Inventors: Jan Larsen, Niels Poulsen, Kit Mogensen, Martin Jeppesen Field.

The invention relates, in general, to methods of processing lignocellulosic biomass to fermentable sugars and, in particular, to methods that rely on hydrothermal pretreatment.

Background.

Historical reliance on petroleum and other fossil fuels has been associated with dramatic and alarming increases in atmospheric levels of greenhouse gases. International efforts are underway to mitigate greenhouse gas accumulation, supported by formal policy directives in many countries. One central focus of these mitigation efforts has been development of processes and technologies for utilization of renewable plant biomass to replace petroleum as a source of precursors for fuels and other chemical products. The annual growth of plant-derived biomass on earth is estimated to approximate 1 x 10 ^Λ 1 1 metric tons per year dry weight. See Lieth and Whittaker (1975). Biomass utilization is, thus, an ultimate goal in development of sustainable economy.

Fuel ethanol produced from sugar and starch based plant materials, such as sugarcane, root and grain crops, is already in wide use, with global production currently topping 73 billion liters per year. Ethanol has always been considered an acceptable alternative to fossil fuels, being readily usable as an additive in fuel blends or even directly as fuel for personal automobiles. However, use of ethanol produced by these "first generation" bioethanol technologies does not actually achieve dramatic reduction in greenhouse gas emission. The net savings is only about 13% compared with petroleum, when the total fossil fuel inputs to the final ethanol outputs are all accounted. See Farrell et al. (2006). Moreover, both economic and moral objections have been raised to the "first generation" bioethanol market. This effectively places demand for crops as human food into direct competition with demand for personal automobiles. And indeed, fuel ethanol demand has been associated with increased grain prices that have proved troublesome for poor, grain- importing countries.

Great interest has arisen in developing biomass conversion systems that do not consume food crops - so-called "second generation" biorefining, whereby bioethanol and other products can be produced from lignocellulosic biomass such as crop wastes (stalks, cobs, pits, stems, shells, husks, etc .), grasses, straws, wood chips, waste paper and the like. In "second generation" technology, fermentable 6-carbon (C6) sugars derived primarily from cellulose and fermentable 5-carbon (C5) sugars derived from hemicellulose are liberated from biomass polysaccharide polymer chains by enzymatic hydrolysis or, in some cases, by pure chemical hydrolysis. The fermentable sugars obtained from biomass conversion in a "second generation" biorefinery can be used to produce fuel ethanol or, alternatively, other fuels such as butanol, or lactic acid monomers for use in synthesis of bioplastics, or many other products.

The total yield of both C5 and C6 sugars is a central consideration in commercialization of lignocellulosic biomass processing. In the case of ethanol production, and also production of lactate or other chemicals, it can be advantageous to combine both C5 and C6 sugar process streams into one sugar solution. Modified fermentive organisms are now available which can efficiently consume both C5 and C6 sugars in ethanol production. See e.g. Madhavan et al. (2012); Dumon et al. (2012); Hu et al. (201 1 ); Kuhad et al.

(201 1 ); Ghosh et al. (201 1 ); Kurian et al. (2010); Jojima et al. (2010); Sanchez et al.

(2010); Bettiga et al. (2009); Matsushika et al. (2009).

Because of limitations of its physical structure, lignocellulosic biomass cannot be effectively converted to fermentable sugars by enzymatic hydrolysis without some pretreatment process. A wide variety of different pretreatment schemes have been reported, each offering different advantages and disadvantages. For review see Agbor et al. (201 1); Girio et al. (2010); Alvira et al. (2010); Taherzadeh and Karimi (2008). From an environmental and "renewability" perspective, hydrothermal pretreatments are especially attractive. These utilize pressurized steam/liquid hot water at temperatures on the order of 160 - 230 °C to gently melt hydrophobic lignin that is intricately associated with cellulose strands, to solubilize a major component of hemicellulose, rich in C5 sugars, and to disrupt cellulose strands so as to improve accessibility to productive enzyme binding.

Hydrothermal pretreatments can be conveniently integrated with existing coal- and biomass-fired electrical power generation plants to efficiently utilize turbine steam and "excess" power production capacity.

In the case of hydrothermal processes, it is well known in the art, and has been widely discussed, that pretreatment must be optimized between conflicting purposes. On the one hand, pretreatment should ideally preserve hemicellulose sugar content, so as to maximize the ultimate yield of monomeric hemicellulose-derived sugars. Yet at the same time, pretreatment should sufficiently expose and pre-condition cellulose chains to susceptibility of enzymatic hydrolysis such that reasonable yields of monomeric cellulose- derived sugars can be obtained with minimal enzyme consumption. Enzyme consumption is also a central consideration in commercialization of biomass processing, which teeters on the verge of "economic profitability" in the context of "global market economies" as these are currently defined. Notwithstanding dramatic improvements in recent years, the high cost of commercially available enzyme preparations remains one of the highest operating costs in biomass conversion.

As hydrothermal pretreatment temperatures and reactor residence times are increased, a greater proportion of C5 sugars derived from hemicellulose is irretrievably lost due to chemical transformation to other substances, including furfural and products of

condensation reactions. Yet higher temperatures and residence times are required in order to properly condition cellulose fibers for efficient enzymatic hydrolysis to monomeric 6-carbon glucose.

In the prior art, an often used parameter of hydrothermal pretreatment "severity" is "R ₀ ," which is typically referred to as a log value. Ro reflects a composite measure of pretreatment temperature and reactor residence time according to the equation: R ₀ = tEXP[T-100/14.75] where t is residence time in minutes and T is reaction temperature in degrees centigrade. Optimization of pretreatment conditions for any given biomass feedstock inherently requires some compromise between demands for high monomeric C5 sugar yields from hemicellulose (low severity) and the demands for high monomeric C6 sugar yields from cellulose (high severity).

A variety of different hydrothermal pretreatment strategies have been reported for maximizing sugar yields from both hemicellulose and cellulose and for minimizing xylo- oligomer inhibition of cellulase catalysis. In some cases, exogenous acids or bases are added in order to catalyse hemicellulose degradation (acid; pH < 3.5) or lignin

solubilisation (base; pH > 9.0). In other cases, hydrothermal pretreatment is conducted using only very mild acetic acid derived from lignocellulose itself (pH 3.5-9.0).

Hydrothermal pretreatments under these mild pH conditions have been termed

"autohydrolysis" processes, because acetic acid liberated from hemicellulose esters itself further catalyses hemicellulose hydrolysis.

Acid catalysed hydrothermal pretreatments, known as "dilute acid" or "acid impregnation" treatments, typically provide high yields of C5 sugars, since comparable hemicellulose solubilisation can occur at lower temperatures in the presence of acid catalyst. Total C5 sugar yields after dilute acid pretreatment followed by enzymatic hydrolysis are typically on the order of 75% or more of what could theoretically be liberated from any given biomass feedstock. See e.g. Baboukaniu et al. (2012); Won et al. (2012); Lu et al. (2009); Jeong et al. (2010); Lee et al. (2008); Sassner et al. (2008); Thomsen et al. (2006); Chung et al. (2005).

Autohydrolysis hydrothermal pretreatments, in contrast, typically provide much lower yields of C5 sugars, since higher temperature pretreatment is required in the absence of acid catalyst. With the exception of autohydrolysis pretreatment conducted at commercially unrealistic low dry matter content, autohydrolysis treatments typically provide C5 sugar yields <40% theoretical recovery. See e.g. Diaz et al. (2010); Dogaris et al. (2009). C5 yields from autohydrolysis as high as 53% have been reported in cases where

commercially unrealistic reactions times and extreme high enzyme doses were used. But even these very high C5 yields remain well beneath levels routinely obtained using dilute acid pretreatment. See e.g. Lee et al. (2009); Ohgren et al. (2007).

As a consequence of lower C5 yields obtained with autohydrolysis, most reports

concerning hydrothermal pretreatment in commercial biomass conversion systems have focused on dilute acid processes. Hemicellulose-derived C5 sugar yields on the order of 85% have been achieved through use of so-called "two-stage" dilute acid pretreatments. In two-stage pretreatments, a lower initial temperature is used to solubilize hemicellulose, whereafter the C5-rich liquid fraction is separated. In the second stage, a higher temperature is used to condition cellulose chains. See e.g. Mesa et al. (201 1); Kim et al. (201 1 ); Chen et al. (2010); Jin et al. (2010); Monavari et al. (2009); Soderstrom et al.

(2005); Soderstrom et al. (2004); Soderstrom et al. (2003); Kim et al. (2001 ); Lee et al. (1997); Paptheofanous et al. (1995). One elaborate "two-stage" dilute acid pretreatment system reported by the US National Renewable Energy Laboratory (NREL) claims to have achieved C5 yields on the order of 90% using corn stover as feedstock. See Humbird et al. (201 1 ).

Notwithstanding the lower C5 yields which it provides, autohydrolysis continues to offer competitive advantages over dilute acid pretreatments on commercial scale.

Most notable amongst the advantages of autohydrolysis processes is that the residual, unhydrolysed lignin has greatly enhanced market value compared with lignin recovered from dilute acid processes. First, the sulphuric acid typically used in dilute acid

pretreatment imparts a residual sulphur content. This renders the resulting lignin unattractive to commercial power plants which would otherwise be inclined to consume sulphur-free lignin fuel pellets obtained from autohydrolysis as a "green" alternative to coal. Second, the sulfonation of lignin which occurs during sulphuric acid-catalysed

hydrothermal pretreatments renders it comparatively hydrophilic, thereby increasing its mechanical water holding capacity. This hydrophilicity both increases the cost of drying the lignin for commercial use and also renders it poorly suited for outdoor storage, given its propensity to absorb moisture. So-called "techno-economic models" of NREL's process for lignocellulosic biomass conversion, with dilute acid pretreatment, do not even account for lignin as a saleable commodity - only as an internal source of fuel for process steam. See Humbird et al. (201 1 ). In contrast, the "economic profitability" of process schemes that rely on autohydrolysis include a significant contribution from robust sale of clean, dry lignin pellets. This is especially significant in that typical soft lignocellulosic biomass feedstocks comprise a large proportion of lignin, between 10 and 40% of dry matter content. Thus, even where process sugar yields from autohydrolysis systems can be diminished relative to dilute acid systems, overall "profitability" can remain equivalent or even better.

Autohydrolysis processes also avoid other well known disadvantages of dilute acid. The requirement for sulphuric acid diverges from a philosophical orientation favouring "green" processing, introduces a substantial operating cost for the acid as process input, and creates a need for elaborate waste water treatment systems and also for expensive anti- corrosive equipment.

Autohydrolysis is also advantageously scalable to modest processing scenarios. The dilute acid process described by NREL is so complex and elaborate that it cannot realistically be established on a smaller scale - only on a gigantic scale on the order of 100 tons of biomass feedstock per hour. Such a scale is only appropriate in hyper-centralized biomass processing scenarios. See Humbird et al. (201 1 ). Hyper-centralized biomass processing of corn stover may well be appropriate in the USA, which has an abundance of genetically-engineered corn grown in chemically-enhanced hyper-production. But such a system is less relevant elsewhere in the world. Such a system is inappropriate for modest biomass processing scenarios, for example, on-site processing at sugar cane or palm oil or sorghum fields, or regional processing of wheat straw, which typically produces much less biomass per hectare than corn, even with genetic-engineering and chemical- enhancements.

Autohydrolysis systems, in contrast with dilute acid, are legitimately "green," readily scalable, and unencumbered by requirements for elaborate waste water treatment systems. It is accordingly advantageous to provide improved autohydrolysis systems, even where these may not be obviously advantageous over dilute acid systems in terms of sugar yields alone. The problem of poor C5 monomer yields with autohydrolysis has generally driven commercial providers of lignocellulosic biomass processing technology to pursue other approaches. Some "two-stage" pretreatment systems, designed to provide improved C5 yields, have been reported with autohydrolysis pretreatments. See WO2010/1 13129; US2010/0279361 ; WO 2009/108773; US2009/0308383; US8,057,639; US20130029406. In these "two stage" pretreatment schemes, some C5-rich liquid fraction is removed by solid/liquid separation after a lower temperature pretreatment, followed by a subsequent, higher temperature pretreatment of the solid fraction. Most of these published patent applications did not report actual experimental results. In its description of two-stage autohydrolytic pretreatment in WO2010/1 13129, Chemtex Italia reports a total of 26 experimental examples using wheat straw with an average C5 sugar recovery of 52%. These C5 recovery values do not distinguish between C5 recovery per se and monomer sugar yields, which is the substrate actually consumed in fermentation to ethanol and other useful products.

The introduction of a second pretreatment stage into a scheme for processing

lignocellulosic biomass introduces additional complexities and costs. It is accordingly advantageous to substantially achieve the advantages of two-stage pretreatment using a simple single-stage autohydrolysis system.

We have discovered that, where single-stage autohydrolysis pretreatment is conducted to very low severity, it is possible to achieve unexpectedly high final C5 monomer yields of 55% theoretical yield and higher, while still achieving reasonable glucose yields. Where biomass feedstocks are pretreated to such low severity that the undissolved solids content of pretreated material retains a residual xylan content of at least 5.0% by weight, loss of C5 during pretreatment is minimized. Yet contrary to expectations, this very high residual xylan content can be enzymatically hydrolysed to monomer xylose, with high recovery, while sacrificing only a very small percentage of cellulose conversion to glucose, provided that sufficiently high xylanase and xylosidase activities are employed during enzymatic hydrolysis. At these very low severity levels, the production of soluble by-products that affect cellulase activity or fermentive organisms is kept so low that the pretreated material can be used directly in enzymatic hydrolysis, and subsequent fermentation, typically without

requirement for any washing or other de-toxification step.

