WO2013063478A1

WO2013063478A1 - Bioconversion of biomass to ethanol

Info

Publication number: WO2013063478A1
Application number: PCT/US2012/062236
Authority: WO
Inventors: Renata Bura; Shannon EWANICK
Original assignee: Treefree Biomass Solutions, Inc.
Priority date: 2011-10-28
Filing date: 2012-10-26
Publication date: 2013-05-02
Also published as: WO2013063478A4

Abstract

The present invention provides a method for the conversion of a biomass to ethanol that includes increasing the moisture content of the biomass. The invention may be employed with any of a variety of biomasses, such as Arundo donax.

Description

BYCONVERSION OF BIOMASS TO ETHANOL BACKGROUND Technical Field

The present invention relates to the bioconversion of a biomass, such as Arundo donax, to bioethanol through a three stage process comprising hydration of the biomass, steam explosion, and simultaneous saccharification and fermentation.

Description of the Related Art

Following the success of first generation bioethanol made from corn starch and sugar cane, lignocellulosic ethanol can provide a transition to more sustainable fuel production by using non-food feedstocks. Bioconversion of biomass to ethanol is a rapidly growing field of research that encompasses many different methods for the fractionation, saccharification and fermentation of a number of different feedstocks.

Availability of raw biomass varies widely depending on both the source and the time of year, with woody biomass more likely to be harvested year round and agricultural residues and grasses harvested on a seasonal basis (El Bassam, "Energy Plant Species: Their Use and Impact on

Environment and Development" James & James, London, UK, 1998). This leads to issues with storage of seasonal biomass to minimize energy inputs for drying or freezing while preventing microbial contamination.

Steam pretreatment has been proposed as an efficient

pretreatment for lignocellulosic materials owing to its limited use of chemicals, low energy consumption and short reaction time (Chandra et ai, "Substrate Pretreatment: the Key to Effective Enzymatic Hydrolysis of Lignocellulosics?" in Olsson, L. (Ed), Advances in Biochemical Engineering/Biotechnology, Biofuels, Springer Berlin / Heidelberg, pp. 67-93, 2007; Galbe and Zacchi, "Pretreatment of Lignocellulosic Materials for Efficient Bioethanol Production" in Olsson, L. (Ed), Advances in Biochemical Engineering/Biotechnology, Biofuels, Springer Berlin / Heidelberg, pp. 41 -65, 2007). Steam pretreatment (explosion) combines both physical and chemical elements, causing the rupture of the wood cell wall structure, hydrolysis and solubilisation of the biomass components (Ramos and Saddler, Applied Biochemistry and Biotechnology, 45-46:193-207, 1994). Previous work has shown that steam pretreatment can successfully fractionate agricultural, hardwood and softwood biomass, resulting in the recovery of most of the original hemicellulose and cellulose derived sugars in a hydrolysable and fermentable form (Ewanick et ai, Biotechnology and Bioengineering, 98:737- 746, 2007; Ohgren et al., Bioresource Technology, 98:2503-2510, 2007;

Sassner et ai, Enzyme and Microbial Technology, 39:756-762, 2006).

Sulfuric Acid and sulfur dioxide (SO₂) can be utilized as

pretreatment catalysts when necessary. The impregnation of biomass with SO₂ prior to pretreatment has been shown to reduce the reaction time and temperature and consequently reduce the formation of sugar degradation products such as furfurals and hydroxymethyl furfurals (HMFs) (Chum et ai, Applied Biochemistry and Biotechnology, 24-25:1 -14, 1990). In addition, adding SO₂ prior to steam pretreatment not only increases the hydrolysis rate, it reduces the degree of polymerization of the oligomers and increases the proportion of monomers in the water soluble stream (Clark et ai, Journal of Wood Chemistry and Technology, 9:135-166 1989). Although SO₂-catalyzed steam pretreatment has been shown to be effective for the pretreatment of agricultural and woody biomass it is recognized that different pretreatment conditions are required to treat each type of biomass (Ewanick et ai,

Biotechnology and Bioengineering, 98:737-746, 2007; Excoffier et ai,

Biotechnology and Bioengineering, 38:1308-1317, 1991 ; Ohgren et ai, Applied Biochemistry and Biotechnology, 121 -124:1055-1067, 2005).

While a great deal of research has been performed in the last 30 years on the effects of biomass composition, pretreatment and fractionation technology, and improvements in saccharification and fermentation have been made, there has been little focus on the condition of raw materials entering the process and its characteristics, for example, particle size, bark content, and moisture content. In particular, the effect of moisture content on the

bioconversion properties of a biomass is unknown. In addition, improved storage and techniques for preparing the biomass are needed to provide consistent results and maximize the ethanol yield from any given biomass. The present invention addresses this need and provides further related advantages.

BRIEF SUMMARY

The present invention provides a method for the bioconversion of a biomass to ethanol, which includes increasing the moisture content of the biomass. In an embodiment of the methods described herein, the moisture content of the biomass is increased prior to pretreating the biomass with an acid-catalyzed steam explosion to produce a slurry. The pretreated slurry is then subjected to enzymatic hydrolysis and fermentation by a microorganism to produce ethanol. In certain embodiments the moisture content of the biomass is increased by contacting the biomass with water or an aqueous solution. In other embodiments the moisture content of the biomass is increased by steaming the biomass. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Figure 1 is a process flow diagram for bioconversion of raw biomass into ethanol following soaking and SO2 impregnation.

Figure 2 is a bar graph that shows the percent change in glucan, xylan, glucose and xylose in SO2-catalyzed or uncatalyzed pretreated switchgrass (SG) and sugarcane bagasse (SCB) as a result of increased moisture content.

Figures 3A-3C are line graphs that show the cellulose conversion of pretreated switchgrass (SG) and sugarcane bagasse (SCB) to glucose during enzymatic hydrolysis at 5% solids consistency and 10 FPU/g cellulose cellulase loading. SG is shown in Figure 3A, SCB-B (Brazilian) is shown in Figure 3B and SCB-H (Hawaiian) is shown in Figure 3C.

Figure 4 is a bar graph that shows the percent change in hydrolytic glucan conversion and SSF ethanol yield in SO₂-catalyzed or uncatalyzed pretreated switchgrass (SG) and sugarcane bagasse (SCB) as a result of increased moisture content.

Figure 5 is a line graph that shows the effect of the xylan content of pretreated solids on the enzymatic cellulose conversion after 10 h of hydrolysis and 24 h of simultaneous saccharification and fermentation (SSF).

Figures 6A-6C are line graphs that show ethanol yield as a percent of maximum theoretical ethanol yield following simultaneous

saccharification and fermentation (SSF) at 5% solids consistency, 10 FPU/g cellulose cellulase loading, and 5 g/L Saccharomyces cerevisiae of pretreated switchgrass (SG) and sugarcane bagasse (SCB). SG is shown in Figure 6A, SCB-B (Brazilian) is shown in Figure 6B and SCB-H (Hawaiian) is shown in Figure 6C.

Figures 7A-7B are line graphs that show cellulose to glucose (Figure 7A) and xylan to xylose (Figure 7B) conversion during enzymatic hydrolysis of A. donax (giant reed; L, M and H severity pretreatment conditions) and hybrid poplar at 2% (w/v) solids consistency and 20FPU/g of cellulose enzyme loading and IU:FPU of 2:1 .

Figure 8 is a line graph that shows hexose consumption (dotted lines) and ethanol production (solid lines) during fermentation of WSFs of A. donax (giant reed) pretreated at L, M, and H severities and hybrid poplar pretreated at 200°C, 5min and 3% SO₂.

Figure 9 is a line graph that shows hexose consumption (dotted lines) and ethanol production (solid lines) during SSF of A. donax (giant reed) pretreated at L, M, and H severities and hybrid poplar pretreated at 200°C, 5min and 3% SO₂ at 20 FPU / g cellulose enzymes loadings of the combined WSF and WIFs at 8% (w/v) consistency solids and IU:FPU ratio of 2:1 .

DETAILED DESCRIPTION

The present disclosure relates to the surprising discovery that the moisture content of a biomass affects the ethanol yield. In particular, increasing the moisture content, or hydrating, a biomass prior to acid-catalyzed steam pretreatment and simultaneous saccharification and fermentation (SSF) results in an increased yield of ethanol in comparison to the ethanol yield obtained from a dry biomass.

One embodiment of the invention pertains to soaking or submerging the biomass in liquid, such as water, in order to increase the moisture content prior to ethanol production. Another embodiment of the invention encompasses steaming the biomass to increase the moisture content prior to ethanol production. The methods described herein can be applied to any biomass. In more specific embodiments, the biomass is Arundo donax.

As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise.

The term "about" when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary between 1 % and 15% of the stated number or numerical range.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or

"comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

The term "bioethanol" is used interchangeably herein with "ethanol" and refers to ethanol generated from the bioconversion of plant matter. As used herein, the term "bioconversion" refers to the process of producing ethanol from a biomass.

