FIELD OF INVENTION
The present invention is for a novel Clavispora spp. yeast strain, NRRL Y-50464, that produces ethanol. More specifically, the yeast strain is able to utilize cellobiose as a sole carbon source and produce native β-glucosidase enzyme activity under a one-step simultaneous saccharification and fermentation of cellulose to ethanol.
BACKGROUND OF INVENTION
Over 90% of ethanol biofuel produced in the United States is made from corn starch using Saccharomyces strains to ferment the glucose obtained by hydrolysis of the starch. The United States Environmental Protection Agency has revised the Renewable Fuel Standard (RFS) program as required by the Energy Independence and Security Act of 2007 (EISA). The final rule (RFS2) increases the volume requirements for total renewable fuel to 20.5 billion gallons and for cellulosic biofuel to 3.0 billion gallons by 2015. To meet these mandates, it will be necessary to use cellulosic biomass, an abundant and renewable carbon source, as a feedstock. However, the microbial strains used to ferment the glucose released by hydrolysis of starch are not capable of fermenting the more diverse mixture of sugars released by hydrolysis of lignocellulosic biomass.
The necessary deconstruction of cellulosic polymers, enzymatic hydrolysis, and saccharification require additional processing procedures to use lignocellulosic biomass ultimately increases the cost of lignocellulose to ethanol conversion when compared to current starch-to-ethanol technologies. Reducing the cost of cellulosic ethanol production poses significant challenges both in scientific advances and technological development.
One barrier is that yeast strains are generally capable of fermenting the hexose sugars, glucose and galactose; however, they do not naturally ferment the pentose sugars, xylose or arabinose without any genetic modification.
Corncobs are commonly used for xylose production, and xylose-extracted corncob residue (X-ER) is an abundant byproduct after industrial processing (Zhang et al., 2011). The X-ER contains a significant amount of cellulose and is a potential feedstock for cellulosic ethanol production. However, ideal processing procedures and economic cellulosic ethanol production from X-ER have not been achieved yet on a large scale (Zhang et al., 2011). More efficient, lower-cost, and consolidated processing procedures are needed.
Simultaneous saccharification and fermentation (SSF) using cellobiose fermenting yeast Brettanoinyces custersii, is described in U.S. Pat. No. 5,100,791, by Spindler, et al. In a simultaneous saccharification fermentation process, saccharification involves the breakdown of cellulose into simpler sugars by a cellulase enzyme. One such sugar is cellobiose, a sugar comprised of two glucose molecules that is subsequently broken down into glucose. The cellulase enzyme will typically have an insufficient amount of β-glucosidase, which is the part of the cellulase enzyme that can breakdown cellobiose into glucose. Cellobiose inhibits the endo- and exo-glucanase enzymes, and this retards the overall ethanol production rate and yield in a simultaneous saccharification fermentation process.
Since the commonly used ethanologenic yeast Saccharomyces cerevisiae is unable to utilize cellobiose, β-glucosidase is added to digest cellobiose into glucose in order to be utilized by the fermentation ethanologenic yeast.
Enzymes are one of the major costs of cellulosic ethanol production (Piccolo and Bezzo, 2009). In addition, efficient enzymatic saccharification requires a higher temperature while microbial growth and fermentation function optimally at a lower temperature. Furthermore, inhibitory compounds such as representative 2-furaldehyde (furfural) and 5-(hydroxymethyl)-2-furaldehyde (HMF) are often generated during biomass pretreatments such as commonly used dilute acid pretreatment, that interfere with microbial growth and fermentation (Palmquist and Hahn-Hagerdal, 2000; Liu and Blaschek, 2010). These undesirable elements and redundant processing procedures compromise the efficiency of SSF.
There is a need in the art to develop an ethanologenic yeast strain that is tolerant to both a higher temperature and inhibitors commonly encountered in the SSF. This new yeast produces sufficient native β-glucosidase enzyme activity allowing it to grow on cellobiose as sole source of carbon. Thus, no additional β-glucosidase enzyme needs to be added for cellulosic ethanol conversion from X-ER by SSF. Development of this yeast provides potential consolidated bio-processing means for lower-cost cellulosic ethanol production from industrial byproduct of xylose extracted corncobs and other lignocellulosic biomass materials.



BRIEF DESCRIPTION OF THE DRAWINGS
The present invention together with the above and other objects and advantages may best be understood from the following detailed description of the embodiment of the invention illustrated in the drawings.
FIG. 1A is a graph depicting corresponding ethanol production (open symbols) and glucose consumption (filled symbols) for Y-50464 (squares) and Y-417 (triangles). FIG. 1B is a graph depicting growth cell growth on a medium containing 15 mM each of furfural and 5-hydroxymethylfurfural between strain Y-50464 (filled square) and its parental wild type Y-417 (open square).
FIG. 2 is graph depicting the conversion of furfural (filled triangles) into FM (open triangles), and HMF (filled squares) into FDM (open triangles), for tolerant strain Y-50464 (A) and its parental wild type Y-417 (B) showing improved detoxification capability.
FIG. 3 is a graph depicting ethanol conversion from xylose-extracted corncob residues between strain Y-50464 with (open symbols) or without the addition (filled symbols) of β-glucosidase using a simultaneous saccharification and fermentation. Ethanol recovered by HPLC assay is labeled with a triangle and cellobiose residues by a square.
FIG. 4 is a graph depicting ethanol conversion yield (squares) and conversion efficiency (circles) using xylose-extracted corncob residues at 15, 25, and 35% solids loading by strain Y-50464 with (open symbols) and without (filled symbols) the addition of β-glucosidase in simultaneous saccharification and fermentation.
FIGS. 5A and 5B are graphs depicting cellulosic ethanol production by strain Y-50464. FIG. 5A depicts a 25% solid loading of xylose-extracted corncob residues by simultaneous saccharification and fermentation using 2-L bioreactors, while FIG. 5B depicts a 25% solid loading of xylose-extracted corncob. Ethanol is labeled by open squares; cellobiose, filled squares; and glucose, filled circles.



DEPOSIT OF BIOLOGICAL MATERIAL
Strain Y-50464 is identified as a Clavispora yeast based on variable nucleotide tandem repeat (VNTR) analysis. NRRL Y-50464 was deposited on Feb. 10, 2011, under the provisions of the Budapest Treaty in the Agricultural Research Culture Collection (NRRL) in Peoria, Ill.
The subject culture has been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR §1.14 and 35 USC §122. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of the deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.
Further, the subject culture deposit will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., the culture will be stored with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the culture. The depositor acknowledges the duty to replace the deposit should the depository be unable to furnish a sample when requested, due to the condition of the deposit. All restrictions on the availability to the public of the subject culture deposit will be irrevocably removed upon the granting of a patent disclosing them.
DEFINITIONS
In a simultaneous saccharification fermentation process, saccharification involves the breakdown of cellulose into simpler sugars by a cellulase enzyme. One such sugar is cellobiose, a sugar comprised of two glucose molecules that is subsequently broken down into glucose by the enzyme beta-glucosidase. Beta-glucosidase is added to a fermentation batch—provided that the yeast used in the fermentation process cannot endogenously produce beta-glucosidase.
The term “beta-glucosidase” or “β-glucosidase” is defined herein as a beta-D-glucoside glucohydrolase (E.C. 3.2.1.21), which catalyzes the hydrolysis of terminal nonreducing beta-D-glucose residues with the release of beta-D-glucose. For purposes of the present invention, beta-glucosidase activity is determined according to the basic procedure described Grover et al., 1977, Biophysica Acta, 482, 89-108; Saha et al., 2008, J. Biobased Mater. Bioenergy 2, 210-217).
As used herein, one unit of “beta-glucosidase activity” is defined by one unit of enzyme needed to release 1 μmole of p-nitrophenol per min under the defined conditions. Moreover, beta-glucosidase activity was assayed on a 96-well microtiter plate. Briefly, 100 μl of 5 mM p-nitrophenyl β-D-glucoside in 100 mM citrate buffer at pH 5.5 was pipetted in each well. Then 25 μl of crude or purified enzyme prep was added to each sample to start the reaction. The reaction was carried out in an incubator at 45° C. for 30 min. After incubation, 125 μl ice cold 0.5 M Na2CO3 was added to stop each reaction and the absorbance at 405 nm measured using a plate reader Power Wavex 340 (Bio-Tek Instruments Inc., Winooski, Vt.). Other names for beta-glucosidase enzyme activity include: gentiobiase; cellobiase; emulsin; elaterase; aryl-β-glucosidase; β-D-glucosidase; β-glucoside glucohydrolase; arbutinase; amygdalinase; p-nitrophenyl β-glucosidase; primeverosidase; amygdalase; limarase; salicilinase; and β-1,4-glucosidase.
The terms “culturing” or “cultivation” refer to growing a population of microbial cells under suitable conditions in a liquid or solid medium:
The term “xylose-extracted corncob residue” or “X-ER” refers to corncob residue that have been treated via acid hydrolysis to release xylose, cellulose, and lignin. Typically, the residue has been treated with 1.2 to 1.5% H2SO4 at 125° C. to minimize the production of inhibitory compounds such as furan and 5-hydroxymethylfurfural.
Disclosed herein is an isolated Clavispora spp. having been deposited with the United States Department of Agriculture, Agricultural Research Patent Culture Collection as Accession Deposit Number NRRL Y-50464. In one embodiment of the invention, the Clavispora spp. strain NRRL Y-50464 metabolizes cellobiose and produces ethanol. In another embodiment of the invention, Clavispora spp. strain NRRL Y-50464 produces beta-glucosidase under when fermenting cellobiose.
Also disclosed is a method of producing ethanol from the fermentation of cellulosic material, the method comprising fermenting Clavispora spp. strain NRRL Y-50464 with cellulosic material under suitable conditions for a period of time sufficient to allow fermentation of at least a portion of cellulosic material to ethanol. In one embodiment of the invention, β-glucosidase produced by Clavispora spp. strain NRRL Y-50464 and cellulase are added to the cellulosic material simultaneously for simultaneous saccharification and fermentation. In another embodiment of the invention the cellulosic material comprises a lignocellulosic biomass.
In yet another embodiment of the invention, the lignocellulosic biomass is subjected to a pretreatment to increase the accessible surface area of cellulose, prior to said contact with said β-glucosidase and said cellulase. In another embodiment of the invention, pretreatment is selected from the group consisting of treatment with acid, treatment with alkali, ammonia fiber explosion, treatment with an organic solvent, autohydrolysis by steam explosion, acid steam treatment, treatment with hot, compressed liquid water, pressure cooking, milling, grinding, shearing, and extruding. In another embodiment of the invention, the lignocellulosic material is selected from the group consisting of agricultural residues, wood, municipal solid wastes, paper and pulp industry wastes, and herbaceous crops.
Disclosed herein is a method of producing ethanol from the fermentation of cellulosic material, the method comprising fermenting Clavispora spp. strain NRRL Y-50464 with glucose. In one embodiment of the invention, the conversion of said cellulosic material to glucose and the fermentation of glucose to ethanol are conducted simultaneously. In another embodiment of the invention, the conversion of said cellulosic material to glucose and the fermentation of glucose to ethanol are conducted sequentially.
DETAILED DESCRIPTION OF THE INVENTION
Disclosed herein is an isolated Clavispora spp. having been deposited with the United States Department of Agriculture, Agricultural Research Patent Culture Collection as Accession Deposit Number NRRL Y-50464. Strain Y-50464 was derived from a parent strain and the parent strain is designed Y-417 herein. S. cerevisiae NRRL Y-12632 from the ARS Culture Collection was also used as a comparison yeast study. Cell cultures were maintained and pre-cultured on YP media consisting of 50 g glucose, 3.0 g yeast extract, 5.0 g Peptone.
Analysis by high performance liquid chromatography (HPLC) as detailed in the examples was conducted using a Shimadzu LC-20AD (Shimadzu Corporation, Kyoto, Japan) equipped with an HPX-87H Aminex ion exclusion column (Bio-Rad, Hercules, Calif.) kept at 65° C. Sugar consumption and ethanol production were detected using a Shimadzu RID-10A Refractive index detector while furfural and HMF and their conversion products were detected using a Shimadzu SPD-m20A PDA. Samples were run isocratically using 0.0017N H2SO4 as mobile phase at a flow rate of 0.6 mL/min.
Strain Identification
An ethanologenic yeast strain affiliated with sweet sorghum was isolated and examined by sequence of 26S ribosomal RNA gene as previously described by Kurtzman et al., 1998, Anton. Leeuw. 73, 331-371. Based on comparison of NCBI DNA sequence database (www.ncbi.nlm.nih.gov) it was identified as a strain of Clavispora sp. and designated as Y-417. A laboratory adaptation using evolutionary engineering was performed to obtain an inhibitor-tolerant and thermo-tolerant strain that can grow rapidly at 37° C. using procedures similar as previously described in Liu et al., 2005, where pressure and temperature conditions were increase to apply selection pressure. The newly adapted tolerant yeast strain was designated as NRRL Y-50464.
Example 1
Cell Growth of Y-50464 on Cellobiose as Sole Carbon Source
A comparison of growth on cellobiose as sole source of carbon was performed between strains Y-50464 and S. cerevisiae Y-12632. A 100-ml Nalgene culture bottle was filled with 50 ml of YP media amended with 5% cellobiose. Each culture was inoculated with a pre-culture at a starting OD(600 nm) reading of 0.03 and incubated with agitation for 48 h. The lid of Nalgene bottles was kept tight to allow for minimal air exchange. Samples were taken for OD(600 nm) reading over the course of growth. The experiment was carried out in triplicate.
On a medium containing 5% cellobiose as sole carbon source, strain Y-50464 quickly established a culture at 37° C. and the cell growth reached to a stationary phase in no more than 24 h. In contrast, strain Y-12632 of S. cerevisiae at its optimum growth temperature of 30° C., was unable to grow on cellobiose. The minimum background of the OD reading observed was likely attributed to residue C-6 sugars in the medium.
Example 2
Beta-Glucosidase Enzyme Activities
Yeast strains Y-50464 and Y-12632 were grown on 250 ml of YP media with either 5% glucose or 5% cellobiose as a carbon source with a 2% inoculum from an overnight culture. The cultures were incubated with agitation at 225 rpm at 37° C. for strain Y-50464 and 30° C. for strain Y-12632. After 17 h, cells were harvested and lysed using Y-PER plus, dialyzable yeast protein extraction reagent (Thermo Scientific, Rockford, Ill.) following manufacturer''s instructions. The supernatant for each sample was then diluted 1,000 times and used as crude enzyme prep for enzyme assays.
When Y-50464 was grown on cellobiose as sole source of carbon as detailed in Example 1, a large amount of β-glucosidase activity was observed in crude cell protein extracts by in vitro assay (Table 1). This enzyme activity was also observed when Y-50464 was grown on glucose but at a considerably lower level. In contrast, strain Y-12632 of S. cerevisiae produced no detectable β-glucosidase activity induced by either sugar.