Brief description of the figures.

Figure 1 shows xylan number as a function of pretreatment severity factor for soft lignocellulosic biomass feedstocks subject to autohydrolysis pretreatment.

Figure 2 shows the percentage by weight xylan in undissolved solids as a function of xylan number for pretreated feedstocks.

Figure 3 shows C5 recovery in soluble and insoluble form as a function of xylan number for soft lignocellulosic biomass feedstocks subject to autohydrolysis pretreatment.

Figure 4 shows total C5 recovery as a function of xylan number for soft lignocellulosic biomass feedstocks subject to autohydrolysis pretreatment.

Figure 5 shows production of acetic acid, furfural and 5-HMF as a function of xylan number for soft lignocellulosic biomass feedstocks subject to autohydrolysis pretreatment.

Figure 6 shows the effect of removal of dissolved solids on cellulose conversion for soft lignocellulosic biomass feedstocks subject to very low severity autohydrolysis

pretreatment.

Figure 7 shows HPLC characterization of liquid fraction from soft lignocellulosic biomass feedstocks subject to very low severity autohydrolysis pretreatment.

Figure 8 shows C5 sugar recovery as a function of time where solid fraction is subject to enzymatic hydrolysis followed by introduction of liquid fraction for post-hydrolysis. Figure 9 shows fermentation profile of ethanol fermentation by a modified yeast strain using wheat straw that was pretreated by very low severity autohydrolysis, enzymatically hydrolysed and used as combined liquid and solid fraction without de-toxification to remove fermentation inhibitors.

Figure 10 shows a process scheme for one embodiment.

Figure 1 1 show cellulose conversion as a function of time - C5 bypass.

Figure 12 shows Xylan conversion as a function of time - C5 bypass.

Figure 13 show cellulose conversion as a function of time - whole slurry.

Figure 14 shows xylan conversion as a function of time - whole slurry.

Figure 15 shows total C6 and C5 recovery after pretreatment and hydrolysis as a function of hydrolysis time - whole slurry.

Detailed description of embodiments.

In some embodiments the invention provides methods of processing lignocellulosic biomass comprising:

- Providing soft lignocellulosic biomass feedstock,

- Pretreating the feedstock at pH within the range 3.5 to 9.0 in a single-stage

pressurized hydrothermal pretreatment to log severity Ro 3.75 or lower so as to produce a pretreated biomass slurry in which the undissolved solids comprise at least 5.0% by weight xylan, and

- Hydrolysing the pre-treated biomass with or without addition of supplemental water content using enzymatic hydrolysis for at least 24 hours catalysed by an enzyme mixture comprising endoglucanase, exoglucanase, β-glucosidase, endoxylanase, and β-xylosidase activities at activity levels in nkat/g glucan of endoglucanase of at least 1 100, exoglucanase of at least 280, β-glucosidase of at least 3000, endoxylanase of at least 1400, and β-xylosidase of at least 75, so as to produce a hydrolysate in which the yield of C5 monomers is at least 55% of the original xylose and arabinose content of the feedstock prior to pretreatment.

In some embodiments, the pretreated biomass is hydrolysed as a whole slurry comprising both solid and liquid fractions.

As used herein, the following terms have the following meanings:

"About" as used herein with reference to a quantitative number or range refers to +/- 10% in relative terms of the number or range referred to.

"Autohydrolysis" refers to a pretreatment process in which acetic acid liberated by hemicellulose hydrolysis during pretreatment further catalyzes hemicellulose hydrolysis, and applies to any hydrothermal pretreatment of lignocellulosic biomass conducted at pH between 3.5 and 9.0.

"Commercially available cellulase preparation optimized for lignocellulosic biomass conversion " refers to a commercially available mixture of enzyme activities that is sufficient to provide enzymatic hydrolysis of pretreated lignocellulosic biomass and that comprises endocellulase (endoglucanase), exocellulase (exoglucanase), endoxylanase, xylosidase and B-glucosidase activities. The term "optimized for lignocellulosic biomass conversion" refers to a product development process in which enzyme mixtures have been selected and/or modified for the specific purpose of improving hydrolysis yields and/or reducing enzyme consumption in hydrolysis of pretreated lignocellulosic biomass to fermentable sugars.

Conducting pretreatment "at" a dry matter level refers to the dry matter content of the feedstock at the start of pressurized hydrothermal pretreatment. Pretreatment is conducted "at" a pH where the pH of the aqueous content of the biomass is that pH at the start of pressurized hydrothermal pretreatment. "Dry matter," also appearing as DM, refers to total solids, both soluble and insoluble, and effectively means "non-water content." Dry matter content is measured by drying at 105°C until constant weight is achieved.

"Fiber structure" is maintained to the extent that the average size of fiber fragments following pretreatment is >750 urn.

"Glucan" as used herein refers to cellulose as well as other gluco-oligomers.

"Hydrothermal pretreatment" refers to the use of water, either as hot liquid, vapor steam or pressurized steam comprising high temperature liquid or steam or both, to "cook" biomass, at temperatures of 120° C or higher, either with or without addition of acids or other chemicals.

"Single-stage pressurized hydrothermal pretreatment" refers to a pretreatment in which biomass is subject to pressurized hydrothermal pretreatment in a single reactor configured to heat biomass in a single pass and in which no further pressurized hydrothermal pretreatment is applied following a solid/liquid separation step to remove liquid fraction from feedstock subject to pressurized hydrothermal pretreatment.

"Solid/liquid separation" refers to an active mechanical process whereby liquid is separated from solid by application of force through pressing, centrifugal or other force.

"Soft lignocellulosic biomass" refers to plant biomass other than wood comprising cellulose, hemicellulose and lignin.

"Solid fraction" and "Liquid fraction" refer to fractionation of pretreated biomass in solid/liquid separation. The separated liquid is collectively referred to as "liquid fraction." The residual fraction comprising considerable insoluble solid content is referred to as "solid fraction." A "solid fraction" will have a dry matter content and typically will also comprise a considerable residual of "liquid fraction." "Theoretical yield" refers to the molar equivalent mass of pure monomer sugars obtained from polymeric cellulose, or from polymeric hemicellulose structures, in which constituent monomeric sugars may also be esterified or otherwise substituted. "C5 monomer yields" as a percentage of theoretical yield are determined as follows: Prior to pretreatment, biomass feedstock is analysed for carbohydrates using the strong acid hydrolysis method of Sluiter et al. (2008) using an HPLC column and elution system in which galactose and mannose co-elute with xylose. Examples of such systems include a REZEX™

Monossacharide H+ column from Phenomenex and an AMINEX HPX 87C™ column from Biorad. During strong acid hydrolysis, esters and acid-labile substitutions are removed. Except as otherwise specified, the total quantity of "Xylose" + Arabinose determined in the un-pretreated biomass is taken as 100% theoretical C5 monomer recovery, which can be termed collectively "C5 monomer recovery." Monomer sugar determinations are made using HPLC characterization based on standard curves with purified external standards. Actual C5 monomer recovery is determined by HPLC characterization of samples for direct measurement of C5 monomers, which are then expressed as a percent of theoretical yield.

"Xylan number" refers to a characterization of pretreated biomass determined as follows: Pretreated biomass is obtained at about 30% total solids, typically after a solid/liquid separation to provide a solid fraction and a liquid fraction. This pretreated biomass having about 30% total solids is then partially washed by mixing with 70° C water in the ratio of total solids (DM) to water of 1 :3 wt:wt. The pretreated biomass washed in this manner is then pressed to about 30% total solids. Xylan content of the pretreated biomass washed in this manner and pressed to about 30% total solids is determined using the method of A. Sluiter, et al., "Determination of structural carbohydrates and lignin in biomass," US

National Renewable Energy Laboratory (NREL) Laboratory Analytical Procedure (LAP) with issue date April 25, 2008, as described in Technical Report NREL/TP-510^2618, revised April 2008, which is expressly incorporated by reference herein in entirety. An HPLC column and elution system is used in which galactose and mannose co-elute with xylose. Examples of such systems include a REZEX™ Monossacharide H+ column from Phenomenex and an AMINEX HPX 87C™ column from Biorad. This measurement of xylan content as described will include some contribution of soluble material from residual liquid fraction that is not washed out of solid fraction under these conditions. Accordingly, "xylan number" provides a "weighted combination" measurement of residual xylan content within insoluble solids and of soluble xylose and xylo-oligomer content within the "liquid fraction."

The xylan content of undissolved solids is determined by taking a representative sample of pretreated biomass, subjecting it to solid/liquid separation so as to provide a solid fraction having at least 30% total solids. This pretreated biomass having at least 30% total solids is then partially washed by mixing with 70° C water in the ratio of total solids (DM) to water of 1 :3 wt:wt. The pretreated biomass washed in this manner is then pressed to at least 30% total solids and again washed by mixing with 70° C water in the ratio of total solids (DM) to water of 1 :3 wt:wt. This washed material is then again pressed to at least 30% total solids and again washed by mixing with 70° C water in the ratio of total solids (DM) to water of 1 :3 wt:wt. This washed material is then again pressed to at least 30% total solids and used as the sample of undissolved solids to be analysed. Xylan content of the undissolved solids sample is then determined as described in the explanation above concerning determination of xylan number and expressed as a percentage by weight of the total solids content of the sample.

Any suitable soft lignocellulosic biomass may be used, including biomasses such as at least wheat straw, corn stover, corn cobs, empty fruit bunches, rice straw, oat straw, barley straw, canola straw, rye straw, sorghum, sweet sorghum, soybean stover, switch grass, Bermuda grass and other grasses, bagasse, beet pulp, corn fiber, or any combinations thereof. Lignocellulosic biomass may comprise other lignocellulosic materials such as paper, newsprint, cardboard, or other municipal or office wastes. Lignocellulosic biomass may be used as a mixture of materials originating from different feedstocks, may be fresh, partially dried, fully dried or any combination thereof. In some embodiments, methods of the invention are practiced using at least about 10 kg biomass feedstock, or at least 100 kg, or at least 500 kg.

Lignocellulosic biomass comprises crystalline cellulose fibrils intercalated within a loosely organized matrix of hemicellulose and sealed within an environment rich in hydrophobic lignin. While cellulose itself comprises long, straight chain polymers of D-glucose, hemicellulose is a heterogeneous mixture of short, branched-chain carbohydrates including monomers of all the 5-carbon aldopentoses (C5 sugars) as well as some 6- carbon (C6) sugars including glucose and mannose. Lignin is a highly heterogeneous polymer, lacking any particular primary structure, and comprising hydrophobic

phenylpropanoid monomers.

Suitable lignocellulosic biomass typically comprises cellulose in amounts between 20 and 50 % of dry mass prior to pretreatment, lignin in amounts between 10 and 40 % of dry mass prior to pretreatment, and hemicellulose in amounts between 15 and 40%.

In some embodiments, biomass feedstocks may be subject to particle size reduction and/or other mechanical processing such as grinding, milling, shredding, cutting or other processes prior to hydrothermal pretreatment. In some embodiments, biomass feedstocks may be washed and/or leached of valuable salts prior to pressurized pretreatment, as described in Knudsen et al. (1998). In some embodiments feedstocks may be soaked prior to pressurized pretreatment at temperatures up to 99° C.

In some embodiments the feedstock is first soaked in an aqueous solution prior to hydrothermal pretreatment. In some embodiments, it can be advantageous to soak the feedstock in an acetic acid containing liquid obtained from a subsequent step in the pretreatments, as described in US 8,123,864, which is hereby incorporated by reference in entirety. It is advantageous to conduct treatment at the highest possible dry matter content, as described in US 12/935,587, which is hereby incorporated by reference in entirety. Conducting pretreatment at high dry matter avoids expenditure of process energy on heating of unnecessary water. However, some water content is required to achieve optimal eventual sugar yields from enzymatic hydrolysis. Typically it is advantageous to pretreat biomass feedstocks at or close to their inherent water holding capacity. This is the level of water content that a given feedstock will attain after soaking in an excess of water followed by pressing to the mechanical limits of an ordinary commercial screw press - typically between 30 and 45% DM. In some embodiments, hydrothermal pretreatment is conducted at DM content at least 35%. It will be readily understood by one skilled in the art that DM content may decrease during hydrothermal pretreatment as some water content is added during heating. In some embodiments, feedstocks are pretreated at DM content at least 20%, or at least 25%, or at least 30%, or at least 40%, or 40% or less, or 35% or less, or 30% or less.

In some embodiments, soaking/wetting with an aqueous solution can serve to adjust pH prior to pretreatment to the range of between 3.5 and 9.0, which is typically advantageous for autohydrolysis. It will be readily understood that pH may change during pretreatment, typically to more acidic levels as acetic acid is liberated from solubilized hemicellulose.

In some embodiments, hydrothermal pretreatment is conducted without supplemental oxygen as required for wet oxidation pretreatments, or without addition of organic solvent as required for organosolv pretreatment, or without use of microwave heating as required for microwave pretreatments. In some embodiments, hydrothermal pretreatment is conducted at temperatures of 140° C or higher, or at 150° C or higher, or at 160° C or higher, or between 160 and 200° C, or between 170 and 190°C, or at 180°C or lower, or at 170° C or lower.