The term "biomass", as used herein, refers to plant matter for use in the production of ethanol. The biomass may include a part or a piece of any of a variety of plant species. In certain embodiments, the biomass comprises plant material from more than one species of plants. In particular

embodiments, the biomass has been dried for transport and/or storage. The term "dry biomass" refers to a biomass in which the moisture content has not been increased. Accordingly, a dry biomass is not necessarily one that has been actively dried, e.g., in an oven. In certain embodiments, a dry biomass may have 5% to 25% moisture.

The terms "hydration", "hydrate" and "hydrating", as used herein, refer to an increase in and/or increasing the moisture content of a biomass. A biomass may be hydrated by, e.g., soaking or steaming.

The term "slurry", as used herein, refers to the liquid and solid components of the biomass generated from pretreatment of the biomass.

Any range described herein is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

As disclosed herein, the method for the bioconversion of a biomass includes a hydrating step prior to the bioconversion process, e.g., steam pretreatment, hydrolysis and fermentation. Modifying the Moisture Content of a Biomass

Little is known about what effect the condition {e.g., particle size, bark content and moisture content) of the biomass entering the bioconversion process has on the ethanol yield. Whereas moisture content can be relatively easily controlled and modified, other physical characteristics are often dictated by the equipment available for particle size reduction and debarking. As described herein, control and alteration of moisture content represents a means of increasing ethanol yields without modification of existing infrastructure.

The moisture content of a biomass can be modified by, for example, soaking or steaming the biomass. The biomass can be soaked in a liquid, such as water, for a period of time to increase the moisture content. For example, the biomass can be soaked overnight. In particular embodiments, the biomass can be soaked for about 6, about 8, about 10, about 12, about 16, about 20, about 24, about 36, or more hours. The biomass can be soaked in any variety of rigid {e.g., tank or reservoir) or flexible {e.g., plastic bag) container. In one embodiment, the biomass is completely submerged in the liquid. In another embodiment, the biomass is partially submerged in the liquid.

Steaming a biomass can be performed in a variety of ways, including, for example, using a steam gun. Using a steam gun to increase the moisture content of the biomass can be achieved in significantly less time than soaking the biomass. For example, a biomass may be steamed for about 15, about 30, about 45, about 60, about 75, about 90, about 120 or more seconds. In certain embodiments, steaming is the preferred method of increasing the moisture content of a biomass.

The moisture content of a biomass is calculated by dividing the weight of dry biomass by the weight of the wet biomass and multiplying by 100. The moisture content of a biomass may be increased by at least about 10%, 20%, 30%, 40%, 50%, or 60%. After hydration, the biomass may have 10%- 60% moisture. Pretreatment

After the moisture content of the biomass has been modified {e.g., by soaking or steaming), the hydrated biomass is subjected to steam explosion pretreatment. Steam pretreatment (i.e., steam explosion) is often chosen due to its ability to fractionate a wide variety of biomass types, from softwood to hardwood to agricultural residues (Bura et al., Biotechnol Prog, 25:315-322, 2009; Carrasco et al., Enzyme Microb Tecnhol, 46:64-73, 2010; Ewanick et al., Biotechnol Bioeng, 98:737-746, 2007). Biomass can be added to the reactor in a wet state, and the reaction conditions remain the same with minimal extra time required to heat in comparison to a dry biomass. Steam explosion can be performed using, e.g., a steam gun.

It has been shown that by employing a low pretreatment severity, defined by the severity factor Ro {Ro=t^*exp (T-100)/14.75} which links the effects of time (t, min) and temperature (T, °C) (Overend and Chornet, Phil. Trans. R. Soc. Lond, 321 :523-5361987), only limited chemical and physical changes occur in the pretreated biomass (Bura et al., Applied Biochemistry and Biotechnology, 105:319-335, 2003; Ohgren et al., Applied Biochemistry and Biotechnology, 121 -124:1055-1067, 2005; Sassner et al., Enzyme and

Microbial Technology, 39:756-762, 2006; Stenberg et al., Journal of Chemical Technology and Biotechnology, 71 :299-308, 1998). As the severity of pretreatment increases, first the hydrolysis of hemicellulose occurs, releasing oligomeric and monomeric sugars. Eventually, as the severity is raised, the cellulose will begin to hydrolyze. As sugars are hydrolyzed during the steam pretreatment process, there is a concomitant increase in the concentration of lignin in the solid fraction (Bura et al., Applied Biochemistry and Biotechnology, 105:319-335, 2003).

In particular embodiments, the temperature applied to the biomass during steam pretreatment is from about 150 to about 230°C. In certain embodiments, the temperature is about 190 to about 210°C. In specific embodiments, the duration of steam pretreatment is from about 1 to about 9 minutes. In particular embodiments, the duration of steam pretreatment is about 3 to about 5 minutes.

In various embodiments described herein, the steam pretreatment is acid-catalyzed. The acids that can be used include, but are not limited to, sulfuric acid (H₂SO₄) and sulfur dioxide (SO₂). For pretreatment of many biomass types such as softwoods and hardwoods, sulfur dioxide impregnation is necessary prior to steam pretreatment to improve hydrolyzability of solids and hemicelluloses removal (Bura et ai, Biotechnol Prog, 25:315-322, 2009;

Ewanick et ai, Biotechnol Bioeng, 98:737-746, 2007).

In one embodiment, gaseous SO₂ is added to the biomass in a closed container {e.g., a plastic bag or a container with a lid). The biomass is impregnated with the SO₂ for a period of time, e.g., overnight. In certain embodiments, the biomass is impregnated with SO₂ for about 4, about 6, about 8, about 10, about 12, about 16, about 20, about 24, or more hours. In specific embodiments, the concentration of SO₂ is from about 1 % to about 6%. In certain embodiments, the concentration of SO₂ is about 3%.

As a gas, SO₂ diffuses more rapidly in water than in air.

Accordingly, and without wishing to be bound by theory, SO₂ uptake and effectiveness may be improved by saturating biomass void volumes with water. Reduced chemical permeability in dried biomass may also be explained by hornification, the fusing of cellulose fibrils upon removal of water from less ordered and more swollen areas (Krassig, Cellulose: Structure, Accessibility and Reactivity, Gordon & Breach, Yverdon, Switzerland, 1993). The result of hornification is a decrease in free surface area, and interstitial areas through which diffusion can occur. While hornification is said to be irreversible (Krassig, Cellulose: Structure, Accessibility and Reactivity, Gordon & Breach, Yverdon, Switzerland, 1993), it is demonstrated herein that re-wetting dried fibers can restore at least some of the permeability. Hydrolysis and Fermentation

Enzymatic hydrolysis (i.e., saccharification) of the slurry resulting from steam pretreatment breaks down the plant polymers (e.g., cellulose and hemicelluose) into sugars (e.g., glucose and xylose). Hydrolysis can be performed by adding one or more enzymes to the pretreated biomass.

Enzymes that can be used include, for example, cellulase, β-glucosidase, gluco-amylase, and a-amylase.

The sugars obtained from both the liquid and solid fractions of the slurry are then fermented by a microorganism to produce ethanol. Various strains of bacteria and yeast that can be used in the fermentation step include, for example, Clostridium sporogenes, C. indolis, C. sphenoides, C. sordelli, Zymomonas mobilis, Spirochaeta aurantia, S. stenostrepta, S. Iitoralis, Erwinia amylovora, Leuconostoc mesenteroides, Streptococcus lactis, Sarcina ventricula, and Saccharomyces cerevisiae. In preferred embodiments, the microorganism is S. cerevisiae.

The hydrolysis and fermentation steps can be performed separately, but in Simultaneous Saccharification and Fermentation (SSF), the two steps are performed at the same time. SSF reduces the cost and complexity of the hydrolysis and fermentation procedure. The microorganism is able to ferment the sugar molecules as they become available during hydrolysis. In certain preferred embodiments, the production of ethanol is via SSF.

The amount of ethanol produced from the biomass may be determined using, e.g., high-performance liquid chromatography (HPLC). In certain embodiments, increasing the moisture content of the biomass increases the ethanol yield by at least about 10%, 20%, or 30% in comparison to a biomass in which the moisture content was not modified. Biomass

The types of biomass that may be used in the disclosed methods include, e.g., softwood, hardwood, agricultural, and grass. Examples of biomass include, but are not limited to, switchgrass, sugarcane bagasse, corn fibers, corn stover, Arundo donax, napiergrass, bermudagrass, lodgepole pine, hybrid poplar, wheat straw, rice straw, maple, spruce pine, and Douglas fir. In certain embodiments, the biomass is stored for a period of days, weeks, or months prior to entering the bioconversion process. In particular embodiments, the biomass comprises a single type of plant species, while in other

embodiments, the biomass comprises a mixture of two or more different plant species.

In specific embodiments, the biomass comprises A. donax. A perennial rhizomatous grass, giant reed (Arundo donax L.) is a promising source of biomass for energy production. A. donax is a tall, perennial C3 grass and it is one of the largest of the herbaceous grasses (Lewandowski et al., Biomass and Bioenergy, 25:335-361 , 2003). Giant reed native from East Asia is widely diffused in Mediterranean environment where it is frequently found in riparian habitats. Usually it does not set fruit because the pollen results unfruitful; consequently, the better propagation method, for this species, is the use of rhizomes (Angelini et ai, Biomass and Bioenergy, 33:635-643, 2009). Throughout the United States, from northern California to Maryland A. donax is an invasive weed, growing in water and is classified as an emergent aquatic plant (Bell, "Ecology and Management of Arundo donax, and Approaches to Riparian Habitat Restoration in Southern California" in J.H, B., M, W., P, P. and D, G. (Eds), Plant Invasions: Studies from North America and Europe, pp. 103- 13, 1997). Currently, in Europe this species has been identified as one of the most promising for energy production for the Southern areas of Europe

(Angelini et al., Biomass and Bioenergy, 33:635-643, 2009; Lewandowski et al., Biomass and Bioenergy, 25:335-361 , 2003). Its high biomass productivity has been observed while also reducing crop inputs, such as fertilization and plant density, and this high yield is furthermore stable over the long-term (Angelini et ai, European Journal of Agronomy, 22:375-389, 2005). A. donax has high biomass productivity such as 37.7 t/ha/year (Angelini et ai, Biomass and Bioenergy, 33:635-643, 2009). Other yields reported in Spain showed 45.9 t/ha/year (Lewandowski et ai, Biomass and Bioenergy, 25:335-361 , 2003). Although there is much interest in utilizing A. donax for pulp and paper production not much research has been done in converting A. donax to bioethanol.

EXAMPLES

An example of the bioconversion process sequence is shown in Figure 1 . Unless specified otherwise, the composition of the biomass and products generated in the Examples was analyzed according to the methods below.

HPLC

Carbohydrates were measured by pulsed amperometric electrochemical detection on a Dionex ICS 3000 HPLC. The method used a flow rate of 1 ml/min and mobile phase of deionized water for the first 30 min followed by 10 min of 0.2 M NaOH, followed by 10 min of deionized water. Samples were diluted as appropriate, spiked with fucose as an internal standard and filtered through 0.22 μιτι syringe filters. 10 μί of sample were injected onto the column, a Dionex Carbopac PA1 fitted with a guard column. After separation of the injected sample on the column, 0.2 M NaOH was added to a T-junction at 0.5 ml/min using a post-column AXP pump and mixed with the sample prior to electrochemical detection. Samples were measured against standards consisting of arabinose, galactose, glucose, xylose, and mannose.

Ethanol, glycerol, acetic acid, and furfurals were measured using refractive index detection on a Shimadzu Prominence LC. Samples were diluted as appropriate, filtered through 0.22 μηη syringe filters and 20 μΙ_ of sample were injected run on a Phenomenex Rezex RHM H⁺ column at 63 °C with an isocratic mobile phase elution of 0.05 mM H₂SO₄. Standards were prepared and used to quantify the unknown samples. Ash

Ash content of raw biomass samples was measured gravimetrically by heating 20-mesh-milled dry biomass to 550 °C for 20 h (Sluiter et al., Determination of Ash in Biomass, Golden, CO: NREL/TP-510- 42622, 2008). Insoluble Carbohydrates and Lignin

Solids were analyzed gravimetrically for lignin content,

photometrically for soluble lignin, and by HPLC for carbohydrate content using the TAPPI method T-222 om-98 (TAPPI Standard Methods, T-222 om-98. TAPPI Journal, TAPPI Press, 1998). Briefly, 0.2 g of 40-mesh ground oven dried sample was mixed with 3 ml of 72% H₂SO₄ for 120 min, diluted with water to 120 ml total volume, and autoclaved at 121 °C for 60 min. The samples were then filtered through tared glass fritted crucibles which were oven dried and weighed to determine acid insoluble lignin. Since the acid insoluble material included ash, the ash content was subtracted from the total acid insoluble lignin amount. The filtrate was analyzed by HPLC for carbohydrate composition and by UV at 205 nm for acid-soluble lignin content.

Soluble Carbohydrates

Monomeric and oligomeric soluble carbohydrates were determined using NREL LAP TP-510-42623 (Sluiter et al., Determination of Sugars, Byproducts, and Degradation Products in Liquid Fraction Process Samples. Golden, CO: NREL/TP-510-42623 2004). Briefly, samples were diluted by half and 72% H₂SO added to reach a pH of 0.07. These samples were then autoclaved at 121 °C for 60 min to determine the total sugar concentration. Monomeric sugars were determined by analyzing the original samples by HPLC without acid hydrolysis. Oligomeric sugar was calculated by subtracting monomeric sugar content from total sugar content.

EXAMPLE 1

PRODUCTION OF BIOETHANOL FROM SOAKED AND UNSOAKED BIOMASS

The moisture content of two types of biomass was altered by soaking the material in water to determine the implications for subsequent enzymatic hydrolysability and overall ethanol yield. In particular, this study aimed to assess whether increasing the moisture content of the biomass prior to impregnation and subsequent pretreatment could stimulate an increase in overall ethanol yield. It is well known that the hydrolysability of some types of biomass is improved by addition of SO2 (Bura et al., Biotechnol. Prog. 25:315- 322, 2009); however, it was not known whether similar improvements can be achieved by altering the moisture content. Switchgrass and two types of sugarcane bagasse were used to determine if any observed effects were consistent across different types of biomass.

Pretreatment and Processing Conditions

Three types of biomass were used. Air dried switchgrass was kindly provided by Weyerhaeuser. Air dried and washed sugarcane bagasse from Hawaii and Brazil was provided by Novozymes, Inc. Both switchgrass and bagasse arrived cut to 1-2 inches in length, 1/8-1/4 inches in diameter.

Prior to pretreatment, half of each type of biomass was submerged in water for 48 h. Each was then vacuum filtered to remove as much excess water as possible and the moisture content calculated and presented in Table 1 . Gaseous sulfur dioxide (3% w/w) was added by weight to half of the soaked biomass and half of the unsoaked biomass based on the dry weight of the material. Specifically, for 200 g of dry biomass, 6 g of SO₂ was added by weight from a cylinder of gas to a plastic bag containing the biomass. 200 g dry weight of each type of biomass were impregnated, and both the catalyzed and uncatalyzed material were divided into 50 g portions which were pretreated sequentially using a 1 .5 L batch steam gun (HM3 Energy, Inc., Gresham OR) at the time and temperature shown in Table 1 . After the specified reaction time had elapsed for each portion of biomass, a pneumatic valve was opened between the pressurized reaction vessel and the collection vessel, blowing the pretreated slurry into the collection vessel. After all 4 shots had been discharged; the slurry of material was collected by opening a valve at the bottom of the collection vessel and allowing the material to drain into a bucket.

Table 1 . Biomass moisture content prior to pretreatment and subsequent pretreatment conditions for switchgrass and sugarcane bagasse

Moisture content (%) Pretreatment conditions

Temperature Time (min)

Initial Soaked

(°C)

SG 9 80 195 7.5

SCB-B 13 79 205 10

SCB-H 9 79 205 10

The liquid and solid fractions were separated from the slurry by vacuum filtration, analyzed as described below, and used to construct a complete mass balance of carbohydrates and lignin. Solids were water-washed (with water equal to ten times the mass of solids) prior to analysis and saccharification. Saccharification

Enzymatic hydrolysis of washed solids was done at 5% w/v solids in a total volume of 50 ml in 125 ml Erlenmeyer flasks. The solution was buffered at pH 4.8 with 0.05 M sodium acetate buffer and the hydrolysis was completed at 50 °C and 150 rpm shaking on an orbital shaking incubator (New Brunswick). Cellulase (Spezyme- CP, 26 FPU/ml, Sigma) was added at 10 FPU/g cellulose and supplemental beta-glucosidase (Novozym 188, 492 CBU/ml, Sigma) was added at 20 CBU/g cellulose. 1 ml samples were periodically removed and analyzed for glucose and xylose.

Simultaneous Saccharification and Fermentation (SSF)

Saccharomyces cerevisiae ATCC 96581 isolated from spent sulfite liquor (Linden et ai, Appl. Environ. Microbiol. 58:1661 -1669, 1992)

(obtained from ATCC) was streaked onto YPD agar plates and allowed to grow for 48 h. Prior to fermentation, preculture cells were grown by adding one colony from the plate to liquid media containing 10 g/L each of glucose, yeast extract and peptone. After 24 h of growth at 30 °C and 150 rpm shaking, the cells were centrifuged and the spent supernatant removed and replaced with fresh media. The cells were then grown for another 24 h under the same conditions; the cells were again spun down, washed twice in water, and then resuspended in a small volume of 0.9% sodium chloride. Cell concentration was determined by measuring the optical density of the suspension at 600 nm and comparing to a calibration curve prepared using oven dried cells at different optical densities.

