysis of cellulose was carried out using commercially available cellulase cocktail for biofuel applications, CMAX (Alternafuel) from Diadick. Dried lignocellulosic substrates as described above were subjected to enzymatic hydrolysis at 5% consistency in 100 mM Succinic acid (pH 5.0) using manufacturer recommended concentration of cellulases. CotA laccase (where indicated) was added to the hydrolysis reactions at concentration 1 microkatal/g of dry feedstock (which corresponds to 60 units/g. One katal is defined as the amount of enzyme needed to convert 1 mole of substrate (ABTS) in 1 sec, catalytic unit is defined as the amount of enzyme needed to convert 1 micromole of substrate (ABTS) in 1 min). Hydrolysis was carried out at 60 degrees Celsius for 72 h.

(12) After the hydrolysis, reducing sugar levels were determined by Dinitrosalicylic Acid Method (DNS method, Sadasivam, 2005). Results are shown in table 1.

(13) It was concluded that the yields of the cellulase were greatly improved upon the addition of a CotA laccase to the reaction mixture, regardless of the source of the lignocellulose.

(14) It was remarkably found that a biomass containing virtually no lignin or no lignin at all (such as eucalyptus pulp) could also serve as a substrate in a method according to the invention. Even with those materials, the yield of the cellulose was greatly improved when a CotA laccase was used in combination with the cellulase. As shown in table 1, the yield from eucalyptus pulp increased with 157% to a yield that was 92% of the theoretical yield.

(15) TABLE-US-00001 TABLE 1 Improvement of sugar yield with CotA laccase. Cellulase Cellulase + CotA Theoretical Percentage [mg sugar/ Laccase yield [mg of Lignocellulose gram [mg sugar/ Improvement sugar/gram theoretical material feedstock) gram feedstock) of yield feedstock) yield Steam exploded 433 556 128% 650 85% wheat straw Old Corrugated 83 214 259% 690 31% Cardboard (OCC) Pine Pulp 219 532 243% 750 71% Eucalyptus pulp 586 922 157% 1000 92% Blow Pulp (Softwood) 429 840 196% 970 86% Blow Pulp (Hardwood) 542 950 175% 970 98%