In some embodiments, some C5 content may be removed by a soaking step prior to pressurized pretreatment. In some embodiments, the single reactor may be configured to heat biomass to a single target temperature. Alternatively, the single reactor may be configured to affect a temperature gradient within the reactor such that biomass is exposed, during a single passage, to more than one temperature region. In some embodiments, it may be advantageous to partially remove some solubilized biomass components from within the pressurized reactor during the course of pretreatment.

Suitable hydrothermal pretreatment reactors typically include most pulping reactors known from the pulp and paper industry. In some embodiments, hydrothermal pretreatment is administered by steam within a reactor pressurized to 10 bar or lower, or to 12 bar or lower, or to 4 bar or higher, or 8 bar or higher, or between 8 and 18 bar, or between 18 and 20 bar. In some embodiments, the pretreatment reactor is configured for a continuous inflow of feedstock.

In some embodiments, wetted biomass is conveyed through the reactor, under pressure, for a certain duration or "residence time." Residence time is advantageously kept brief to facilitate higher biomass throughput. However, the pretreatment severity obtained is determined both by temperature and also by residence time. Temperature during hydrothermal pretreatment is advantageously kept lower, not only because methods of the invention seek to obtain a very low pretreatment severity, but also because lower temperatures can be accomplished using lower steam pressures. To the extent that pretreatment temperature can be at levels of 180° C or lower, and accordingly, saturated steam pressures kept to 10 bar or lower, lower tendency for corrosion is experienced and much lower grade pressure fittings and steel compositions may be used, which reduces plant capital costs. In some embodiments, the reactor is configured to heat biomass to a single target temperature between 160 and 200° C, or between 170 and 190° C.

Residence times in some embodiments are less than 60, or less than 30, or less than 20, or less than 5, or less than 14, or less than 13, or less than 12, or less than 10, or less than 8, or less than 5 minutes.

Biomass feedstocks may be loaded from atmospheric pressure into a pressurized reactor by a variety of means. In some embodiments, a sluice-type "particle pump" system may be used to load biomass feedstocks, such as the system described in US 13/062,522, which is hereby incorporated by reference in entirety. In some embodiments, it may be advantageous to load a pretreatment reactor using a so-called "screw plug" feeder.

Pretreated biomass may be unloaded from a pressurized reactor by a variety of means. In some embodiments, pretreated biomass is unloaded in such manner as to preserve the fiber structure of the material. Preserving the fiber structure of the pretreated biomass is advantageous because this permits the solid fraction of the pretreated material to be pressed during solid/liquid separation to comparatively high dry matter levels using ordinary screw press equipment, and thereby avoiding the added expense and complexity of membrane filter press systems.

Fiber structure can be maintained by removing the feedstock from the pressurized reactor in a manner that is non-explosive. In some embodiments, non-explosive removal may be accomplished and fiber structure thereby maintained using a sluice-type system, such as that described in US 13/043,486, which is hereby incorporated by reference in entirety. In some embodiments, non-explosive removal may be accomplished and fiber structure thereby maintained using a hydrocyclone removal system, such as those described in US 12/996,392, which are hereby incorporated by reference in entirety. In some embodiments, pretreated biomass can be removed from a pressurized pretreatment reactor using "steam explosion," which involves explosive release of the pretreated material. Steam-exploded, pretreated biomass does not retain its fiber structure and accordingly requires more elaborate solid/liquid separation systems in order to achieve dry matter content comparable to that which can be achieved using ordinary screw press systems with pretreated biomass that retains its fiber structure.

The biomass feedstock is pretreated to very low severity log Ro 3.75 or less. This will typically result in the pretreated biomass having a xylan number of 10% or higher. The parameter "xylan number" refers to a composite measurement that reflects a weighted combination of both residual xylan content remaining within insoluble solids and also the concentration of soluble xylose and xylo-oligomers within the liquid fraction. At lower Ro severity, xylan number is higher. Thus, the highest xylan number refers to the lowest pretreatment severity. Xylan number provides a negative linear correlation with the conventional severity measure log R ₀ even to very low severity, where residual xylan content within undissolved solids is higher. The relationship between pretreatment severity log Ro and xylan number of the resulting pretreated biomass for a variety of different feedstocks is shown in Figure 1 , which is explained in detail in Example 1 .

Xylan number is particularly useful as a measure of pretreatment severity in that different pretreated biomass feedstocks having equivalent xylan number exhibit equivalent C5 monomer recovery. In contrast, conventional Ro severity is simply an empirical description of pretreatment conditions, which does not provide a rational basis for comparisons between different biomass feedstocks. For example, as shown in Figure 1 , single-stage autohydrolysis to severity log R ₀ = 3.75 provides pretreated sugar cane bagasse and corn stover having a xylan number of between 6-7%, while with typical wheat straw strains, the resulting xylan number of pretreated feedstock is about 10%.

One skilled in the art can readily determine an appropriate pretreatment severity log Ro for any given feedstock to produce a pretreated biomass having the desired xylan number. In some embodiments biomass feedstock is pretreated to very low severity log Ro 3.75 or less, or 3.74 or less, or 3.73 or less, or 3.72 or less, or 3.71 or less, or 3.70 or less, or 3.69 or less, or 3.68 or less, or 3.67 or less, or 3.66 or less, or 3.65 or less, or 3.64 or less, or 3.63 or less, or 3.62 or less, or 3.61 or less, or 3.60 or less, or 3.59 or less, or 3.58 or less, or 3.57 or less, or 3.56 or less, or 3.55 or less, or 3.54 or less, or 3.53 or less, or 3.52 or less, or 3.51 or less, or 3.50 or less, or 3.45 or less, or 3.40 or less. In some

embodiments, the biomass is pretreated to a low severity log Ro so as to produce a pretreated biomass having a xylan number of 1 1 % or higher, or 12% or higher, or 13% or higher, or 4% or higher, or 15% or higher, or 16% or higher, or 17% or higher.

Xylan number is useful as a practical measure in production scale biomass processing because it can be readily measured on-line based on simple measurements such as are provided by near infrared spectroscopic monitors. Xylan number can further be used as an end-point measure for control of pretreatment, as described in WO2013/120492.

As an alternative to xylan number, which reflects a composite measure of both soluble xylan content and also residual xylan content in undissolved solids, the effect of

pretreatment can be expressed in terms of residual xylan content in undissolved solids. The relationship between residual xylan content in undissolved solids and xylan number is shown in Figure 2, which is explained in Example 1 . One skilled in the art can readily determine an appropriate pretreatment severity log Ro for any given feedstock to produce a pretreated biomass having the desired residual xylan content in undissolved solids. For example, as shown in Figure 2, single-stage autohydrolysis at a severity sufficient to produce pretreated biomass having a xylan number of 10% will typically produce pretreated biomass having a residual xylan content in undissolved solids of about 5.0% by weight for soft lignocellulosic feedstocks, including but not limited to wheat straw, sugar cane bagasse, epty fruit bunches, and corn stover. In some embodiments, the biomass feedstock is pretreated to severity log Ro appropriate to produce a pretreated biomass in which the residual xylan content of undissolved solids is at least 5.0% by weight, or at least 5.1 %, or at least 5.2%, or at least 5.3%, or at least 5.4%, or at least 5.5%, or at least 5.6%, or at least 5.7%, or at least 5.8%, or at least 5.9%, or at least 6.0%, or at least 6.1 %, or at least 6.2%, or at least 6.3%, or at least 6.4%, or at least 6.5%, or at least 6.6%, or at least 6.7%, or at least 6.8%, or at least 6.9%, or at least 7.0%, or at least 7.1 %, or at least 7.2%, or at least 7.3% or at least 7.4%, or at least 7.5%, or at least 7.6%, or at least 7.7%, or at least 7.8%, or at least 7.9% or at least 8.0%, or at least 8.1 %, or at least 8.2%, or at least 8.3%, or at least 8.4%, or at least 8.5%. It is advantageous that biomass feedstocks be pretreated to very low severity wherein xylan number of the pretreated feedstock is 10% or greater, or wherein the residual xylan content of undissolved solids in the pretreated feedstock is at least 5.0% or greater. This very low severity level corresponds to a process in which the total hemicellulose content of the feedstock before pretreatment that is either solubilized or irretrievably lost during pretreatment is minimized. We have unexpectedly discovered that very high final C5 monomer yields of at least 55% theoretical can be obtained without appreciable loss of C6 monomer yields with typical strains of wheat straw, sugar cane bagasse, sweet sorghum bagasse, corn stover, and empty fruit bunches after enzymatic hydrolysis of feedstocks pretreated to very low severity by single-stage autohydrolysis, provided that sufficiently high xylanase and xylosidase activities are employed during enzymatic hydrolysis. At very low severity levels, a large fraction of the feedstock's hemicellulose content remains within the solid fraction after pretreatment, where it can subsequently be hydrolysed to C5 monomers with high recovery using enzymatic hydrolysis, where sufficient xylanase and xylosidase activities are used.

It should be noted that reports concerning "xylose recovery" are often expressed in terms that are not comparable to the xylose recoveries reported here. For example, Ohgren et al. (2007) and Lee et al. (2009) report high xylose recoveries. But these values refer only to xylose recovery from pretreated biomass, not expressed as a percentage of the original hemicellulose content of the feedstock prior to pretreatment. Or for example

WO2010/113129 refers to hemicellulose recovery as a percentage of hemicellulose content of the feedstock prior to pretreatment, but does not specify the monomer yield, which is invariable smaller than the total hemicellulose recovery. In some embodiments, C5 monomer yields of at least 56% theoretical can be obtained in hydrolysate after enzymatic hydrolysis, or at least 57%, or at least 58%, or least 59%, or at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%, or at least 65%.

Enzymatic hydrolysis may be conducted in a variety of different ways. In some

embodiments, the pretreated biomass is hydrolysed as a whole slurry, meaning that substantially all of the solids from the pretreated biomass are subject to enzymatic hydrolysis in one reaction mixture comprising both dissolved and undissolved solids. As used herein the term "whole slurry" refers to an enzymatic hydrolysis reaction mixture in which the ratio by weight of undissolved to dissolved solids at the start of enzymatic hydrolysis is less than 2.2:1 .

Undissolved solids" and "dissolved solids" content of pretreated biomass slurry is determined as follows:

The "total solids" and "filtrated total solids" content are determined according to the procedure described in Weiss et al. (2009). From those values the "undissolved solids" and "dissolved solids" content can be calculated according to the following formulas:

[Undissolved solids] (wt-%) = ([Total solids] (wt-%) - [Filtrated total solids] (wt-%))/(1 - {Filtrated total solids] (wt-%))

[Dissolved solids] (wt-%) = [Total solids] (wt-%) - [Undissolved solids] (wt-%)

In some embodiments, prior to enzymatic hydrolysis, the pretreated biomass is subject to a solid/liquid separation step to produce a separate solid fraction and liquid fraction. Such a separation is generally advantageous in that some component of the dissolved solids in the pretreated biomass typically acts to inhibit activities of one or more enzymes used in enzymatic hydrolysis. For example, removal of dissolved solids content from the pretreated biomass whole slurry clearly improves cellulose conversion achieved with feedstocks pretreated as described here that were subject to enzymatic hydrolysis at high dry matter content using commercially available cellulase preparations optimized for lignocellulosic biomass conversion provided either by GENENCOR™ under the trademark ACCELLERASE TRIO™ or by NOVOZYMES™ under the trademark CELLIC CTEC3™, as is shown in Figure 6 and as explained in Example 4.

Surprisingly, however, where the pretreated biomass slurry is sufficiently diluted to lower dry matter content, the concentrations of the inhibiting substances present in the pretreated biomass slurry are sufficiently diluted that equivalent conversion yields can be obtained in whole slurry hydrolysis, and even at lower enzyme dose levels, as explained in Example 10.

In embodiments which employ a solid/liquid separation step prior to enzymatic hydrolysis, in which enzymatic hydrolysis is desired to be conducted at high dry matter content, it is advantageous to achieve the highest practicable levels of dry matter content in the solid fraction or, alternatively, to remove the highest practicable amount of dissolved solids from the solid fraction. In some embodiments, solid/liquid separation achieves a solid fraction having a DM content of at least 40% by weight, or at least 45%, or at least 50%, or at least 55%. Solid/liquid separation using ordinary screw press systems can typically achieve DM levels as high as 50% in the solid fraction, provided the biomass feedstock has been pretreated in such manner that fiber structure is maintained. In some embodiments, it may be advantageous to incur higher plant capital expenses in order to achieve more effective solid/liquid separation, for example, using a membrane filter press system. In some embodiments, dissolved solids can be removed from a solid fraction by serial washing and pressing or by displacement washing techniques known in the pulp and paper art. In some embodiments, either by solid/liquid separation directly, or by some combination of washing and solid/liquid separation, the dissolved solids content of the solid fraction is reduced by at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%.