SSF was carried out at 5% w/v washed, never dried solids, 5 g/L yeast, and enzyme loading of 10 FPU/g cellulose and 20 CBU/g cellulose. The total reaction volume was 50 ml in 125 ml Erlenmeyer flasks. Ammonium phosphate (2 g/L), sodium phosphate (0.2 g/L) and sodium nitrate (2 g/L) were added to each flask. Prior to mixing with the solids, the pretreated liquid stream was adjusted to pH 5.5 with 10% NaOH. The pH-adjusted liquid stream was added to each flask along with yeast, enzymes, and nutrients such that the final volume including the moisture in the pretreated solids was 50 ml. Flasks were incubated at 37 °C with 150 rpm orbital shaking. 1 ml samples were removed periodically for ethanol and glucose analysis. Compositional Analysis Raw Biomass

Switchgrass (SG) and two subtypes of sugar cane bagasse (SCB- B and SCB-H) were chosen for their similar composition (Table 2) and for their potential use as sustainable bioethanol feedstocks. The relatively similar composition was important, as it enabled any differences in characteristics after pretreatment to be seen as a result of the pretreatment or characteristics of the biomass rather than chemical composition. Switchgrass and bagasse differ in terms of their level of pre-processing; switchgrass is either dried in the field or harvested and then dried (El Bassam, Energy Plant Species: Their Use and Impact on Environment and Development. James & James, London, UK, 1998). Sugar cane is harvested, mechanically pressed to extract sucrose, and the remaining fiber, the bagasse, can be dried for transport or used immediately (El Bassam, Energy Plant Species: Their Use and Impact on Environment and Development. James & James, London, UK, 1998).

Table 2. Composition of raw switchgrass (SG) and sugarcane bagasse (SCB) in % of total biomass analyzed prior to pretreatment

Lignin (%)

SG 2.8 0.9 35.2 21 .7 0.2 3.7 24.1 3.3

SCB-

1 .8 0.5 41 .3 21 .8 0.3 4.1 20.5 2.9 B

SCB-

1 .4 0.3 40.5 21 .9 0.3 1 .4 23.6 2.9 H

The total polysaccharide content of all of the biomass proved to be very high (61-66%) with only 23-27% lignin, making both the switchgrass and sugar cane bagasse attractive material for saccharification and

fermentation processes. The composition of the biomass was similar to compositions observed by other investigators (Carrasco et ai, Enzyme Microb. Technol. 46:64-73, 2010; Jensen et ai, Bioresour. Technol. 101 :2317-2325, 2010). Glucan was shown to be the most abundant component in the

feedstocks as determined by secondary acid hydrolysis of constituent polysaccharides with the remainder of the biomass composed of 35-41 % lignin, 22% xylan, 1 .5-3% arabinan and minor amounts of galactan and mannan. As glucose and xylose made up the majority of carbohydrates in the raw material, only their behavior was reported in subsequent analysis.

Solids and Liquid Composition and Sugar Recovery After Pretreatment Switchgrass and sugarcane bagasse, while similar in chemical composition, differed in their response to steam pretreatment. Initial

experiments (data not shown) showed that more severe conditions were required to pretreat the bagasse samples to an acceptable level of enzymatic digestibility (10 min at 205 °C for sugar cane bagasse compared to 195 °C for 7.5 min for switchgrass). As a result, there were significant differences between the two feedstocks in the amount of sugar remaining in solids and liquids after pretreatment (sugar recovery), enzymatic cellulose conversion of the solids (digestibility) and subsequent overall ethanol yield after simultaneous

saccharification and fermentation (SSF).

Following pretreatment and liquid-solid separation of all 12 samples, the compositions of the solid, water-insoluble fraction and the liquid, water-soluble fraction, were analyzed. The glucan content of the resulting solids for all samples was between 45.8 and 51 .9 g glucan/100 g pretreated solids (Table 3). Along with lignin, this comprised at least 80% of the pretreated solids with the majority of the hemicellulosic sugars solubilized into the liquid fraction. Xylan content in all of the samples was low; only uncatalyzed switchgrass contained more than 5% xylan (1 1-13%). Both presoaking and the addition of SO2 had a significant impact on the composition of the resulting biomass (Table 3). Simply adding SO2 to dry biomass had the expected result with a large decrease in xylan, a small decrease in glucan and resulting increase in lignin. However, by soaking prior to adding SO₂, the effect of SO₂ was enhanced; xylan was further reduced in the solids with minimal change to glucan (Figure 2, Table 3). In the liquid, soluble glucose was increased by 56-170% in samples treated with SO2 following soaking compared to dry samples (Figure 2,

Table 3).

Table 3. Composition of pretreated switchgrass (SG) and sugarcane bagasse (SCB) determined by gravimetric analysis of solids and acid hydrolysis and analysis of liquids

1 «

% oligomeric" describes the percentage (w/v) of soluble sugar not present in monomeric form.

² 5-hydroxymethyl furfural

The pretreatment conditions were chosen to be severe enough to produce a solid substrate that could be relatively quickly and completely hydrolyzed but not so severe that the sugars in the starting material were degraded. As glucose recovery was over 80% in the two bagasse samples, and over 96% in the switchgrass samples (Table 4), it indicated that the

pretreatment conditions were adequate and not overly severe.

Table 4. Overall carbohydrate recovery as a percentage of each component present in the original material following pretreatment of switchgrass (SG) and

Furfural and 5-hydroxymethyl furfural (HMF), degradation products of pentoses and hexoses respectively, were present in low

concentrations (Table 3). Furfural was below 2.6 and HMF was less than 0.7 g/100 g original biomass in all twelve samples.

These relatively low levels of furan formation are a result of minimal sugar degradation during pretreatment. Minimizing their formation serves to improve sugar yields and increase potential ethanol yield by reducing microbial inhibition during fermentation. Carrasco et al. (Enzyme Microb.

Technol. 46:64-73, 2010) pretreated sugar cane bagasse using steam

pretreatment under similar conditions on high moisture (75-77%) biomass with and without SO2, with the major difference being almost twice as much SO2 added to the biomass. Despite this additional SO2, pretreated liquid

hydrolysates in this study had less than 0.7 dry material of furfural and nearly no HMF present. Comparable samples in this study contained at least 3 times as much furfural and 0.7 g/100 g of HMF, a result of either a lack of further degradation to formic and levulinc acids or different pretreatment equipment. Hydrolysis

Following pretreatment, all 12 samples were enzymatically hydrolyzed at 5% consistency and 10 FPU/g cellulose cellulase enzyme loading (Figure 3). The extent of cellulose conversion highlighted the differences in digestibility between soaked and unsoaked, and catalyzed and uncatalyzed. In all of the samples, the maximum cellulose conversion after 10 h was 92-94%, for all three soaked and SO2 catalyzed substrates. For each of the three feedstocks, soaking improved the extent of conversion after 10 h by 10-19% for SO2-catalyzed samples, whereas soaking reduced digestibility in uncatalyzed samples by 1-9% (Figure 4). In switchgrass, the addition of SO₂ to dry biomass increased the hydrolysis conversion by 43%, while soaking prior to SO2 addition increased the yield by 82% over the soaked, uncatalyzed sample. Similarly, the two bagasse samples showed a 2% increase in conversion for Brazilian and 28% increase for Hawaiian bagasse following the addition of SO2 to dry biomass. A far more substantial increase in conversion was generated by adding SO2 to soaked biomass; the glucose yield increased by 22-42% for Brazilian and Hawaiian, respectively.

The increase in glucan conversion in soaked, SO2-catalyzed samples appears to correlate to the increased reduction in xylan content in these samples (Figure 5). Conversely, as xylan content in the pretreated solids increases, the extent of glucan conversion decreases. The relationship between these two variables has an R² value of 0.781 , indicating a strong correlation. While xylan removal has been previously shown to improve the cellulose digestibility (Bura et ai, Biotechnol. Prog. 25:315-322, 2009), many other factors influence hydrolyzability, including lignin content, particle size, available surface area and cellulose crystal I in ity. With so many variables it is difficult to determine the extent of the role that xylan plays (Chandra et ai, Biotechnol. Prog. 24:1 178-1 185, 2008; Mansfield et ai, Biotechnol. Prog. 15:804-816, 1999), but it is possible that increased moisture content allows better penetration of SO₂ into the cell wall. Xylan present in cell wall hemicelluose is susceptible to acid hydrolysis and is solubilized by the SO₂ and other acids formed during steam pretreatment (Bura et ai, Biotechnol. Prog. 25:315-322, 2009). Removal of xylan from the cellulose matrix increases cellulose accessibility and subsequently improves enzymatic saccharification. Simultaneous Saccharification and Fermentation

SSF was carried out using the same enzyme loading and solids consistency as for enzymatic hydrolysis with the addition of the pH-adjusted pretreated liquid stream and 5 g/L of S. cerevisiae. Only hexoses were utilized by this organism, and since galactose and mannose made up 4% or less of the six-carbon sugars present in the reaction, only glucose was measured. The production of ethanol and consumption of glucose were analyzed over time and compared after 24 h of saccharification and fermentation.

