The United States Government has rights in this invention under Contract No. DE-AC02- 83CH 10093 between the United States Department of Energy and the National Renewable Energy Laboratory, a Division of the Midwest Research Institute.

Technical Field

The present invention relates generally to a process for treating a lignocellulosic slurry with a proteinaceous material capable of binding the lignins in the lignocellulosic material and thereby occupying sites that cellulase would normally bind upon addition of cellulase to the lignocellulosic slurry. More specifically, the present invention relates to a process for treatment of a lignocellulosic substrate with a proteinaceous material in order to bind the lignins in the lignocellulosic material and thereby occupy sites that cellulase would normally bind when using cellulase to convert a lignocellulosic biomass to ethanol in a simultaneous saccharification fermentation process. By use of this treatment process, the amount of cellulase available to hydrolyze cellulose to glucose in a process for conversion of lignocellulosic biomass to ethanol will not be lessened.

Background Art

Cellulose is the most widely occurring organic compound on earth, and is essentially composed of repeating subunits of the disaccharide of D-cellobiose, linked by β-(l-4)-glycosidic bonds. Total hydrolysis yields D-glucose, and partial hydrolysis gives the disaccharide cellobiose, which is β-D-glucopyranosyl-β- glucopyranosyl-β-(l-4)-D-glycopyranose. Therefore, cellulose is a β-l,4-glucan. Cellulose constitutes the major storage form of photosynthesized glucose and the major component of solar energy which has been converted to biomass. As worldwide demand for energy and food supplies increases, cellulose has become an attractive raw material. The glucose subunits of cellulose can be used for production of energy or for use in the production of protein.

However, a major impediment to cellulose utilization is the difficulty of obtaining glucose in good yield from ceUulose at reasonable costs in terms of energy input equipment requirements. For example, chemical hydrolysis is attendant by drawbacks of high costs of equipment, high processing costs, low yields, production of complex product mixtures and inability to stop degradation of cellulose at a point which produces primarily the sought product of glucose.

Therefore, enzyme-catalyzed saccharification of cellulose is a more promising alternative to chemical degradation which can achieve a high efficiency conversion of cellulose to glucose.

Enzymatic conversion of cellulose to glucose using cellulase enzymes is superior to chemical dissolution because it proceeds at moderate temperature and pressure, provides recyclable catalysts and frees the environment from undesirable side products associated with chemical hydrolysis.

Cellulase is a complex of enzymes which act cooperatively, or synergistically, in degrading crystalline cellulose. These enzymes are endoglucanase (EC 3.2.1.4), cellobiohydrolase (EC 3.2.1.91) or glucohydrolase, and cellobiase (β-glucosidase EC 3.2.1.21).

However, because cellulase binds to the lignins in lignocellulosic substrates, this lessens the amount of cellulase available to hydrolyze cellulose to glucose in a process for conversion of lignocellulosic biomass to ethanol. Lignins are complex, irregular phenylpropane polymers that represent approximately 20% by weight of the available polymeric content of hardwood tree stems. Wood lignin precursors are the compounds 4-hydroxy-3-methoxycinnamyl (coniferyl) alcohol, 4-hydroxy-3,5-dimethoxycinnamyl (sinapyl) alcohol, and p-hydroxycinnamyl (p-coumaryl) alcohol. The proportions of these alcohols vary with the species, location in the cell wall, and the age of the tree. Hardwood lignins, the group most widely studied to date, are primarily composed of sinapyl and coniferyl alcohol. The major type of bond present in these lignins links the central carbon atom (β -position) of the side chain to the aryl group through an ether (β-O-4-alkyl aryl ether).

Unlike other natural polymers, lignins cannot be degraded to yield structurally intact precursors. The presence of many reactive sites in these molecules results in condensation reactions during hydrolytic treatments used to depolymerize polysaccharide structures often associated with lignins, such as the commonly used dilute acid hydrolysis pretreatment of hardwoods and agricultural residues prior to SSF.

Lignin is known for its ability to interact with a wide variety of chemical compounds, including chromatography supports, materials of containment, and other biomacromolecules, primarily proteins. The mechanism of adsorption is that of classical hydrophobic interaction, via van der Waals type atomic forces. Enzymes having hydrophobic surfaces are therefore especially susceptible to loss on native lignin surfaces.