The liquid fraction obtained from such a solid/liquid separation can then be maintained separately from solid fraction during enzymatic hydrolysis of the solid fraction. We term this temporary separation "C5 bypass." The C5-rich "bypass" material can then be added back into the hydrolysis mixture, after enzymatic hydrolysis of the solid fraction has reached a desired degree of glucan conversion. We refer to this as "poshydrolysis" of the "C5 bypass" material. In this manner, interference that would otherwise be caused by the enzyme-inhibitory dissolved solids present in the pretreated biomass slurry is minimized, without loss of C5 monomer yield. Liquid fraction obtained from soft lignocellulosic biomass feedstocks such as typical strains of wheat straw, sugar cane bagasse, sweet sorghum bagasse, corn stover, and empty fruit bunches pretreated by single-stage autohydrolysis to very low severity so as to provide pretreated material having xylan number 10% or higher, or having undissolved solids xylan content of 5.0% or greater, typically comprise a small component of C6 monomers (1x), primarily glucose with some other sugars; a larger component of soluble C6 oligomers (about 2x - 7x); a larger component of C5 monomers (about 4x - 8x), primarily xylose with some arabinose and other sugars; and a much larger component of soluble xylo-oligomers (about 18x - 30x). Soluble xylo-oligomers typically include primarily xylohexose, xylopentose, xylotetraose, xylotriose and xylobiose with some higher chain oligomers. These xylose oligomers (or xylo-oligomers) are typically enzymatically hydrolysed during "posthydrolysis" by enzyme activities used in the enzymatic hydrolysis of solid fraction. Or in other words, by mixing the separated liquid fraction and the hydrolysed solid fraction, xylo-oligomers in the liquid fraction are degraded to xylose monomers by the action of enzyme activities remaining within the hydrolysed solid fraction.

Alternatively, in some embodiments, the separated liquid fraction can be used for other purposes. In some embodiments, the separated liquid fraction can be blended with thin stillage obtained after recovery of ethanol from fermentation of hydrolysates. The blended liquid fraction and thin stillage can then be used as a biomethane substrate. Or alternatively, the thin stillage and liquid fractions can be used as separate biomethane substrate. Surprisingly, biomethane potential of thin stillage and also of liquid fraction is increased after very low severity pretreatment that produce pretreated biomass having xylan number 10% or greater. Consequently in some embodiments it is advantageous to conduct C6 fermentation at very low severity, in that increased biomethane yields can offset somewhat decreased ethanol yields. In some embodiments, a biomethane yield of at least 75 NM3 methane/ton biomass feedstock, or at least 78, or at least 80, or at least 82, or at least 85, or at least 87, or at least 90, or at least 92, or at least 95 can be produced from a biomethane substrate comprising combined liquid fraction and thin stillage, where feedstock has been pretreated to low severity log Ro so as to produce a pretreated biomass having xylan number at least 10%, or in which the undissolved solids comprise at least 5.0% by weight xylan. In some embodiments, the whole slurry hydrolysate itself is used as a biomethane substrate.

In some embodiments the invention provides methods of processing lignocellulosic biomass comprising: - Providing soft lignocellulosic biomass feedstock,

- Pretreating the feedstock at pH within the range 3.5 to 9.0 in a single-stage

pressurized hydrothermal pretreatment to very low severity such that the pretreated biomass is characterized by having a xylan number of 10% or higher,

- Separating the pretreated biomass into a solid fraction and a liquid fraction,

- Hydrolysing the solid fraction with or without addition of supplemental water content using enzymatic hydrolysis catalysed by an enzyme mixture comprising

endoglucanase, exoglucanase, B-glucosidase, endoxylanase, and xylosidase activities, and

- Subsequently mixing the separated liquid fraction and the hydrolysed solid fraction, whereby xylo-oligomers in the liquid fraction are degraded to xylose monomers by the action of enzyme activities remaining within the hydrolysed solid fraction.

In some embodiments, the enzymatic hydrolysate, either from a separated solid fraction or from a hydrolysed solid fraction to which separated liquid fraction has been added for posthydrolysis, or from a whole slurry, is subsequently subject to fermentation to produce ethanol.

In some embodiments, thin stillage recovered after fermentation of a hydrolysate is used as a substrate for biomethane production. In some embodiments, separated liquid fraction is blended with thin stillage recovered after fermentation of a hydrolysate and used as a combined biomethane substrate.

It will be readily understood that "solid fraction" and "liquid fraction" may be further subdivided or processed. In some embodiments, biomass may be removed from a pretreatment reactor concurrently with solid/liquid separation. In some embodiments, pretreated biomass is subject to a solid/liquid separation step after it has been unloaded from the reactor, typically using a simple and low cost screw press system, to generate an solid fraction and a liquid fraction.

As is well known in the art, enzymatic hydrolysis using cellulase activity is more efficient where hydrolysis is conducted at lower dry matter content. Higher solids concentration effectively inhibits cellulase catalysis, although the precise reasons for this well known effect are not fully understood. See e.g. Kristensen et al. (2009). While the prevailing view in the art is that hydrolysis at the highest practical dry matter content is

advantageous, this is necessarily associated with increased enzyme consumption. The same enzymatic hydrolysis effects can be achieved using the same enzyme preparations at lower dry matter content with savings of enzyme costs.

One skilled in the art will readily determine, through routine experimentation, a DM level at which to conduct enzymatic hydrolysis that is appropriate to achieve given process goals, for any given biomass feedstock and enzyme preparation. In some embodiments, it may be advantageous to conduct hydrolysis at very high DM > 20%, notwithstanding some resulting increase in enzyme consumption. It is generally considered advantageous to conduct hydrolysis at the highest practicable dry matter level, for a variety of reasons including minimizing water consumption and sparing energy costs in ethanol distillation, where higher sugar concentrations produced by enzymatic hydrolysis at higher dry matter levels result in higher ethanol concentrations, which in turn reduces distillation costs. In some embodiments, enzymatic hydrolysis of a separated solid fraction or of whole slurry may be conducted at 8% DM or greater, or at 9% DM or greater, or at 10% DM or greater, or at 11% DM or greater, or at 12% DM or greater, or at 13% DM or greater, or at 14% DM or greater, or at 15% DM or greater, or at 16% DM or greater, or at 17% DM or greater, or at 18% DM or greater or at 19% DM or greater, or at 20% DM or greater, or at 21% DM or greater, or at 22% DM or greater, or at 23% DM or greater, or at 25% DM or greater, or at 30% DM or greater, or at 35% DM or greater.

In some embodiments, solid fraction is recovered from solid/liquid separation of pretreated biomass at 40% DM or greater, but additional water content is added so that enzymatic hydrolysis may be conducted at lower DM levels. It will be readily understood that water content may be added in the form of fresh water, condensate or other process solutions with or without additives such as polyethylene glycol (PEG) of any molecular weight or surfactants, salts, chemicals for pH adjustment such as ammonia, ammonium hydroxide, calcium hydroxide, or sodium hydroxide, anti-bacterial or anti-fungal agents, or other materials. In order to achieve C5 monomer yields in hydrolysate of at least 55% or greater, according to methods of the invention, enzymatic hydrolysis is conducted using a variety of enzyme activities. The mixture of enzymes used in enzymatic hydrolysis should comprise at least the following activities endoglucanase, exoglucanase, β-glucosidase, endoxylanase, and β-xylosidase. It will be readily understood by one skilled in the art that various different enzyme dose levels may be applied, depending on the dry matter content under which hydrolysis is conducted, the glucan conversion yields desired as process goals, and the hydrolysis times desired as process goals. Thus, an enzyme dose level appropriate for higher dry matter, rapid hydrolysis may be greatly reduced and used in a lower dry matter, longer hydrolysis time frame.

As a general rule, C5 monomer yields in hydrolysate of at least 55% or greater can be achieved relatively quickly, typically in time frames as low as 24 hours, and generally within the range 18 hours to 60 hours. It is advantageous to achieve these very high C5 monomer recoveries as quickly as practicable, because once xylan content is substantially removed from undissolved solids, endo- and exo-glucanases are comparatively less hindered in approach to productive binding. In some embodiments, it can be

advantageous to supplement an enzyme mixture with excess endo- and exo- glucanase activity after high C5 monomer yields are achieved. The hydrolysis time frames over which these high C5 monomer yields can typically be achieved are indicated, for example, in Figure 1 1 , and as explained in Example 10, showing xylan conversion in whole slurry hydrolysis at 12% DM, where total C5 recovery after pretreatment was about 77%. As shown, even at very low enzyme dose levels, C5 monomer yields of at least 55% can be achieved within 40 hours.

Appropriate levels of the various enzyme activities suitable for practicing methods of the invention so as to achieve C5 monomer yields of 55% or greater are typically as follows:

Endoglucanase refers to 4-(1 ,3;1 ,4)- -D-glucan 4-glucanohydrolase also known as β-1 ,4- glucanase (EC 3.2.1.4). For purposes of defining limiting values, endoglucanase activities are determined using the method of Ghose (1987) using hydroxyl-ethyl cellulose (HEC) as substrate and expressed in nkat/g enzyme preparation. Typically endoglucanase levels should be at least 1 100 nkat/g glucan at the start of enzymatic hydrolysis. Depending on process goals, such as hydrolysis speed, conversion degree and dry matter content, endoglucanase activity levels may vary within the range 1 100 - 30000 nkat/g glucan. In some embodiments, the range may be between 1 100-2832, or between 1 130-1529, or between 2317-3852, or between 3000-5120, or between 4000-8000, or between 7000- 10000, or between 1 1000-20000.

Exoglucanase refers to 4-3-D-glucan cellobiohydrolase (EC 3.2.1.91 ). For purposes of defmining limiting values, exoglucanase activities are determined using the method of Bailey and Tahtiharju (2003) using 4-methyl-umbelliferyl^-D-lactoside as substrate and expressed in nkat/g enzyme preparation. Typically exoglucanase levels should be at least 280 nkat/g glucan at the start of enzymatic hydrolysis. Depending on process goals, such as hydrolysis speed, conversion degree and dry matter content, activity levels may vary within the range 280-20000 nkat/g glucan. In some embodiments, the range niay be between 280-690, or between 370-560, or between 400-932, or between 700-1240, or between 1200-2000, or between 3000-5000. β-glucosidase refers to β-D-glucoside glucohydrolase (EC 3.2.1.21 ). For purposes of definining limiting values, β-glucosidase activities are determined using the method of Berghem and Pettersson (1974) using 4-nitrophenyl- -D-glucopyranoside as substrate and expressed in nkat/g enzyme preparation. Typically β-glucosidase levels should be at least 3000 nkat/g glucan at the start of enzymatic hydrolysis. Depending on process goals, such as hydrolysis speed, conversion degree and dry matter content, activity levels may vary within the range 3000-50000 nkat/g glucan. In some embodiments, the range may be between 3000-7500, or between 4000-6010, or between 5000-1000, or between 7000- 14000, or between 15000-25000.

Endoxylanase refers to 4^-D-xylan xylanohydrolase (EC 3.2.1 .8). For purposes of definining limiting values, endoxylanase activities are determined using the method of Bailey et al. (1992) using birchwood xylan as substrate and expressed in nkat/g enzyme preparation. Citrate buffer may be used to adjust pH to an appropriate level for the tested activity. Typically endoxylanase levels should be at least 1400 nkat/g glucan at the start of enzymatic hydrolysis. Depending on process goals, such as hydrolysis speed, conversion degree and dry matter content, activity levels may vary within the range 1400-70000 nkat/g glucan. In some embodiments, the range may be between 1400-3800, or between 4000- 5000, or between 6000-7000, or between 7000-8000, or between 9000-12000, or between 11000- 5000, or between 15000-20000, or between 18000-30000. β-xylosidase refers to 4-p-D-xylan xylohydrolase (EC 3.2.1 .37). For purposes of definining limiting values, β-xylosidase activities are determined using the method of Poutanen and Puis (1988) using para-nitrophenyl-p-D-xylanopyranoside as substrate and expressed in nkat/g enzyme preparation. Typically β-xylosidase levels should be at least 75 nkat/g glucan at the start of enzymatic hydrolysis. Depending on process goals, such as hydrolysis speed, conversion degree and dry matter content, activity levels may vary within the range 75-124, or between 100-300, or between 250-500, or between 400-800, or between 700-20000 nkat/g glucan. In some embodiments, the range may be between 700- 900, or between 800-1400, or between 1 100-1700, or between 1500-2500, or between 2000-3500, or between 3000-5000, or between 4000-10000, or between 8000-20000.

Any of the assays indicated above to be used for activity determinations may be modified in appropriate ways, including that samples may be adjusted to appropriate dilution for purposes of measurements. The assay may be adapted for measurements in

representative samples taken from a hydrolysis mixture by comparison with standard curves where known activities are added to samples with similar background dry matter content.

As will be readily understood by one skilled in the art, the composition of enzyme mixtures suitable for practicing methods of the invention may vary within comparatively wide bounds. Suitable enzyme preparations include commercially available cellulase

preparations optimized for lignocellulosic biomass conversion. Selection and modification of enzyme mixtures during optimization may include genetic engineering techniques, for example such as those described by Zhang et al. (2006) or by other methods known in the art. Commercially available cellulase preparations optimized for lignocellulosic biomass conversion are typically identified by the manufacturer and/or purveyor as such. These are typically distinct from commercially available cellulase preparations for general use or optimized for use in production of animal feed, food, textiles detergents or in the paper industry. In some embodiments, a commercially available cellulase preparation optimized for lignocellulosic biomass conversion is used that is provided by GENENCOR™ and that comprises exoglucanases, endoglucanases, endoxylanases, xylosidases, and beta glucosidases isolated from fermentations of genetically modified Trichoderma reesei, such as, for example, the commercial cellulase preparation sold under the trademark

ACCELLERASE TRIO™. In some embodiments, a commercially available cellulase preparation optimized for lignocellulosic biomass conversion is used that is provided by NOVOZYMES™ and that comprises exoglucanases, endoglucanases, endoxylanases, xylosidases, and beta glucosidases, such as, for example, the commercial cellulase preparations sold under either of the trademarks CELLIC CTEC2™ or CELLIC CTEC3™.