As shown in Figure 6, soaking prior to adding SO₂ produced the highest yield of ethanol, while adding SO₂ to dry biomass generated higher yields than uncatalyzed samples. Switchgrass showed the greatest increase in ethanol production, with over twice as much ethanol produced after soaking and SO₂ than soaking without SO₂. This result is likely a result of the reduced enzymatic digestibility of the unsoaked samples (Figure 3) rather than a difference in fermentability of the liquid stream; analysis of the liquid stream (Table 3) showed that levels of 5-hydroxymethyl furfural and furfural were below levels shown to be inhibitory to S. cerevisiae (Keating et al., Biotechnol. Bioeng. 93:1 196-1206, 2006). In all samples, glucose was consumed after 6 h (Figure 6) while the small amount of galactose and mannose present took up to 24 h to be consumed (data not shown).

Linear regression of the relationship between xylan content in the pretreated solids and the ethanol yield following SSF had a slope very close to the plot of hydrolysis glucan conversion, with an R² value of 0.869 (Figure 5). This indicated that the difference in ethanol yield between all of the samples was due to the same factors that affect hydrolytic conversion, likely the extent of xylan removal.

Overall ethanol yields were calculated based on the sugar recovery following pretreatment and the amount of ethanol produced after 24 h of SSF of the pretreated material. Not surprisingly, based on the above results, overall ethanol yields for all three feedstocks were higher for SO2-catalyzed than uncatalyzed. For SO2-catalyzed samples, overall yields were as much as 28% higher for soaked biomass than for dry. In uncatalyzed samples, soaking had a negligible or negative effect on overall ethanol yield (Table 5). The highest ethanol yield, from Brazilian sugarcane bagasse was 52 gallons per ton of raw biomass, followed by 51 gal/ton for Hawaiian bagasse and 45 gal/ton for switchgrass. Since only hexoses were utilized, these yields are lower than if both pentoses and hexoses were used. The maximum yields for each biomass, assuming 100% recovery of sugars after pretreatment and full conversion of only hexoses to ethanol is 63, 72, and 71 gal ethanol/ton for switchgrass, Brazilian bagasse, and Hawaiian bagasse, respectively. The highest ethanol yield for each feedstock is therefore 71 % of the maximum possible. Table 5. Theoretical ethanol yields from raw biomass following pretreatment of switch rass SG and su arcane ba asse SCB followin 24 h of SSF

The surprising increase in overall ethanol yield brought on by increasing the moisture content of SO₂-catalyzed biomass is hypothesized to be due to an increase in permeability, allowing improved penetration of SO₂.

Soaking alone did not produce an increase in ethanol yield, so it seems that the improved yields are due to increased efficacy of the added SO₂. The lower ethanol yield of dried biomass compared to soaked is thought to be due at least partially to hornification of the biomass during drying preventing thorough uptake of SO₂. The cause of hornification is not well understood, but the effects are reduced pore size and surface area (Diniz et ai, Wood Sci. Technol.

37:489-494, 2004), both of which could be responsible for reduced efficacy of pretreatment and subsequent reduced enzymatic hydrolyzability. The most established definition of hornification is that cellulose fibrils are brought closer together upon drying and crossl inked by formation of hydrogen bonds (Jayme, Cellulosechemie, 21 :73-86, 1943). Suchy et al. (Biomacromolecules, 1 1 :515- 520, 2010) propose an alternative mechanism for hornification to explain ultrastructural rearrangements seen after drying, an irreversible stiffening of the hemicellulose-lignin matrix in regions that typically swell when exposed to water (Suchy et al., Biomacromolecules, 1 1 :515-520, 2010). Hornification has been well studied on pulp fibers, but is less understood with regard to a whole biomass. It is thought to be less likely that untreated biomass (such as the raw switchgrass and bagasse used in this study) would experience cellulose microfibril aggregation due the substantial presence of lignin and hemicellulose (Suchy et al., Biomacromolecules, 1 1 :515-520, 2010), and would experience only low level hornification and stiffening of the hemicellulose-lignin matrix. This would still allow penetration of water into the cell walls and improve the transfer of SO₂ throughout the biomass, thus increasing hemicelluose solubilization and improving enzymatic hydrolysis. Full reversal of hornification requires harsh treatments such as beating, addition of bulking agents, or derivitization (Diniz et ai, Wood Sci. Technol. 37:489-494, 2004). As such, increasing moisture content is likely not fully reversing hornification, but improving the passage of SO₂ through the biomass.

By increasing the moisture content of biomass prior to SO₂- catalysis and steam pretreatment, the yield of ethanol was increased by over 25%. Improved solids digestibility also represents a potential cost savings in that reduced enzyme loadings are required for the same ethanol yield.

In summary, the moisture content of biomass at the time of SO₂ impregnation and subsequent steam pretreatment has a major impact on the final ethanol yield, with water soaked, SO₂-catalyzed biomass providing an 18- 28% increase in the amount of ethanol produced after SSF. These higher ethanol yields are thought to be due to improved efficacy of SO₂ catalysis.

Enhanced SO₂ catalysis efficacy results in increased xylan removal, leading to increased cellulose accessibility and eventual hydrolyzability of the pretreated biomass. These results can reduce costs at the industrial scale by reducing enzyme loadings and improving ethanol yields. EXAMPLE 2

PRODUCTION OF BIOETHANOL FROM ARUNDO DONAX

Little is known about the conversion efficiency of A. donax to ethanol via pretreatment, hydrolysis and fermentation processes. In order to determine pretreatment conditions for A. donax that are severe enough to produce a solid substrate that can be relatively quickly and completely hydrolyzed but not so severe that the sugars in the starting material are degraded, different steam pretreatment conditions were tested, and the hydrolyzability and fermentability of steam pretreated A. donax was compared with hybrid poplar.

Raw Material

A. donax was provided by TreeFree Biomass Solutions, Inc. and hybrid poplar was supplied by HM3 Company. The moisture contents of A. donax and hybrid poplar were -33 and 64% (w/w), respectively. A. donax (the whole plant, including leaves) was chipped, and screened to approximately 2 x 2 x 0.5cm³. The debarked hybrid poplar chips were screened to approximately 2 x 2 x 0.5cm³. The biomass was stored at -4°C.

Pretreatment

The conditions for steam pretreatment for A donax and hybrid polar are shown in Table 6. The names for each pretreatment condition for A donax represent severity of the pretreatment conditions in terms of the applied severity factor log R₀. The severity range for A donax ranged from log R₀ 3.4 to 4.2. Prior to steam explosion, A donax and hybrid poplar were impregnated with sulfur dioxide in the amount as shown in Table 6 by adding SO₂ to plastic bags containing 300 g d.w. of biomass. The bags were weighed and left at room temperature overnight. The impregnated biomass was added to the reactor of a 2-L Stake Tech II gun (Stake Technologies, Norvall, ON, Canada) in 50 g aliquots which were treated at the specified temperature and time shown in Table 6. After 300 g d.w. had been discharged to the collecting vessel, the resulting slurry was removed and stored at 4°C. The water soluble fraction (WSF) was separated from water insoluble fraction (WIF) using vacuum filtration. The WIF was then washed with a volume of water equivalent to 20 times the dry weight of the sample. Monomeric and oligomeric sugar

concentrations were determined for the wash liquid, WIF, and WSF to calculate the overall sugar recovery.

Table 6. Steam pretreatment conditions for hybrid poplar and Arundo donax.

Severity Conditions

Pretreatment

(Ro)

Hybrid poplar 3.6 200°C, 5 min, 3% SO₂

A. donax (Low) 3.4 190°C, 5 min, 3% SO₂

A. donax (Med) 3.6 200°C, 5 min, 3% SO₂

A. donax (High) 4.2 210°C, 5 min, 3% SO₂ Enzymatic Hydrolysis

All experiments were carried it out in 125ml_ Erlenmeyer flasks in triplicate and the range values were reported. Total solution volume was 50ml_. Washed solids were diluted to 2% (w/v) consistency with acetate buffer (50mM, pH 5) at 50°C and 150rpm. Enzymes were added in the form of cellulase at 20 FPU/ g cellulose (Celluclast 1 .5L, Franklinton, NC, U.S.) with the protein content of 123mg/mL , and β-glucosidase at 20 CBU/g of cellulose (Novozymes 188, Franklinton, NC, U.S.) with the protein content of 12.3mg/ml_. 500μΙ_ samples were taken periodically over 72 hours, boiled for 5min and stored at - 20°C. Fermentation

The S. cerevisiae strain (strain purchased from the grocery store "QFC") was maintained on YPG solid medium (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose, and 18g/L agar, Difco, Becton Dickinson, MD) at 4°C and transferred to fresh plates on a bimonthly basis. Cells were grown to high cell density (culminating in average 600 nm absorbance values of

approximately 10) in foam-plugged 1 L Erlenmeyer flasks containing 500ml YP- sugar liquid media (10 g/L yeast extract and 10 g/L peptone, supplemented with 10 g/L glucose) in an orbital shaker for 2 days at 30°C and 150 rpm, with concurrent transfer to fresh medium performed every 24 h.

Fermentation of the liquid sugar fractions (water soluble) was conducted in 125 mL flasks containing 50 mL medium pre-adjusted to pH 6.0 with 0.5 M sodium hydroxide. Control fermentations were run in parallel using glucose-based media. The fermentation vessels were maintained at 30°C with continuous agitation (150 rpm). Samples (0.5 mL) were withdrawn aseptically by syringe, centrifuged for 5 min at 15000 x g and 4°C and the supernatant was filtered by using a 0.45 mm syringe filter (Restek Corp., Bellefonte, PA, U.S.) and then stored at -20°C until analysis. Sugars, ethanol, 5-HMF and furfurals were determined periodically from the aliquot culture samples during the course of the fermentation. Each experiment was run in triplicate and the range values were reported.

Simultaneous Saccharification and Fermentation

The SSF experiments were performed under nonsterile conditions in 125 mL flasks, with S. cerevisiae as the sugar fermenting microorganism. The water insoluble fraction at 8% (w/v) concentrations was supplemented with the water soluble streams during SSF experiments. The fermentation vessels were maintained at 37°C with continuous agitation (200 rpm). The SSF experiments were performed at enzyme concentrations of 20 and FPU g cellulose^"1 and an IU: FPU ratio of 2:1 , for 48 hours. There was neither nutrient, nor antibiotic supplementation during most of the SSF experiments. The reaction vessel contained only S. cerevisiae at a cell concentration of 5 g/L, the enzymes, and the pretreated slurry. Samples (0.5 mL) were withdrawn aseptically by syringe, kept on ice until centrifugation (5 min at 15000 x g and 4°C) and the supernatant filtered through a 0.45 mm syringe filter (Restek Corp., Bellefonte, PA, U.S.) and then stored at -20°C until further analysis. In addition, during each experiment, controls were run in parallel (enzymes in buffer with yeast, water insoluble stream with yeast). Each experiment was run in triplicate and the range value reported. Sample Analysis

The concentration of monomeric sugars (arabinose, galactose, glucose, xylose and mannose) was measured on a Dionex (Sunnyvale, CA, U.S.) HPLC (ICS-3000) system equipped with an AS autosampler, ED electrochemical detector, dual pumps, and anion exchange column (Dionex, CarboPac PA1 ) across a gold electrode. Deionized water at 1 mL/min was used as an eluent, and postcolumn addition of 0.2 M NaOH at a flow rate of 0.5 mL/min ensured optimization of baseline stability and detector sensitivity. After each analysis, the column was reconditioned with 0.25 M NaOH. Twenty microliters of each sample were injected after filtration through a 0.45 mm syringe filter (Restek Corp., Bellefonte, PA, U.S.). Standards were prepared containing sufficient arabinose, galactose, glucose, xylose, and mannose to encompass the same range of concentrations as the samples. Fucose (0.2 g/L) was added to all samples and standards as an internal standard.

Ethanol and concentrations of sugar degradation products such as 5-hydroxymethylfurfural (5-HMF) and furfural were determined using

Shimadzu Prominence HPLC chromatograph (Shimadzu Corporation,

Columbia, MD, U.S.). Separation of ethanol and degradation products was achieved by an anion exchange column (REZEX RHM-Mono saccharide H+ (8%), Phenomenex, Inc., and Torrance, CA) with isocratic mobile phase that consisted of 5 μΜ H₂SO₄ at a flow rate of 0.6 mL/min. The column oven temperature was maintained at 63°C constantly. Twenty microliters of each sample were injected after being appropriately diluted in deionized water and filtered through a 0.45 mm syringe filter (Restek Corp., Bellefonte, PA, U.S.). Standards were prepared containing sufficient concentration of a desired compound to encompass the same range of concentrations as the samples. Each experiment was run in triplicate and the standard deviation values were reported.

Posthydrolysis analysis of all liquid samples allowed quantification of the amount of oligomeric sugars present. 0.7 ml_ of 70% H₂SO₄ was added to 15 mL of the liquid samples and the volume made up to 20 ml_ with water. Samples were autoclaved at 121 °C for 1 h and analysed by HPLC as described above.

Solid samples were analysed in triplicate for insoluble (Klason) lignin and sugars using the modified Tappi T-222 on-88 method as previously described (Bura et ai, Applied Biochemistry and Biotechnology, 105:319-335, 2003). The hydrolysate from this analysis was retained and analysed for soluble lignin by absorbance at 205 nm and sugars by HPLC was described above. Arundo donax and Hybrid Poplar Chemical Composition

The carbohydrate and Klason lignin content of the original untreated A. donax and hybrid poplar is shown in Table 7. The total

polysaccharides content of A. donax and hybrid poplar proved to be very high (-59.5% and -59.2%, respectively), as observed by other investigators

(Joseleauand Barnoud, Phytochemistry, 14:71 -75, 1975; Shatalov and Pereira, Carbohydrate Polymers, 49:331 -336, 2002; Shatalov et ai, TAPPI Journal, 84:96-107, 2001 ) making this perennial grass an attractive material for saccharification and fermentation processes. Glucose, followed by xylose and arabinose were shown to be the most abundant components of A. donax as determined by secondary acid hydrolysis of constituent polysaccharides. A. donax and hybrid poplar are characterized by different types of hemicellulose. It has been suggested that arabinoxylan is the major hemicellulose found in A. donax with branches containing xylose, arabinose and galactose in descending order of abundance (Joseleauand Barnoud, Phytochemistry, 14:71 -75, 1975; Shatalov et al., TAPPI Journal, 84:96-107, 2001 ). The most abundant hemicellulose constituent of hybrid poplar is glucuronoxylan (Polizeli et al., Applied Microbiology and Biotechnology, 67:577-591 , 2005). Acid-insoluble lignin (Klason lignin) content for A. donax and hybrid poplar was determined to be -26.2% and 23.6%, respectively, which concurs with previous findings using similar quantification techniques (Bura et ai, Biotechnology Progress, 25:315- 322, 2009; Shatalov et ai, TAPPI Journal, 84:96-107, 2001 ).

% total dry weight (w/w)

Hybrid A. donax poplar

Arabinan (%) 0.4 1 .2

Galactan (%) 0.5 0.4

Glucan (%) 42.8 39.3

Xylan (%) 13.6 18.4

Mannan (%) 1 .9 0.2

Total lignin (%) 23.6 26.2 Chemical Composition of Solid and Liquid Fractions

Since the conversion of A. donax to ethanol has not been previously studied, Arundo donax was pretreated at low (L), medium (M) and high (H) severities (Table 6) to determine the optimum steam pretreatment conditions for maximum sugar recovery in hydrolysable and fermentable form. The optimum steam pretreatment condition for hybrid poplar of 210°C, 5 min and 3%, SO₂ was established in the previous study by Bura et al.

(Biotechnology Progress, 25:315-322, 2009).

The chemical compositions of WIFs after steam pretreatment of A. donax and hybrid poplar are shown in Table 8. Since arabinose, galactose and mannose make up only a small portion of total sugars in biomass tests, only the two main sugars, glucan and xylan, were considered (Table 7, Table 8). As the severity of the pretreatment for A donax was increased, the concentration of glucan in WIF increased from 55.6% to 63.8%. The increase in glucan content was most likely due to the fact that at increased pretreatment severity, the xylan content decreased from 5.8% to 1 .6% (Table 8). The increased glucan and decreased xylan content with increased severity of steam pretreatment correlated well with results described previously for similar pretreatment severities of corn fiber, barley straw, hybrid poplar and wheat straw (Bura et al., Applied Biochemistry and Biotechnology, 105:319-335, 2003; Bura et al., Biotechnology Progress, 25:315-322, 2009; Garcia-Aparicio et al., Applied Biochemistry and Biotechnology, 137:353-365, 2007; Kabel et ai, Bioresource Technology, 98:2034-2042, 2007).

Table 8. Chemical composition of hybrid poplar and Arundo donax \N\Fs pretreated at different severities.

Glucan Xylan (%) Total lignin

Pretreatment

(%) (%)

Hybrid poplar 68.0 1 .9 30.8

A. donax (Low) 55.6 5.8 30.9

A. donax (Med) 61 .9 3.0 33.0

A. donax (High) 63.8 1 .6 34.6

It was determined that as the pretreatment severity for A donax was increased, the concentration of glucose in WSF increased (Table 9).

Similar results were reported for wheat straw, corn fiber and corn stover (Bura et al., Applied Biochemistry and Biotechnology, 105:319-335, 2003; Bura et al., Biotechnology Progress, 25:315-322, 2009; Kabel et ai, Bioresource

Technology, 98:2034-2042, 2007; Ohgren et ai., Applied Biochemistry and Biotechnology, 121 -124:1055-1067, 2005). However, the concentration of xylan in WSF decreased with increased severity of the pretreatment. In addition, more xylose than glucose was found in the liquid stream since hemicellulose is more labile and becomes solubilized during the pretreatment. Since it is known that as pretreatment severity increases, the overall sugar recovery, and hemicellulose in particular, decreases due to sugar degradation (Kabel et ai., Bioresource Technology, 98:2034-2042, 2007; Ohgren et ai., Applied Biochemistry and Biotechnology, 121 -124:1055-1067, 2005), overall sugar recoveries were calculated during optimization of stream pretreatment of A. donax.

Table 9. Chemical composition of hybrid poplar and Arundo donax WSFs pretreated at different severities. Glucose and xylose concentrations shown g/L.

Glucose Xylose

Pretreatment

(g/L) (g/L)

Hybrid poplar 10.3 14.8

A. donax (Low) 2.5 18.7

A. donax (Med) 2.7 14.0

A. donax (High) 3.2 9.4

When the severity of pretreatment increased, less xylan remained within WIF, and thus the combined (WSF+WIF), overall xylan recovery decreased for A donax from 85.1 % to 58.5% (Table 10). Moreover, the glucan recovery decreased with increased severity of the pretreatment from 99.1 % to 92.0%.

Table 10. The combined glucan and xylan recovery of WSF and WIF after steam pretreatment of hybrid poplar and Arundo donax (assuming 100% as maximum recovery for each sugar).

Glucan Xylan

Pretreatment recovery recovery

(%) (%)

Hybrid poplar 99.9 77.7

A. donax (Low) 99.1 85.1

A. donax (Med) 95.4 70.1

A. donax (High) 92.0 58.5

Based on the sugar recovery, it was determined that the optimum pretreatment condition is of 190°C, 5 min and 3% SO₂. At this condition, the majority of glucan and over 85% of xylan can be recovered. The recovered sugars had to be in hydrolysable and fermentable form. Therefore, ease of hydrolysis of the WIF from A. donax and hybrid poplar was assessed next. Enzymatic Hydrolysis of Arundo donax and Hybrid Poplar

Recovered pretreated, water washed A. donax and hybrid poplar solids were subject to enzymatic hydrolysis for 48 hours. Complete cellulose to glucose conversion could be achieved after 24 hours of saccharification for M and H pretreated A. donax solids and hybrid poplar, whereas 100% cellulose conversion was observed after 48 hours of hydrolysis for L pretreated A. donax (Figure 7A). Faster cellulose to glucose conversion was seen with increasing pretreatment severity for A donax, which has been previously observed for corn stover (Bura et ai, Biotechnology Progress, 25:315-322, 2009). It has been shown that agricultural biomass is more responsive to steam pretreatment and results in a solid substrate that is easier to hydrolyze than hardwood biomass (Ekiund et al., Bioresource Engineering, 52:225-229, 1995; Sassner et ai, Applied Biochemistry and Biotechnology, 121 :1 101 -1 1 17, 2005). However, surprisingly, the cellulose to glucose conversion occurred at a faster rate for steam pretreated hybrid poplar compared to steam pretreated A. donax at L severity. Although lignin contents in steam pretreated hybrid poplar and L pretreated A. donax samples were identical (-31 %), the three times lower xylan content in poplar compared to L pretreated A. donax (Table 6) might have contributed to the higher saccharification rate of the pretreated poplar, since it has been shown that the percentage of residual xylan appears to be a good indicator concerning cellulose digestibility (Jeoh et al., Biotechnology and Bioengineering, 98:1 12-122, 2007; Kabel et al., Bioresource Technology, 98:2034-2042, 2007). It has also been shown that xylan, when absorbed onto the surface of cellulose can hinder the action of cellulases (Kabel et ai,

Bioresource Technology, 98:2034-2042, 2007).

An increase in the xylan to xylose conversion was seen with increasing process severity for A donax (Figure 7B). As shown in Figure 7B, xylan in A donax pretreated at highest severity was most efficiently hydrolyzed with 100% of xylan being converted to xylose after 48 h. The L and M severity conditions provided 80 and 91 % conversion, respectively. For steam pretreated poplar 61 % conversion of xylan to xylose was achieved after 48 hours of hydrolysis (Figure 7B).

Fermentation of Arundo donax and Hybrid Poplar

As steam explosion severity increases, so does the degradation of monomeric sugars, by dehydration and condensation reactions that occur at higher temperatures and longer cooking times (Palmqvist et al., Bioresource Technology, 74:25-33, 2000). Therefore, WSFs obtained from all of the pretreatment conditions were assessed for their feasibility as a medium for effective fermentation to ethanol (Figure 8). In this study rather than using genetically modified microorganisms which can utilize both 5 and 6C sugars for bioethanol production, the toxicity of water soluble streams using S. cerevisiae was tested. Fermentation of L, M and H-pretreated WSF from A. donax showed complete uptake of glucose and mannose after 10 h and 90, 85 and 84% of the theoretical maximum ethanol yield was achieved (Figure 8). For WSF from hybrid poplar with 6 times higher concentration of six carbon sugar and over 1 g/L of galactose all the six carbon sugars were converted to ethanol after 20 hours of fermentation 86% of the theoretical maximum ethanol yield was achieved. The delayed uptake of galactose is characteristic of the strain of S. cerevisiae used (Keating et al., Journal of Industrial Microbiology &

Biotechnology, 31 :235-244, 2004). To increase ethanol concentration in the fermentation media, WSF and WIF were mixed during simultaneous

saccharification and fermentation (SSF) process.

SSF (Figure 9) was carried out at 8% (w/v) solids consistency in WSFs. All four substrates resulted in rapid initial uptake of glucose and mannose present in the WSF. Again, slower uptake of galactose was observed for steam pretreated hybrid poplar. The experimental ethanol yields after SSF of steam pretreated A. donax at L, M, and H severities were 84%, 87%, and 89% of the theoretical maximum based on starting hexose concentration in pretreated materials. Simultaneous saccharification and fermentation of steam pretreated hybrid poplar provided an overall ethanol yield of 85% of the theoretical yield.

After examination of sugar recovery, hydrolytic efficiency, and ethanol yields using SSF, it was determined that, of the conditions tested, the L severity condition (190°C, 5 min, 3% SO₂) is the ideal steam pretreatment for A donax, of the three conditions tested. A. donax pretreated using these conditions provided 269 g ethanol/kg raw material after SSF, corresponding to 79% of the theoretical maximum ethanol yield. M and H severity steam pretreatments and SSF of A donax provided overall ethanol yields of 76% and 72% of the theoretical yield from raw which corresponds to 257 and 244 g ethanol/kg raw material within 32 h. Hybrid poplar pretreated at 200°C, 5 min, 3% SO₂ provided 274 g ethanol/kg raw material after SSF, corresponding to 80% of the theoretical maximum ethanol yield. The lower overall ethanol yields for A donax pretreated at M and H severities were due to the low glucan and xylan recovery after pretreatment, since the theoretical ethanol yields based on starting hexose in pretreated materials during SSF were quite high, 87 and 89%, respectively.

In summary, L, M, and H severity steam pretreatment conditions were applied to A donax to select the optimum set of conditions. Since there is no information about A donax to ethanol conversion yields, in this study, bioconversion of hybrid poplar to ethanol was analysed. SSF of steam pretreated mixed of WSF and WIFs provided 79, 76, and 72% of the theoretical maximum ethanol yield from the raw material pretreated at L, M, and H severity. Due to the high ethanol yield achieved, the L severity condition of 190°C, 5 min, and 3% SO₂ was chosen as the optimum pretreatment condition of the three assessed. Hybrid poplar pretreated at 200°C, 5 min, 3% SO₂ provided 274 g ethanol/kg raw material after SSF, corresponding to 80% of the theoretical maximum ethanol yield. These results demonstrate the technical feasibility of producing ethanol from A donax using two stages processing of SO₂-catalyzed steam explosion and SSF. When comparing ethanol yields based on kg of ethanol produced from kg of biomass, the conversion yields for A donax pretreated at 190°C, 5 min, 3% SO₂ are identical to poplar, 0.26kg/kg of biomass. However, due to the higher biomass yields per hectare per year for A donax compared with poplar, the ethanol yields are 3 times higher for A donax if the ethanol yields are based on t/ha/year.