Cellulases are enzymes that fall into this category, as cellulases have a spear-shaped terminal peptide referred to as the cellulose binding domain (CBD). This peptide is known to have one side, or face, which is very hydrophobic, thus permitting efficient binding of the enzyme to the surface of cellulose (Kraulis et al. 1989. Biochemistry. 28:7241-7257). In this regard, it is interesting to note that although a polysaccharide, the surface of crystalline cellulose is quite hydrophobic (Immergut. 1975, in The Chemistry i2l- ΩΩd, Krieger Publishing: Huntington, NY, pp. 103-190).

During the course of biomass saccharification in SSF, the Klason lignin fraction in pretreated biomass increases from an approximate starting value of 20%, to a final level of about 65% (Tatsumoto et al. 1988. Appl. Biochem. Biotechnol. 18:159-174). This fraction with a high native lignin content is known to adsorb enzymes with hydrophobic surfaces or domains, such as cellulases (Tatsumoto et al. 1988, suprah

Therefore, it would be beneficial to discover low-cost additives, more precisely adsorbents, for addition to the post pretreatment process stream, prior to SSF, that would block the cellulase-lignin binding sites existing in pretreated biomass.

A process for microbial saccharification of a cellulosic substrate is disclosed in U.S. Patent 4,628,029, wherein the saccharification proceeds by inoculating an aqueous nutrient medium with a cellulase- producing microorganism culture. This patent also pretreats the cellulosic substrate by strong acid delignification in order to enhance microbial digestion.

U.S. Patent 3,862,901 discloses purifying waste water containing high molecular weight organic compounds such as protein polypeptides by bringing a waste water solution containing said polypeptides into contact with a sulfite pulp resulting from sulfite digestion of lignocellulosic material.

A process of using a thermophilic bacterial microorganism to digest an aqueous mixture containing lignocellulose is disclosed in U.S. Patent 4,292,328; however, this process attacks the lignin in the lignocellulosic material and frees the cellulose cells for digestion.

U.S. Patent 4,938,972 disclose a process for microbial bioconversion of cereal milling bi-products into proteinaceous material for human consumption by aerobically fermenting these by-products in a culture of the fungus Neurosp ra sitophila. Pretreatment with caustic solution in conjunction with high temperatures partially delignifies the lignocellulose so that the biodegradability of the lignocellulose is increased because of either partial or full removal of lignin.

U.S. Patent 4,957,599 disclose a process for delignifying and bleaching lignocellulosic materials into products that are digestible by ruminants and ingestible by humans.

Accordingly, a need is extant in the art of hydrolyzing lignocellulose to glucose to find a means for pretreatment of a lignocellulosic slurry with a low cost material that will bind to the lignin and occupy the sites that cellulase would ordinary bind when cellulase is added to a lignocellulosic substrate to enzymatically convert cellulose to glucose in the lignocellulosic biomass conversion of a lignocellulosic substrate to ethanol.

Disclosure of Invention

Accordingly, one object of the invention is provide a low-cost additive to inactivate binding sites on native lignin, a major component of biomass, that otherwise binds valuable cellulase enzymes normally used in simultaneous saccharification and fermentation. A further object of the invention is to provide a low-cost proteinaceous material to inactivate binding sites on native lignin, a major component of biomass, that otherwise binds values cellulase enzymes normally used in simultaneous saccharification and fermentation.

A yet further object of the invention is to provide low -cost proteinaceous, non-cellulase ligninases to bind native lignins, a major component of biomass, that otherwise binds valuable cellulase enzymes normally used in simultaneous saccharification and fermentation.

In general, the process of the invention is accomplished by the addition of ligninases blocking agents to the pretreatment pulp stream before delivery to the SSF tanks, or after delivery to the tanks, but before initiation of SSF; however, the characteristics of ligninases (lignin peroxidases) are such that fungal biomass preparation cannot be added to the pretreated wood pulp at temperatures above about 40 °C, nor at pH conditions outside the range of about pH 3 to about 5. The time of exposure of the pretreated biomass to the ligninases must be a minimum of about 30 minutes.

Brief Description of Drawings

Figure 1 depicts a flow chart of the biomass to ethanol process incorporating a lignin blocking agent of the invention.