The enzyme activities represented in three commercially available cellulase preparation optimized for lignocellulosic biomass conversion were analysed in detail. For each of these commercial cellulase preparations, levels of twelve different enzyme activities were characterized and expressed per gram protein. Experimental details are provided in Example 8. Results are shown in Table 1 . It should be noted that the assay methods used in this example are not the same as those relied upon for determinations of activities herein. These results provide only a generalized, qualitative comparison and should not be viewed as limiting regarding claims to methods of the invention.

Table 1. Selected activity measurements in commercial cellulase preparations optimized for lignocellulosic biomass conversion.

Activity Substrate Unit definition

(formation)

CTEC 3 ACTrio CTEC2

CBH I 454±2.5 U/g 171+0.4 U/g 381+21 U/g MeUmb-3- cellobioside 1 umole MeUmd equivalent/min

CBH II* Not measurable Not measurable Not measurable

Endo-1 ,4-p-glucanase 466±31 U/g 149+21 U/g 173+15 U/g Avicel PH-101 1 umole glucose equivalent/ min.

2 umole glucose/min.

β-glucosidase 3350175 U/g 891+60 U/g 2447+70 U/g Cellobiose

(Conversion of 1 umole ceUobiose/rn n)

Endo-1 ,4-p-xylanase 278110 U/g 799+55 U/g 306+41 U/g EAX (medium vise.) 1 umole glucose equivalent/min. β-xylosidase 27917.0 U/g 431+22 U/g 87+0.2 U/g EAX (medium vise.) 1 umole xylose/ min. β-L-arabinofuranosidase 2011.0 U/g 9.4+0.4 U/g 12+0.1 U/g WEAX (medium vise.) 1 umole arabinose/ min.

Laccase No activity No activity No activity Syringaldazine -

Amyloglucosidase (AMG) 18+3.6 U/g 29+0.1 U/g 18+1.5 U/g Corn starch (soluble) 1 mole glucose/min. o-amylase 2.710.1 U/g 3.4+0.5 U/g 4.7+1.4 U/g Corn starch (soluble) 1 umole glucose equivalent/ min.

Acetyl xylan esterase 3.8-10 +9-10 u g 3.1-10-Ί1-10 ⁴ U/g 4.2-10 +4.2-10 U/g pNP-acetate 1 umole pNP equivalent/ min.

Ferulic acid esterase No activity No activity No activity Methyl ferulate -

Enzyme mixtures that are effective to hydrolyse lignocellulosic biomass can alternatively be obtained by methods well known in the art from a variety of microorganisms, including aerobic and anaerobic bacteria, white rot fungi, soft rot fungi and anaerobic fungi. See e.g. Singhania et al. (2010). Organisms that produce cellulases typically secrete a mixture of different enzymes in appropriate proportions so as to be suitable for hydrolysis of lignocellulosic substrates. Preferred sources of cellulase preparations useful for conversion of lignocellulosic biomass include fungi such as species of Trichoderma, Penicillium, Fusarium, Humicola, Aspergillus and Phanerochaete.

One fungus species in particular, Trichoderma reesei, has been extensively studied. Wild type Trichoderma reesei secretes a mixture of enzymes comprising two exocellulases (cellobiohydrolases) with respective specificities for reducing and non-reducing ends of cellulose chains, at least five different endocellulases having differing cellulose recognition sites, two B-glucosidases as well as a variety of endoxylanases and exoxylosidases. See Rouvinen, J., et al. (1990); Divne, C, et al. (1994); Martinez, D., et al. (2008). Commercial cellulase preparations typically also include alpha-arabinofuranosidase and acetyl xylan esterase activities. See e.g. Vinzant, T., et al. (2001).

An optimized mixture of enzyme activities in relative proportions that differ from the proportions presented in mixtures naturally secreted by wild type organisms has previously been shown to produce higher sugar yields. See Rosgaard et al. (2007). Indeed, it is has been suggested that optimizations of enzyme blends including as many as 16 different enzyme proteins can be advantageously determined separately for any given biomass feedstock subject to any given pretreatment. See Billard, H., et al. (2012); Banerjee, G., et al. (2010). As a commercial practicality, however, commercial enzyme providers typically seek to produce the smallest practicable number of different enzyme blends, in order that economies of scale can be obtained in large-scale production.

In some embodiments, it can be advantageous to supplement a commercially available cellulase preparation optimized for lignocellulosic biomass conversion with one or more additional or supplemental enzyme activities. In some embodiments, it may be advantageous simply to increase the relative proportion of one or more component enzymes present in the commercial preparation. In some embodiments, it may be advantageous to introduce specialized additional activities. For example, in practicing methods of the invention using any given biomass feedstock, particular unhydrolysed carbohydrate linkages may be identified that could be advantageously hydrolysed through use of one or more supplemental enzyme activities. Such unhydrolysed linkages may be identified through characterization of oligomeric carbohydrates, using methods well known in the art, in soluble hydrolysates or in insoluble unhydrolysed residual. Unhydrolysed linkages may also be identified through comprehensive microarray polymer profiling, using monoclonal antibodies directed against specific carbohydrate linkages, as described by Nguema-Ona et al. (2012). In some embodiments it can be advantageous to supplement a commercially available cellulase preparation optimized for lignocellulosic biomass conversion using any one or more of additional endoxylanase, B-glucosidase, mannanase, glucouronidase, xylan esterase, amylase, xylosidase, glucouranyl esterase, or

arabinofuranosidase.

In some embodiments, it can alternatively be advantageous to produce enzymes on-site at a lignocellulosic biomass processing facility, as described by Humbird et al. (201 1 ). In some embodiments, a commercially available cellulase preparation optimized for lignocellulosic biomass conversion may be produced on-site, with or without customized supplementation of specific enzyme activities appropriate to a particular biomass feedstock.

In some embodiments the enzyme mixture may further include any one or mor of mannosidases (EC 3.2.1.25), a-D-galactosidases (EC 3.2.1.22), a-L-arabinofuranosidases (EC 3.2.1.55), a-D-glucuronidases (EC 3.2.1.139), cinnamoyl esterases (EC 3.1 .1 .-), or feruloyl esterases (EC 3.1.1.73), acetyl xylan esterases (EC 3.1 .1.72); B-1 ,3 xylosidase activity (EC 3.2.1.72); alpha 1 ,3 and/or alpha 1 , 5 arabinofuranosidase activity (EC

3.2.1 .23); or other activities. Another startling feature of biomass that has been pretreated by single-stage

autohydrolysis to very low severity levels is that the concentrations of pretreatment byproducts that serve as inhibitors of fermentive organisms are kept to very low levels. As a consequence, it is typically possible to use hydrolysed biomass obtained by methods of the invention directly in fermentations, without requirement for any washing or other detoxification step.

As is well known in the art, autohydrolysis hydrothermal pretreatment typically produces a variety of soluble by-products which act as "fermentation inhibitors," in that these inhibit growth and/or metabolism of fermentive organisms. Different fermentation inhibitors are produced in different amounts, depending on the properties of the lignocellulosic feedstock and on the severity of pretreatment. See Klinke et al. (2004). At least three categories of fermentation inhibitors are typically formed during autohydrolysis pretreatment: (1) furans, primarily 2-furfural and 5-HMF (5 hydroxymethylfurfural) which are degradation products from mono- or oligo-saccharides; (2) monomeric phenols, which are degradation products of the lignin structure; and (3) small organic acids, primarily acetic acid, which originate from acetyl groups in hemicelluloses, and lignin. The mixture of different inhibitors has been shown to act synergistically in bioethanol fermentation using yeast strains, see e.g. Palmquist et al. (1999), and, also, using ethanolic Escherichia coli, see e.g. Zaldivar et al. (1999). In some embodiments, it can be advantageous to subject pretreated biomass to flash evaporation, using methods well known in the art, in order to reduce levels of volatile inhibitors, most notably furfural. Using autohydrolysis with typical strains of biomass feedstocks such as wheat straw, sweet sorghum bagasse, sugar cane bagasse, corn stover, and empty fruit bunches, pretreated to xylan number 10% or higher, in our experience only acetic acid and furfural levels are potentially inhibitory of fermentive organisms. Where biomass feedstocks are pretreated at DM 35% or higher to xylan number 10% or higher, and where solid fraction is subsequently hydrolysed enzymatically at 25% or lower DM, with added water to adjust DM but without washing steps, furfural levels in the hydrolysate can typically be kept under 3 g/kg and acetic acid levels beneath 9 g/kg. These levels are typically acceptable for yeast fermentations using specialized strains. During enzymatic hydrolysis, some additional acetic acid is released from degradation of hemicellulose in the solid fraction. In some embodiments, it may be advantageous to remove some acetic acid content from liquid fraction and/or hydrolysed solid fraction using electrodialysis or other methods known in the art.

Different feedstocks can be pretreated using single-stage autohydrolysis to low severity log Ro sufficient to produce pretreated biomass having xylan number 10% or greater using a variety of different combinations of reactor residence times and temperatures. One skilled in the art will readily determine through routine experimentation an appropriate pretreatment routine to apply with any given feedstock, using any given reactor, and with any given biomass reactor-loading and reactor-unloading system. Where feedstocks are pretreated using a continuous reactor, loaded by either a sluice-system or a screw-plug feeder, and unloaded by either a "particle pump" sluice system or a hydrocyclone system, very low severity of 10% or greater xylan number can be achieved using typical strains of wheat straw or empty fruit bunches by a temperature of 180° C and a reactor residence time of 24 minutes, or temperatures within the range 175oC to 185oC for residence times within the range 18 to 35 minutes, or temperatures within the range 170oC to 190oC for residence times within the range 13 to 40 minutes. For typical strains of corn stover, sugar cane bagasse, and sweet sorghum bagasse, very low severity of 10% or greater xylan number can typically be achieved using within the range 175oC to 185oC for residence times within the range 8 to 25 minutes, or temperatures within the range 170oC to 190oC for residence times within the range 6 to 35 minutes. It will be readily understood by one skilled in the art that residence times and temperatures maybe adjusted to achieve comparable levels of R ₀ severity.

Enzymatic hydrolysis of feedstocks pretreated to xylan number 10% or higher can typically be conducted at high DM > 20% with commercially reasonable enzyme consumption, without requirement for specific washing or de-toxification steps, where the solid fraction is pressed to at least 40% DM, or where dissolved solids content of the solid fraction is reduced by at least 50%.

In some embodiments the enzyme mixture may further include any one or more of mannosidases (EC 3.2.1 .25), a-D-galactosidases (EC 3.2.1 .22), a-L-arabinofuranosidases (EC 3.2.1 .55), a-D-glucuronidases (EC 3.2.1.139), cinnamoyl esterases (EC 3.1 .1 .-), or feruloyl esterases (EC 3.1.1 .73), acetyl xylan esterases (EC 3.1 .1 .72); B-1 ,3 xylosidase activity (EC 3.2.1.72); alpha 1 ,3 and/or alpha 1 , 5 arabinofuranosidase activity (EC

3.2.1 .23); or other activities.

One skilled in the art will readily determine an appropriate dose level of any given enzyme preparation to apply, and appropriate pH and temperature conditions as well as an appropriate duration for enzymatic hydrolysis. As mentioned previously, duration of hydrolysis may vary depending on process goals. Longer hydrolysis leads to better ultimate glucose conversion yields, but imposes greater capital and operating costs at production scale. Hydrolysis duration in some embodiments is at least 24 hours, or at least 36 hours, or at least 48 hours, or at least 64 hours, or at least 72 hours, or at least 96 hours, or for a time between 24 and 150 hours. It is generally advantageous to maintain lower enzyme dose levels, so as to minimize enzyme costs. In some embodiments, it can be advantageous to use a high enzyme dose. In practicing methods of the invention, one skilled in the art can determine an economic optimisation of enzyme dose in consideration of relevant factors including local biomass costs, market prices for product streams, total plant capital costs and amortization schemes, and other factors. In embodiments where a commercially available cellulase preparation optimized for lignocellulosic biomass conversion is used, a general dose range provided by manufacturers can be used to determine the general range within which to optimize.

In some embodiments, after a separated solid fraction has been enzymatically hydrolysed to a desired degree of conversion, the liquid fraction, which has been maintained in C5 bypass, is mixed with the hydrolysate mixture for post-hydrolysis. In some embodiments, all of the recovered liquid fraction may be added at one time, while in other embodiments, some component of the liquid fraction may be removed and/or liquid fraction may be added incrementally. In some embodiments, prior to mixing with liquid fraction, the solid fraction is hydrolysed to at least 50%, or at least 55%, or at least 60% cellulose

conversion, meaning that at least the specified theoretical yield of glucose monomers is obtained in the hydrolysate. A substantial portion of xylo-oligomers present in liquid fraction can typically be hydrolysed to xylose monomers by action of xylanase and other enzymes that remain active within the hydrolysate mixture. In some embodiments post- hydrolysis is conducted for at least 6 hours, or for a time between 15 and 50 hours, or for at least 24 hours. In some embodiments, at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% by mass of xylo- oligomers present in the liquid fraction are hydrolysed to xylose monomers during post- hydrolysis by action of xylanase and other enzymes that remain active within the hydrolysate mixture. In some embodiments, the liquid fraction is mixed with hydrolysate directly, without further addition of chemical additives. In some embodiments, some components of liquid fraction such as acetic acid, furfural or phenols may be removed from liquid fraction prior to mixing with hydrolysate.

In some embodiments, enzymatic hydrolysis of the solid fraction and/or post-hydrolysis of the liquid fraction may be conducted as a simultaneous saccharification and fermentation (SSF) process. As is well known in the art, when SSF can be conducted at the same temperature as that which is optimal for enzymatic hydrolysis, enzyme consumption can be minimized because a fermentive organism introduced during the course of enzymatic hydrolysis consumes glucose and xylose monomers and thereby reduces product inhibition of enzyme catalyzed reactions. In some embodiments, post-hydrolysis is only conducted after the fiber fraction has been hydrolysed, without addition of fermentive organism, to at least 60% cellulose conversion. In some embodiments SSF can be conducted after an initial period of enzymatic hydrolysis, that is a fermentive organisms added after an initial period of enzymatic hydrolysis, and both fermentation and hydrolysis continued, optionally at a temperature that is not optimal for enzymatic hydrolysis.

Where biomass feedstocks such as typical strains of wheat straw, sugar cane bagasse, sweet sorghum bagasse, corn stover or empty fruit bunches are pretreated at 35% or greater DM by single-stage autohydrolysis to severity log Ro sufficiently low so as to produce pretreated biomass having xylan number 10% or greater, where solid fraction of the pretreated biomass is obtained having at least 40% DM or having at least 50% removal of dissolved solids, where solid fraction is subsequently subject to enzymatic hydrolysis at DM between 15 and 27% using a commercially available cellulase preparation optimized for lignocellulosic biomass conversion, where enzymatic hydrolysis is conducted for at least 48 hours, where liquid fraction is added to the solid fraction hydrolysate after at least 50% glucose conversion has been obtained, and where the added liquid fraction is subject to post-hydrolysis for a period of at least 6 hours, it is typically possible to achieve C5 monomer concentrations in the combined C5/C6 hydrolysate that correspond to C5 monomer yields of 60% or greater of the theoretical maximal C5 monomer yield.

In some embodiments, a combined C5/C6 hydrolysate can be directly fermented to ethanol using one or more modified yeast strains.

Figure 9 shows a process scheme for one embodiment. As shown, soft lignocellulosic biomass is soaked, washed or wetted to DM 35% or greater. The biomass is pretreated at pH within the range of 3.5 to 9.0 using pressurized steam in single-stage autohydrolysis to a severity characterized by xylan number 10% or greater. The pretreated biomass is subject to solid/liquid separation producing a liquid fraction and a solid fraction having DM content 40% or greater. The solid fraction is adjusted to an appropriate DM content then subject to enzymatic hydrolysis at DM content 15% or greater to a degree of cellulose conversion 60% or greater. The separated liquid fraction is subsequently mixed with the hydrolysed solid fraction and subject to post-hydrolysis, whereby a substantial quantity of xylo-oligomers present in the liquid fraction are hydrolysed to monomeric xylose. After the end of hydrolysis and post-hydrolysis as described, the C5 monomer yield is typically at least 60% while the cellulose conversion is similarly at least 60%.

In alternative embodiments, the pretreated biomass is subject to enzymatic hydrolysis as a whole slurry. In still other embodiments, a liquid fraction is separated and a solid fraction subject to enzymatic hydrolysis and subsequent fermentation. In some embodiments, following ethanol recovery from such a fermentation, the remaining thin stillage can be blended with separated liquid fraction and used as biomethane substrate.

Examples:

Example 1. "Xylan number" characterization of solid fraction as a measure of pretreatment severity. Wheat straw (WS), corn stover (CS) , Sweet sugarcane bagasse (SCB) and Empty Fruit Bunches (EFB) were soaked with 0-10 g acetic acid/kg dry matter biomass, pH > 4.0, prior to pretreatment at 35-50% dry matter About 60 kg DM/h biomass was pretreated at temperatures from 170-200°C with a residence time of 12-18 minutes. The biomass was loaded into the reactor using a sluice system and the pretreated material unloaded using a sluice system. The pressure within the pressurized pretreatment reactor corresponded to the pressure of saturated steam at the temperature used. The pretreated biomass was subject to solid/liquid separation using a screw press, producing a liquid fraction and a solid fraction having about 30% dry matter. The solid fraction was washed with about 3 kg water/kg dry biomass and pressed to about 30% dry matter again. Details concerning the pretreatment reactor and process are further described in Petersen et al. (2009).

Raw feedstocks were analysed for carbohydrates according to the methods described in Sluiter el al. (2005) and Sluiter et al. (2008) using a Dionex Ultimate 3000 HPLC system equipped with a Rezex Monossacharide H+ column from Phenomenex. Samples of liquid fraction and solid fraction were collected after three hours of continuous pretreatment and samples were collected three times over three hours to ensure that a sample was obtained from steady state pretreatment. The solid fractions were analysed for carbohydrates according to the methods described in Sluiter et al. (2008) with an Ultimate 3000 HPLC system from Dionex equipped with a Rezex Monossacharide H+ Monosaccharide column. The liquid fractions were analysed for carbohydrates and degradation products according to the methods described in Sluiter et al. (2006) with an Ultimate 3000 HPLC system from Dionex equipped with a Rezex Monossacharide H+ Monosaccharide column. Degradation products in the solid fraction were analysed by suspension of the solid fraction in water with 5mM sulphuric acid in a ratio of 1 :4 and afterward analysed according to the methods described in Sluiter et al. (2006) with an Ultimate 3000 HPLC system from Dionex equipped with a Rezex Monossacharide H+ column. The dry matter content and the amount of suspended solids was analysed according to the methods described in Weiss et al. (2009). Mass balances were set up as described in Petersen et al. (2009) and cellulose and hemicellulose recoveries were determined. The amount of sugars which were degraded to 5-HMF or furfural and the amount of acetate released from hemicelleulose during pretreatment per kg of biomass dry matter was quantified as well, although loss of furfural due to flashing is not accounted for.

The severity of a pretreatment process is commonly described by a severity factor, first developed by Overend et al. (1987). The severity factor is typically expressed as a log value such that log(Ro)=t*eksp((T-Tref)/14.75), where Ro is the severity factor, f is the residence time in minutes, 7 ^" is the temperature and is the reference temperature, typically 100°C. The severity factor is based on kinetics of hemicellulose solubilisation as described by Belkecemi et al. (1991), Jacobsen and Wyman (2000) or Lloyd et al. (2003). The severity of a pretreatment is thus related to residual hemicellulose content remaining in the solid fraction after pretreatment.

Solid fractions prepared and washed as described were analysed for C5 content according to the methods described by Sluiter et al. (2008) with a Dionex Ultimate 3000 HPLC system equipped with a Rezex Monossacharide H+ column from Phenomenex. The xylan content in the solid fraction produced and washed as described above is linearly depended upon the severity factor for soft lignocellulosic biomasses such as for example wheat straw, corn stover of EFB when pretreating by hydrothermal autohydrolysis. The definition of severity as the xylan content in a solid fraction prepared and washed as described above is transferable between pretreatment setups. Xylan number is the measured xylan content in the washed solid fractions, which includes some contribution from soluble material. The dependence of xylan number on pretreatment severity log(R ₀ ) is shown in Figure 1 for wheat straw, corn stover, sugarcane bagasse and empty fruit bunches from palm oil processing.

As shown, there exists a clear, negative linear correlation between xylan number and pretreatment severity for each of the tested biomass feedstocks pretreated by single-stage autohydrolysis.

The xylan content of undissolved solids in the experiments was also calculated as the total xylan content in fibre fraction from which is subtracted the content of dissolved xylan in the liquid between the fibres (oligomers and monomers). [Solid Xylan in fibres](wt-%) = [Total xylan in fibre fraction] (wt-%)- [Dissolved xylan in fibre fraction] (wt-%)

The dissolved xylan content is calculated by [(dissolved solids/ total solids) as wt% in fibre fraction] x [dissolved xylan concentration in liquid fraction].

Calculated xylan content of undissolved solids in wt% is shown as a function of Xylan number in Figure 2 for pretreated wheat straw (PWS), corn stover (PCS), sugarcane bagasse (SCB) and empty fruit bunches from oil palm (PEFB).

Example 2. C5 recovery as a function of pretreatment severity.

Biomass feedstocks were pretreated and samples characterized as described in example 1 . Figure 3 shows the C5 recoveries (xylose + arabinose) as a function of xylan number for experiments where wheat straw was pretreated by single-stage autohydrolysis. C5 recoveries are shown as water insoluble solids (WIS), water soluble solids (WSS) and total recovery. As shown, C5 recovery as both water insoluble and water soluble solids increases as xylan number increases. As xylan number increases over 10%, C5 recovery as water soluble solids diminishes while C5 recovery as water insoluble solids continues to increase

Typical strains of wheat straw tested contained about 27% hemicellulose on dry matter basis prior to pretreatment. Figure 4 shows total C5 recovery after pretreatment as a function of Xylan number for wheat straw, corn stover, sugarcane bagasse and EFB pretreated by autohydrolysis. Typical strains of corn stover, sweet sugarcane bagasse and EFB tested contained about 25%, 19% and 23% respectively of C5 content on dry matter basis prior to pretreatment. As shown, for all feedstocks, total C5 recovery after pretreatment is dependent upon pretreatment severity as defined by the xylan number of pretreated biomass. As shown, where 90% of C5 content recovered after pretreatment can be fully hydrolysed to C5 monomer, at least 60% final C5 monomer yield after enzymatic hydrolysis can typically be expected where pretreatment severity is characterized by producing a xylan number of 10% or higher.

Example 3. Production of degradation products that inhibit enzymes and yeast growth as a function of pretreatment severity.

Biomass feedstocks were pretreated and samples characterized as described in example 1 . Figure 5 shows the dependence of acetic acid release and production of furfural and 5- hydroxy-methyl-fufural (5-HMF) as a function of xylan number for experiments where wheat straw was pretreated by single-stage autohydrolysis. As shown, production of these degradation products, which are well known to inhibit fermentive yeast and which in some cases also inhibit cellulase enzymes, exhibits an exponential increase at xylan numbers lower than 10%. At xylan number 10% and higher, the levels of furfural and acetic acid fall within ranges that permit fermentation of pretreated biomass without requirement for detoxification steps. In the case of acetic acid, levels are further increased during enzymatic hydrolysis of biomass pretreated to xylan number 10% and higher, although typically to levels that are well tolerated by yeast modified to consume both C5 and C6 sugars.

Example 4. Inhibition of cellulase enzymes by material remaining in solid fraction as a function of DM% of solid fraction.

Experiments were conducted in a 6-chamber free fall reactor working in principle as the 6- chamber reactor described and used in WO2006/056838. The 6-chamber hydrolysis reactor was designed in order to perform experiments with liquefaction and hydrolysis at solid concentrations above 20 % DM. The reactor consists of a horizontally placed drum divided into 6 separate chambers each 24 cm wide and 50 cm in height. A horizontal rotating shaft mounted with three paddles in each chamber is used for mixing/agitation. A 1.1 kW motor is used as drive and the rotational speed is adjustable within the range of 2.5 and 16.5 rpm. The direction of rotation is programmed to shift every second minute between clock and anti-clock wise. A water-filled heating jacket on the outside enables control of the temperature up to 80°C. The experiments used wheat straw, pretreated by single-stage autohydrolysis using the system described in example 1 . The biomass was wetted to a DM of > 35% and pretreated at pH > 4.0 by steam to severity log Ro approximately 3.7, producing pretreated material having xylan number 10.5%. The pretreatment was conducted in the Inbicon pilot plant in Skaerbaek, Denmark. The biomass was loaded into the pretreatment reactor using a sluice system and the pretreated biomass removed from the reactor using a sluice system. The pretreated biomass was, in some cases, subject to solid/liquid separation using a screw press, producing a liquid fraction and a solid fraction. The solid fraction had a DM content of about 30%, contained the majority of initial cellulose and lignin, part of the hemicellulose and a total of about 25% of the dissolved solids.

The chambers of the 6 chamber reactor were filled with either total pretreated biomass comprising all dissolved and undissolved solids or pressed solid fraction comprising about 25% of total dissolved solids. Dry matter content was adjusted to 19 % DM. The pretreated biomass was then hydrolyzed at 50°C and pH 5.0 to 5.3 using 0.08 ml CTec2 ™ from Novozymes / g glucan or 0.2-0.3 ml Accellerase TRIO™ from Dupont, Genencor / g glucan. These dose levels of these commercially available cellulase preparations optimized for lignocellulosic biomass conversion were well within the range suggested by the manufacturers. Enzymatic hydrolysis experiments were conducted for 96 hours at a mixing speed of 6 rpm.

It can be shown that the enzyme activities in the experiment with Accellerase TRIO as measured as described herein were initially within the range exoglucanase 280-5000 nkat/g glucan, endoglucanase 1 100-20000 nkat/g glucan, β-glucosidase 3000-25000 nkat/g glucan, endoxylanase 1400-30000 nkat/g glucan, β-xylosidase 75-25000 nkat/g glucan nkat/g glucan.

Figure 6 shows cellulose conversion after enzymatic hydrolysis under these conditions as a function of % dissolved solids removed prior to enzymatic hydrolysis. As shown, removal of 75% dissolved solids at these enzyme dose levels improves cellulose conversion by 10-20% in absolute terms. Thus, in cases where enzymatic hydrolysis is to be conducted using a separated solid fraction, it is advantageous to press the solid fraction to DM content at least 40% or to otherwise reduce dissolved solids content by at least 50% prior to enzymatic hydrolysis, since this will typically provide improved enzyme

performance.

Example 5. Sugar content and hydrolysis of liquid fraction from biomass pretreated to xylan number > 10%.

Wheat straw, corn stover, and sugar cane bagasse were pretreated to severity log Ro 3.63, producing pretreated wheat straw (WS) having xylan number 11.5%, to log Ro 3.51 producing pretreated sugar cane bagasse (SCB) having xylan number 12.3% and to log Ro 3.35, producing pretreated corn stover (CS) having xylan number 15.5%. The pretreated feedstocks were subject to solid/liquid separation to produce a liquid fraction and a solid fraction, as described in example 4. The liquid fractions were analysed for carbohydrates and degradation products according to the methods described in (Sluiter, Hames et al. 2005) using a Dionex Ultimate 3000 HPLC system equipped with a Rezex Monosaccharide column. Table 2 shows the sugar content of liquid fractions expressed as a percent of DM content broken down into categories of oligomeric and monomeric glucose/glucan, xylose/xylan and arabinose/arabinan. As shown, while some glucose content is present in both monomeric and oligomeric form, the bulk of the sugar content is oligomeric xylan. The predominance of xylan oligomers in liquid fraction obtained using autohydrolysis is in noted contrast with the liquid fraction obtained using dilute acid pretreatment. In biomass pretreated by dilute acid hydrothermal pretreatment, the liquid fraction is typically hydrolysed to monomeric constituents by actions of the acid catalyst.

Table 2. Sugar content of liquid fractions in biomass pretreated to xylan number > 0%.

The liquid fraction from pretreated wheat straw was further characterized by HPLC analysis using a Thermo Scientific Dionex CarboPacTM PA200 column using a modular Dionex ICS-5000 chromatographic system. The analytes were separated using NaOH/NaOAc-gradient conditions and measured by integrated and pulsed amperometric detection (IPAD) using a gold electrode. Figure 7 shows an HPLC chromatogram in which the elution profile of xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (Χβ) standards is super-imposed as the upper trace over the lower trace, which depicts the elution profile of liquid fraction. As shown, liquid fraction of the autohydrolysed biomass contains a mixture comprising a small amount of xylose monomer and comparatively larger amounts of xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (Χβ), along with other materials.

Example 6. Enzymatic hydrolysis of solid fraction and addition of liquid fraction after the fibre hydrolysis from biomass pretreated to xylan number > 10% and pressed to > 40% DM followed by post hydrolysis.

Experiments were conducted in a 6-chamber free fall reactor as described in example 4.

The experiments used wheat straw, corn stover, or sugar cane bagasse pretreated by single-stage autohydrolysis to severity log Ro between about 3.19 and 3.73 to produce pretreated biomass having xylan numbers ranging from 1 1.5 to 15.6%. The biomass was cut and wetted to a DM of > 35% and pretreated by steam at 170-190°C for 12 min. The pretreatment was conducted in the Inbicon pilot plant in Skaerbaek, Denmark. The pretreated biomass was subject to solid/liquid separation using a screw press to produce a solid fraction having > 40% DM. The liquid fraction was saved (C5 bypass) so as to be subsequently added to the hydrolysate (posthydrolysis).

The chambers of the 6 chamber reactor were filled with about 10 kg pressed pretreated biomass solid fraction and adjusted by water addition to 19-22 % DM. The pretreated solid fraction was hydrolyzed at 50°C and pH 5.0 to 5.3 using ACCELLERASE TRIO™ from GENENCOR-DuPONT. The mixing speed was 6 rpm. The hydrolysis experiments were run for 96 hours and afterwards the savewd liquid fraction (C5 bypass) was added and post hydrolysis was run for 48 hours at 50°C and pH 5.0 to 5.3.

HPLC samples were taken daily to follow the conversion of cellulose and hemicellulose and analysed for glucose, xylose and arabinose using a Dionex Ultimate 3000 HPLC system equipped with a Rezex Monosaccharide column with quantification through use of external standard.

Figure 8 shows hydrolysis data for conversion of hemicellulose with addition of liquid fraction after 96 hours hydrolysis of solid fraction using sugar cane bagasse pretreated to xylan number 12.3% and hydrolysed using 0.3 ml Accellerase Trio™ (Genencor) per g glucan. Shown is a typical hydrolysis profile. C5 monomer recovery is expressed as a percent of theoretical yield from the material present in the hydrolysis reaction. Most of the hemicellulose within the solid fraction has been converted to monomeric sugars within the first 24 hours in hydrolysis of the solid fraction. Addition of liquid fraction after 96 hours increases the theoretical potential yield, which explains the drop in C5 conversion observed just after liquid fraction is added. Within the first 24 hours most of the C5 from liquid fraction is converted to monomers. Comparing the C5 conversion just before liquid fraction is added with the end point of the hydrolysis, it is possible to calculate the C5 conversion in the liquid fraction as 90 % when using sugar cane bagasse under these conditions.

Table 3 shows hydrolysis data for different biomasses pretreated under different circumstances and hydrolysed using different dose levels of a commercially available cellulase preparation optimized for lignocellulosic biomass conversion, Accellerase Trio™ (Genencor). All enzyme dose levels used were within the range suggested by the manufacturer. As shown, using single-stage autohydrolysis and enzymatic hydrolysis with C5 bypass and post-hydrolysis, C5 monomer yields of 60% or greater can be achieved using manufacturers' recommended doses of commercially available cellulase

preparations optimized for lignocellulosic biomass conversion while still achieving cellulose conversion of 60% or greater.

It can be shown that the enzyme activities in the experiments referred to in Table 3 with Accellerase TRIO measured as described herein were initially within the range

exoglucanase 280-5000 nkat/g glucan, endoglucanase 1 100-20000 nkat g glucan, β- glucosidase 3000-25000 nkat g glucan, endoxylanase 1400-30000 nkat/g glucan, β- xylosidase 75-25000 nkat/g glucan nkat/g glucan. Table 3. Hydrolysis yields using very low severity single-stage autohydrolysis with C5 bypass and post-hydrolysis.

WS SCB SCB CS CS EFB

Dry matter after soaking [wt%] 40% 39% 39% 40% 40% 39%

Residence time [min] 12.0 12.0 12.0 12.0 12.0 12.0

Temperature [°C] 183.0 182.7 182.7 174.5 174.5 185.2

Pretreatment severity [logRo] 3.52 3.51 3.51 3.27 3.27 3.58

C5 recovery from pretreatment [%] 74% 87% 87% 88% 88% 84%

Xylan number 11.5% 12.3% 12.3% 15.6% 15.6% 15.5%

Enzyme dosage [mL Ac. TRIO/g glucan] 0.2 0.3 0.3 0.3 0.2 0.4

%TS in fiber hydrolysis 22% 22% 22% 19% 22% 22%

Cellulose conversion after hydrolysis

(96h) 78% 64% 66% 68% 58% 69%

Hemicellulose conversion (C5 recovery)

after hydrolysis (96h) 80% 73% 73% 61 % 61 % 75%

%TS in second hydrolysis 18% 17% 17% 16% 18% 18

Cellulose conversion after post hydrolysis

(144h) 78% 65% 67% 67% 61 % 72%

Hemicellulose conversion (C5

recovery)after post hydrolysis (144h) 90% 79% 78% 71 % 68% 83%

Overall cellulose conversion 78% 65% 67% 67% 61 % 72%

Overall C5 monomer yield 67% 69% 68% 63% 60% 70%

Example 7. Co-fermentation to ethanol of C5 and C6 sugars in combined hydrolysate by modified yeast.

As an example on the use of a hydrolysate produced from soft lignocellulosic biomass (in this case wheat straw) prepared by single-stage autohydrolysis pretreatment to a xylan number > 10%, Figure 9 shows data for a fermentation performed without detoxification or any other process steps before fermentation with GMO yeast able to convert both C5 and C6 sugars (strain V1 from TERRANOL™). The solid fraction from pretreated wheat straw separated as described in example 4 was hydrolyzed using Cellic Ctec2™ from Novozymes and then combined with saved liquid fraction and used without any detoxification to remove fermentation inhibitors. The hydrolysate was adjusted to pH 5.5 with KOH pellets before fermentation and supplemented with 3 g/L urea. The fermentation was conducted as a batch fermentation. The initial cell concentration in the reactor was 0.75 g dw/L. The fermentations were controlled at pH 5.5 using automatic addition of 10% NH3. The temperature was kept at 30°C and the stirring rate was 300 rpm. As shown, glucose and xylose are readily consumed and ethanol readily produced, notwithstanding the presence of acetic acid, furfural and other compounds that would typically prove inhibitory at higher levels of pretreatment severity.

Example 8. Experimental determination of activity levels in commercial cellulase preparations.

Commercial preparations of ACCELLERASE TRIO™ from GENENCOR™ and CELLIC CTEC2™ and CELLIC CTEC3™ from NOVOZYMES™ were diluted so that the enzyme preparations would have equivalent density, meaning that equivalent sized aliquots had equivalent mass. Equivalent volumes of diluted enzyme preparations were added and assay determinations made in duplicate or triplicate.

Assay of CBHI (exocellulase) activity was conducted in 50 mM NaOAC buffer at pH 5, 25° C, for 25 minutes. Activity was determined in triplicate by following continuous rate of 4-Methylumbelliferon release (Abs: 347nm) from the model substrate

4-methylumbelliferyl- -cellobioside. Activity unit was 1 umole MeUmb equivalent/minute. Enzyme preparation concentrations were 0.16, 0.14, 0.17mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays. Substrate concentration was 0.5mg/ml.

Assay of Endo-1 ,4- -glucanase activity was conducted in 50 mM NaOAC buffer, pH 5; 50 °C, for 60 minutes. Activity was determined in triplicate by following absorbance change associated with generation of reducing ends from the model substrate Avicel PH-101 . Activity unit was 1 μηιοΐβ glucose equivalent/min. Enzyme preparation concentrations were 0.80, 0.67, 0.79 mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays. Substrate concentration was 80 mg/ml. Assay of β-glucosidase activity was conducted in 50 mM NaOAC buffer, pH 5; 50 °C, for 20 minutes. Activity was determined in triplicate by following absorbance change associated with release of glucose from model substrate cellobiose. Activity unit was 2 pmole glucose/min. Enzyme preparation concentrations were 0.1 , 0.12, 0.12 mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays. Substrate concentration was 1.7 mg/ml.

Assay of Endo-1 ,4- -xylanase activity was conducted in 50 mM NaOAC buffer, pH 5; 50 °C, for 60 minutes. Activity was determined in triplicate by following absorbance change associated with generation of reducing ends from the model substrate water extractable arabinoxylan. Activity unit was 1 μιηοΐΘ glucose equivalent/min. Enzyme preparation concentrations were 1.12, 0.97, 1.12 mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays. Substrate concentration was 10 mg/ml.

Assay of β-xylosidase activity was conducted in 50 mM NaOAC buffer, pH 5; 50 °C, for 60 minutes. Activity was determined in duplicate by following release of xylose associated with hydrolysis of the model substrate water extractable arabionxylan. Activity unit was 1 mole xylose/min. Enzyme preparation concentrations were 1.12, 0.97, 1.12 mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays. Substrate concentration was 10 mg/ml.

Assay of β-L-arabinofuranosidase activity was conducted in 50 mM NaOAC buffer, pH 5; 50 °C, for 60 minutes. Activity was determined in triplicate by following release of arabinoase associated with hydrolysis of the model substrate water extractable

arabionxylan. Activity unit was 1 pmole arabinose/min. Enzyme preparation concentrations were 1.12, 0.97, 1.12 mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays.

Substrate concentration was 10 mg/ml.

Assay of Amyloglucosidase (AMG) activity was conducted in 50 mM NaOAC buffer, pH 5; 50 °C, for 80 minutes. Activity was determined in triplicate by following absorbance change associated with glucose release from the model substrate soluble com starch. Activity unit was 1 pmole glucose/min. Enzyme preparation concentrations were 1.12, 0.97, 1 .12 mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays. Substrate concentration was 10 mg/ml.

Assay of a-amylase activity was conducted in 50 mM NaOAC buffer, pH 5; 50 °C, for 60 minutes. Activity was determined in triplicate by following absorbance change associated with generation of reducing ends from the model substrate soluble corn starch. Activity unit was 1 pmole glucose equivalent/min. Enzyme preparation concentrations were 1.12, 0.97, 1 .12 mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays. Substrate concentration was 10 mg/ml.

Assay of acetyl xylan esterase activity was conducted in 100 mM Succinate buffer, pH 5; 25 °C, for 25 minutes. Activity was determined in triplicate by following continuous rate of 4-Nitrophenyl release (Abs: 410 nm) from the model substrate 4 4-Nitrophenyl acetate. Activity unit was 1 pmole pNP equivalent/min. Enzyme preparation concentrations were 0.48, 0.42, 0.51 mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays. Substrate concentration was 10 mg/ml.

Results of the activity determinations are shown in Table 1.

These results provide a qualitative comparison between the enzyme preparations, but are in most cases not conducted according to the methods used to determine nkat values for enzyme activities for purposes of claims herein.

Example 9. Identification of enzyme activities important for achieving high C5 monomer yield in enzymatic hydrolysis of feedstock pretreated by low severity, single stage autohydrolysis.

Wheat straw was pretreated as described in example 4 to severity log Ro 3.52 (183o C for a residence time of 12 minutes) to produce a pretreated biomass having xylan number 13.5%, with approximately 7.8% by weight xylan remaining in undissolved solids, as estimated from Figure 2. Glucan recovery from pretreatment was 100%. Xylan recovery from pretreatment was 77%. Cellulase activity measurements in Filter Paper Units (FPU) were determined for three separate commercially available cellulase enzyme preparations ACCELLERASE TRIO™ from GENENCOR™, CELLIC CTEC3™ from NOVOZYMES™, and a mixture of

CELLUCLAST and NOVOZYME 188 from NOVOZYMES™ mixed in the ratio by weight 1 :0.2 respectively. FPU activities were determined by the method of Ghose (1987) and found to be 179 FPU/g enzyme preparation for CTEC3, and 60 FPU/g enzyme preparation for CELLUCLCAST/188.

Hydrolysis experiments were conducted essentially as described in example 6, except that initial dry matter content was 22% DM, 1 wt% polyethylene glycol (PEG) was added, the initial hydrolysis of solid fraction was conducted for 94 hours, posthydrolysis with added liquid fraction (C5 bypass) was conducted for 50 hours, and the enzyme used was either CTEC3, ACTRIO, or CELLUCLAST/188 applied at an equivalent dose in FPU/g glucan of 14.3 FPU/g glucan.

The actual dose of enzymes applied was CTEC3 0.08 g/g glucan, AcTRIO 0.24 g/g glucan, CELLUCLAST/188 0.22 g/g glucan.

Enzyme activities in nkat/g glucan, to be measured as described herein, that were used in the experiment were estimated to be as follows:

Exogluc endogluc beta-gluc xylanase xylosidase

Trio 685a 2829b 8016c 12024c 267d

Celluc/188+477 2860 3372 2200 51

CTEC3 607a 2949e 10046f N/A N/A

+ based on values reported by Juhasz et al. (2005)

a measured using 4-methylumbelliferyl-beta-cellobioside as substrate

b based on ACCELLERASE TRIO product information sheet reporting minimal

endoglucanase value using carboxymethylcellulose (CMC) as substrate and assuming that the corresponding value for hydroxyethylcellulose will be approximately 0.35 times the CMC value, such as reported for example by Dori et al. (1995)

c based on ACCELLERASE TRIO product information sheet reporting minimal values d based on alternatively measured ratio of xylosidase activity comparing Trio:Celluclast of 3.86:1 per g enzyme preparation.

e based on alternatively measured ratio of endoglucanase activity comparing

CTEC3:TRIO of 3.12:1 per g enzyme preparation.

f based on alternatively measured ratio of beta-glucosidase activity comparing

CTEC3:TRIO of 3.76:1 per g enzyme preparation.

It can be shown that the enzyme activities in these experiments measured as described herein were initially within the range exoglucanase 280-1240 nkat/g glucan,

endoglucanase 1 100-8000 nkat/g glucan, β-glucosidase 3000-15000 nkat/g glucan, endoxylanase 9000-30000 nkat/g glucan, β-xylosidase 75-1400 nkat/g glucan nkat/g glucan.

Figure 1 1 shows cellulose conversion as a function of time in the various reaction chambers. The cellulose conversion is determined as the glucose concentration divided by the theoretical glucose potential at the moment the sample was taken. When the C5 bypass is added the glucose potential changes as the bypass contains a small amount of glucose oligomers which are not digested resulting in an overall conversion decrease. As shown, the cellulose conversion kinetics are equivalent for the CTEC and TRIO chambers, but not for the CELLUCLAST/188 chamber. This is attributed to appreciably lower levels of xylanase and xylosidase activity.

Figure 12 shows corresponding xylan conversion as a function of time. The xylan conversion is calculated in the same way as for the cellulose conversion, but as the C5- bypass contains a large amount of xylose oligomers the conversion initially dramatically drops when the C5-bypass is added to the hydrolysate. As shown, the xylan conversion kinetics are equivalent for the CTEC and TRIO chambers, but not for the

CELLUCLAST/188 chamber. This is again attributed to appreciably lower levels of xylanase and xylosidase activity. Where xylan recovery from pretreatment was 77%, the xylan recoveries shown in Figure 12 correspond to very high final C5 monomer recovery in the hydrolysate, for example, 80% conversion in Figure 12 corresponds to (0.80)*(0.77)= 61 .6% C5 monomer recovery.

Glucan and xylan contents are determined as described in Sluiter, A., B. Hames, et al. (2005). Determination of Sugars, Byproducts, and Degradation Products in Liquid Fraction Process Samples, NREL - Biomass Program and in Sluiter, A., B. Hames, et al. (2006). Determintation of Structural Carbohydrates and Lignin in Biomass, NREL- Biomass Program.

Example 10. Dilution to lower dry matter permits equivalent conversion yields at lower enzyme dose using whole slurry.

In the experiments described in example 9, two chambers of the 6-chambered hydrolysis reactor were used to compare hydrolysis of whole slurry, in which the liquid fraction separated from solid fraction after pretreatment was not saved as bypass to be later added to the hydrolysate, but was instead blended back with solid fraction and hydrolysed at the same time. The whole slurry was diluted to 12% DM, which renders the concentration of inhibitory dissolved substances to be approximately equivalent with that achieved in the reactions described in example 9, where dissolved solids were removed and held separate (C5 bypass) from hydrolysis of the solid fraction at 22% DM. CTEC3 and ACTRIO were used as enzyme preparation and applied at a lower dose of 10.7 FPU/g glucan.

Enzyme activities in nkat/g glucan, to be measured as described herein, that were used in the experiment were estimated (based on the values given in example 9) to be as follows:

Exogluc endogluc beta-gluc xylanase xylosidase

Trio 514 2122 6012 9018 200

CTEC3 455 2212 7535 N/A N/A It can be shown that the enzyme activities in these experiments measured as described herein were initially within the range exoglucanase 280-1240 nkat/g glucan,

endoglucanase 1100-8000 nkat/g glucan, β-glucosidase 3000-15000 nkat/g glucan, endoxylanase 9000-30000 nkat/g glucan, β-xylosidase 75-1400 nkat/g glucan nkat/g glucan.

Figure 13 shows cellulose conversion as a function of time for both of the whole slurry hydrolysis samples. The cellulose conversion is determined as described in example 9. As shown, the cellulose conversion kinetics are equivalent for the CTEC and TRIO chambers. Further, notwithstanding a lower enzyme dose, the conversion levels are equivalent with those achieved with C5 bypass and posthydrolysis, as described in example 9.

Figure 14 shows corresponding xylan conversion as a function of time for both of the whole slurry hydrolysis samples. As shown, the xylan conversion kinetics are

approximately equivalent for the CTEC and TRIO chambers. Where xylan recovery from pretreatment was 77%, the xylan recoveries shown in Figure 14 correspond to very high final C5 monomer recovery in the hydrolysate, for example, 80% conversion in Figure 12 corresponds to (0.80) ^* (0.77)= 61.6% C5 monomer recovery. As shown, in whole slurry hydrolysis, very high C5 monomer recoveries of at least 55%, corresponding to 71% xylan conversion in Figure 14, are achieved within 41 hours hydrolysis under these conditions.

The various parameters of pretreatment, hydrolysis and recovery for the experiments described in examples 9 and 10 are shown in Table 4.

Table 4: Hydrolysis yields using very low severity single-stage autohydrolysis.

Enzyme dosage [FPU/g

14.3 14.3 14.3 14.3 10.7 10.7 glucan]

%TS in fiber hydrolysis 22 22 22 22 12 12

Cellulose conversion after

77% 78% 49% 74% 71% 70% 96 h hydrolysis

Hemicellulose conversion

after 96h hydrolysis 80% 82% 57% 76% 75% 71 %

%TS in second hydrolysis 18,5 18,5 18,5 18,5

Cellulose conversion after

73% 74% 48% 76% 74% 74% 144h

Hemicellulose conversion

79% 79% 52% 79% 84% 80% after144h

Overall cellulose conversion 73% 74% 48% 76% 74% 74%

Overall C5 monomer yield 61% 61% 40% 61% 65% 62%

Example 11. Whole slurry hydrolysis of pretreated sugar cane bagasse.

Sugar cane bagasse was pretreated as described in example 4 to severity log Ro 3.43 (180o C for a residence time of 12 minutes) to produce a pretreated biomass having xylan number 12.0%, with approximately 6.8% by weight xylan remaining in undissolved solids, as estimated from Figure 2. Xylan recovery from pretreatment was 83%. After leaving the reactor, the slurry was pressed to a fibre fraction of approximately 57 % and a liquid fraction. The pretreated material, fibre fraction as well as liquid fraction, was collected and analysed. The dry matter and composition of the samples were determined as described in previous examples.

The experiments were conducted in the 6-chamber reactor described in example 6. The pretreated bagasse was used as whole slurry mixture, where the fiber fraction was mixed with the liquid fraction before enzyme addition. The dry matter content was then adjusted with water to 18 wt-% DM. The hydrolysis was carried out at 50 °C with a pH adjusted to between 4.7 and 5.3 with the use of 20 wt-% calcium hydroxide solution (Ca(OH)2).

Accellerase Trio was used as enzyme in a concentration of 0.16 mL/g glucan (9.5 FPU/g glucan). Each day a sample was taken and analysed for sugar content. After 118 h the hydrolysis was stopped. The experiments were performed in double determination.

Enzyme activities in nkat/g glucan, to be measured as described herein, that were used in the experiment were estimated (based on the values given in example 9) to be initially as follows: Exogluc endogluc beta-gluc xylanase xylosidase

Trio 478 1973 5591 8387 186

It can be shown that the enzyme activities in these experiments measured as described herein were initially within the range exoglucanase nkat/g glucan, endoglucanase nkat/g glucan, β-glucosidase nkat/g glucan, endoxylanase nkat/g glucan, β-xylosidase nkat/g glucan nkat/g glucan.

The various parameters of pretreatment, hydrolysis and recovery for the experiments described in this examples 1 1 are shown in Table 5.

Table 5. Hydrolysis yields using very low severity single-stage autohydrolysis.

Figure 15 shows total C5 and C6 monomer recovery in hydrolysate as a function of hydrolysis time for the whole slurry hydrolysis of bagasse. As shown, total C5 monomer recovery of at least 55% is achieved within 24 hours under these conditions.

Example 12. Improved biomethane potential of thin stillage remaining after C6 ethanol fermentation of hydrolysate from feedstock pretreated by low severity autohydrolysis. Wheat straw was pretreated as described in example 4 to three different severities so as to produce pretreated biomass having xylan number 3.0%, 9.1% and 13.2%, having respectively approximately 2.0%, 4.3% and 7.8% by weight xylan remaining in undissolved solids, as estimated from Figure 2.

The pretreated material was subject to solid/liquid separation to produce a fiber fraction, which was subsequently used in enzymatic hydrolysis experiments, as well as a liquid fraction, which was kept separate from the hydrolysis. The solid fractions were hydrolysed using CTEC3 in the respective doses 0.052 g/g glucan for the 3.0% xylan number sample, .056 g/g glucan for the 9.1% xylan number sample, and 0.075 g/g glucan for the 3.2% xylan number sample.

It can be shown that the enzyme activities in these experiments measured as described herein were initially within the range exoglucanase nkat/g glucan, endoglucanase nkat/g glucan, β-glucosidase nkat/g glucan, endoxylanase nkat/g glucan, β-xylosidase nkat/g glucan nkat/g glucan.

Hydrolysis was conducted at 22% DM for 144 hours at 50o C with pH adjusted to 5.0. After 144 hours, ordinary bakers' yeast (C6 fermenting) was added to the hydrolysate, the temperature lowered to 37o C and fermentation/hydrolysis continued for another 60 hours. At the end of the hydrolysis/fermentation, ethanol concentrations were approximately equivalent between the different groups, between 61.77 - 63.68 g/kg ethanol.

Biomethane production from the fermented hydrolysates and from the corresponding separated liquid fraction was determined in duplicate batch assays, using inoculum from Fredericia Spildevand, Fredericia, Denmark, at an inoculum/substratio ratio between 5-6.5 (on a volatile solids basis).

Mass balances for each of the pretreatments were carefully determined and used to estimate the methane potential of thin stillage remaining from C6 ethanol fermentation of each of hydrolysates from each of the three different severity levels. Substracting the known contribution of ethanol to observed biomethane potentials, estimate methane production from 1 ton of wheat straw dry matter were determined. Results are shown in Table 6.

Table 6. Methane production from thin stillage and liquid fraction derived from 1 tonne of straw dry matter, depending on severity of pretreatment.

The embodiments and examples are descriptive only and not intended to limit the scope of the claims. Each of the references cited herein is hereby expressly incorporated by reference in entirety.

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