EXAMPLE 3

PRODUCTION OF BIOETHANOL FROM SOAKED AND UNSOAKED ARUNDO DONAX

Arundo donax is a perennial grass that can be harvested two times a year, and it is typically dried for storage. In order to determine if the moisture content of Arundo donax affects the enzymatic hydrolability and overall ethanol yield of the biomass, Arundo donax is soaked in water prior to acid-catalyzed steam explosion and SSF.

Raw Material

A donax is provided by TreeFree Biomass Solutions, Inc. A donax (the whole plant, including leaves) is chipped and screened to

approximately 2 x 2 x 0.5cm³.

Pretreatment and Processing Conditions

Prior to pretreatment, half of the A donax biomass is submerged in water for 48 h. Next, it is vacuum filtered to remove as much excess water as possible and the moisture content is calculated. Gaseous sulfur dioxide (3% w/w) is added by weight to half of the soaked biomass and half of the unsoaked biomass based on the dry weight of the material. Specifically, for 200 g of dry biomass, 6 g of SO2 is added by weight from a cylinder of gas to a plastic bag containing the biomass. 200 g dry weight of the soaked and unsoaked A donax biomass is impregnated, and both the catalyzed and uncatalyzed material is divided into 50 g portions which are then pretreated sequentially using a 1 .5 L batch steam gun (HM3 Energy, Inc., Gresham OR) using the low severity conditions identified in the previous example (i.e., 190°C, 5 minutes, 3% S0₂).

After the specified reaction time elapses for each portion of biomass, a pneumatic valve is opened between the pressurized reaction vessel and the collection vessel, which blows the pretreated slurry into the collection vessel. After all 4 shots are discharged, the slurry of material is collected by opening a valve at the bottom of the collection vessel and allowing the material to drain into a bucket.

The liquid and solid fractions are separated from the slurry by vacuum filtration, analyzed as described below, and used to construct a complete mass balance of carbohydrates and lignin. Solids are water-washed (with water equal to ten times the mass of solids) prior to analysis and saccharification. Following pretreatment and liquid-solid separation of all 4 samples, the compositions of the solid, water-insoluble fraction and the liquid, water-soluble fraction, are analyzed.

Saccharification

Enzymatic hydrolysis of washed solids is done at 5% w/v solids in a total volume of 50 ml in 125 ml Erlenmeyer flasks. The solution is buffered at pH 4.8 with 0.05 M sodium acetate buffer and the hydrolysis is completed at 50 °C and 150 rpm shaking on an orbital shaking incubator (New Brunswick). Cellulase (Spezyme- CP, 26 FPU/ml, Sigma) is added at 10 FPU/g cellulose and supplemental beta-glucosidase (Novozym 188, 492 CBU/ml, Sigma) is added at 20 CBU/g cellulose. 1 ml samples are periodically removed and analyzed for glucose and xylose.

Simultaneous Saccharification and Fermentation (SSF)

Saccharomyces cerevisiae is streaked onto YPD agar plates and allowed to grow for 48 h. Prior to fermentation, preculture cells are grown by adding one colony from the plate to liquid media containing 10 g/L each of glucose, yeast extract and peptone. After 24 h of growth at 30 °C and 150 rpm shaking, the cells are centrifuged and the spent supernatant is removed and replaced with fresh media. The cells are then grown for another 24 h under the same conditions; the cells are again spun down, washed twice in water, and then resuspended in a small volume of 0.9% sodium chloride. Cell

concentration is determined by measuring the optical density of the suspension at 600 nm and comparing to a calibration curve prepared using oven dried cells at different optical densities.

SSF is carried out at 5% w/v washed, never dried solids, 5 g/L yeast, and enzyme loading of 10 FPU/g cellulose and 20 CBU/g cellulose. The total reaction volume is 50 ml in 125 ml Erlenmeyer flasks. Ammonium phosphate (2 g/L), sodium phosphate (0.2 g/L) and sodium nitrate (2 g/L) are added to each flask. Prior to mixing with the solids, the pretreated liquid stream is adjusted to pH 5.5 with 10% NaOH. The pH-adjusted liquid stream is added to each flask along with yeast, enzymes, and nutrients such that the final volume including the moisture in the pretreated solids is 50 ml. Flasks are incubated at 37 °C with 150 rpm orbital shaking. 1 ml samples are removed periodically for ethanol and glucose analysis.

Overall ethanol yields are calculated based on the sugar recovery following pretreatment and the amount of ethanol produced after 24 h of SSF of the pretreated material.

EXAMPLE 4

PRODUCTION OF BIOETHANOL FROM STEAMED BIOMASS

In order to determine if increasing the moisture content of a biomass by steaming it affects the overall ethanol yield of the biomass, Arundo donax is steamed prior to acid-catalyzed steam explosion and SSF. Raw Material

A. donax is provided by TreeFree Biomass Solutions, Inc. A.

donax (the whole plant, including leaves) is chipped and screened to

approximately 2 x 2 x 0.5cm³. Pretreatment and Processing Conditions

Prior to pretreatment, half of the A. donax biomass is steamed for about 30 seconds to 2 minutes in a steam gun. Gaseous sulfur dioxide (3% w/w) is added by weight to half of the steamed biomass and half of the unsteamed biomass based on the dry weight of the material. Specifically, for 200 g of dry biomass, 6 g of SO₂ is added by weight from a cylinder of gas to a plastic bag containing the biomass. 200 g dry weight of the steamed and unsteamed A. donax biomass is impregnated, and both the catalyzed and uncatalyzed material is divided into 50 g portions which are then pretreated sequentially using a 1 .5 L batch steam gun (HM3 Energy, Inc., Gresham OR) using the low severity conditions identified in Example 2 (i.e., 190°C, 5 minutes, 3% SO₂).

The liquid and solid fractions are separated from the slurry by vacuum filtration, analyzed as described below, and used to construct a complete mass balance of carbohydrates and lignin. Solids are water-washed (with water equal to ten times the mass of solids) prior to analysis and saccharification. Following pretreatment and liquid-solid separation of all 4 samples, the compositions of the solid, water-insoluble fraction and the liquid, water-soluble fraction, are analyzed. Saccharification

Enzymatic hydrolysis of washed solids is done at 5% w/v solids in a total volume of 50 ml in 125 ml Erlenmeyer flasks. The solution is buffered at pH 4.8 with 0.05 M sodium acetate buffer and the hydrolysis is completed at 50 °C and 150 rpm shaking on an orbital shaking incubator (New Brunswick). Cellulase (Spezyme- CP, 26 FPU/ml, Sigma) is added at 10 FPU/g cellulose and supplemental beta-glucosidase (Novozym 188, 492 CBU/ml, Sigma) is added at 20 CBU/g cellulose. 1 ml samples are periodically removed and analyzed for glucose and xylose. Simultaneous Saccharification and Fermentation (SSF)

The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent

applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific

embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

1 . A method for producing ethanol from a biomass comprising: a) increasing the moisture content of the biomass; b) pretreating the biomass with an acid-catalyzed steam explosion to produce a slurry; and

c) subjecting the slurry to i) enzymatic hydrolysis and ii) fermentation by a microorganism to produce ethanol.

2. The method according to claim 1 , wherein step a) comprises soaking the biomass in water for a period of time.

3. The method according to claim 2, wherein the biomass is totally submerged in the water.

4. The method according to claim 2, wherein the period of time is from about 6 to about 36 hours.

5. The method according to claim 4, wherein the period of time is about 24 hours.

6. The method according to claim 2, wherein the acid-catalyzed steam explosion utilizes an acid selected from sulfur dioxide and sulfuric acid.

7. The method according to claim 2, wherein step c) comprises simultaneous saccharification and fermentation (SSF).

8. The method according to claim 2, wherein the microorganism is

S. cerevisiae.

9. The method according to claim 2, wherein the moisture content of the biomass after step (a) is 10-60%.

10. The method according to claim 2, wherein the biomass is Arundo donax.

1 1 . The method according to claim 1 , wherein step a) comprises steaming the biomass for a period of time.

12. The method according to claim 1 1 , wherein the steaming occurs in a steam gun.

13. The method according to claim 1 1 , wherein the period of time is from about 15 seconds to about 120 seconds.

14. The method according to claim 13, wherein the period of time is about 45 seconds to about 90 seconds.

15. The method according to claim 1 1 , wherein the acid-catalyzed steam explosion utilizes an acid selected from sulfur dioxide and sulfuric acid.

16. The method according to claim 10, wherein step c) comprises simultaneous saccharification and fermentation (SSF).

17. The method according to claim 1 1 , wherein the microorganism is S. cerevisiae.

18. The method according to claim 1 1 , wherein the moisture content of the biomass after step (a) is about 10-60%.

19. The method according to claim 1 1 , wherein the biomass is Arundo donax.