Detailed Description of Invention

The process for production of ethanol from biomass is based on the dilute acid hydrolysis of hardwoods and agricultural residues, followed by separation of the hydrolysate liquor and pretreated pulp, and finally, the use of simultaneous saccharification and fermentation technology to produce ethanol.

The current scheme for simple production of ethanol by fermentation from biomass has been modified by the innovation of the present invention, in that, a lignin-blocking agent has been added to provide lignin-binding sites after the acid pretreatment of the pulp stream before delivery to the simultaneous saccharification fermentation tanks, or after delivery to the tanks, but before initiation of simultaneous saccharification and fermentation.

The acid hydrolysis step, according to the invention is practiced using an acid concentration of about 2 to about 10% by volume (preferably sulfuric acid), and the hydrolysis temperature may range from about 120°C or less. The residence time in the acid hydrolysis step is from about 1 to about 3 hours, and the treated biomass slurry will have a biomass solids to hquid ratio of about 20/100 to about 40/100 on a volume basis. While it is not critical, it is preferred that the particle size of the biomass range from about 1 to about 4 mm.

Examples of specific fermentation using the process of the invention are illustrated in the examples which follows.

SSF FERMENTATION 8% Cellulose

2% Peptone Media : 5 ml/L Lipids* 2 ml/L Antibiotics*

*Lipids - Stock: 50 mg Tween 80

50 mg Ergosterol

1) Dissolve ergosterol in minimal volume of 95% ethanol (2-3 ml)

2) Mix ergosterol/ethanol solution into 50 gm Tween 80 (0.8% unesterified oleic acid) 3) Evaluate and flush with N ₂ .

Final medium concentration = Ergosterol 5 ml/L, Oleic Acid 30 ml/L

^♦ Antibiotics Penicillin 10 mg/L (16,500 U)

Streptomycin 10 mg/L

Stock: 500 mg/ Pen

(Filter 500 mg/ Strep

Sterilize) 100 ml H ₂ 0

Example 1 Cellulose media is added to a 6 L vessel containing 1 L of water, and the volume is brought up to about 2,500 ml in order to leave enough room for the inocula. The media is mixed in the fermenter and a lipid stock of 5 ml/L of Ergosterol and 30 ml/L of oleic acid is added, after which the mixture is autoclaved at about 120°C for about 35 to 40 minutes. A mixture of antibiotics containing (500 mg of 10 mg/L) of penicillin and 500 mg (of 10 mg/L) of streptomycin is added and the pH is checked to insure that it is between about 4.5 to about 5.0. A 2% by volume solution of sulfuric acid is added to effect acid hydrolysis for a period of 1 to 3 hours. Next, the acid pretreated slurry is lowered to a temperature of about 40°C and the pH is adjusted to a range between about 3 to 5, whereupon the ligninase or lignin peroxidase from Phanerochaete chryxo.ψorium is added in the amount of 0.1 to 1 mg ligninase to 1 gram pretreated wood for at least 30 minutes. Thereafter, the enzyme cellulase is added in sufficient amount to bring the volume up to the 3L mark with sterile H ₂ 0. The enzyme breaks the cellulose down to glucose sugar which is then fermented to ethanol, and thereafter the ethanol is separated from the fermented substrate by well known techniques in the art.

Fig. 1 shows the biomass to ethanol process incorporating the lignin blocking step to block cellulase binding sites on lignin.

While the proteinacous lignin peroxidases (ligninases) are preferred, it should be understood that there is a second class of low cost materials that will suffice equally as well as a lignin blocking agent in the context of this invention. This second lignin blocking agent can be any low molecular weight, aromatic compound of unidentified structure obtained from the hot water, or alcoholic extraction of biomass (i.e., normally referred to as "extractives"). These extractives are utilized in the same way as the lignin- binding enzymes of ligninases, with the exception that, these low molecular weight aromatic compounds of unidentified structure are less temperature sensitive than the ligninase protein - as such, addition of the second class of agents may be made to the acid pretreated pulp at temperatures as high as 100 °C.

As a result of the invention process, large amounts of ethanol can be economically prepared from an almost unlimited supply of source material. The present invention thus provides a highly economic and useful process for fuel production.

The foregoing description is illustrative only of the principles of the invention. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation shown and described and accordingly all suitable modifications and equivalents may be resorted to within the scope of the invention as defined by the claims which follow.

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows: