Methods for pretreating biomass

[0001] The invention relates to the field of biomass processing, in particular to alkaline pretreatment of biomass.

BACKGROUND OF THE INVENTION [0002] With the constantly growing demand for oil, interest in renewable raw materials for the production of bioethanol is also growing (1). Based on current economic analyzes, modern biorefinery uses about 2,000 tonnes / day of lignocellulosic biomass ("biomass") for the production of biofuels and biochemicals (2). Lignocellulosic fibers have a complex network of cellulose, hemicellulose and lignin (3-4), which gives a compact matrix that is difficult to hydrolyze due to the poor availability of enzymes. To improve the enzymatic availability of the related polysaccharides, a thermochemical treatment (i.e., "pretreatment") is usually required before the enzymatic hydrolysis.

[0003] There are various types of readily available raw materials for the production of biofuels. These include agricultural residues, wood biomass, municipal waste, oil seeds / pulp and seaweed. Commercially available oilseed masses include canola, sunflower, sesame, peanut, palm oil, jatropha and soybeans. Currently, these various agricultural waste and oil cake are used as animal feed, biocompost material or are drained to the ground. Grasses and masses / fodder from oilseeds are rich in proteins, fiber and other nutrients. It is possible to use part of the fiber rich material to obtain bioethanol using appropriate thermochemical treatment, enzymatic hydrolysis and fermentation method. It is quite difficult to conduct economical pre-treatment of the raw material continuously. A detailed economic analysis was carried out for several main pretreatment processes such as dilute acid treatment, concentrated ammonia, AFEX, Steam Explosion and Organosolv (5). Ammonia fiber expansion (AFEX) is the main of the alkaline pretreatment processes that modifies the ultrastructure of the cell wall without physical extraction of lignin and hemicellulose into a separate liquid stream. In addition, inhibitory compounds that play an important inhibitory role during further biological processing formed during the alkaline pretreatment process are negligible compared to the treatment with dilute acid. The first advantage of using ammonia during pre-treatment is the relatively simple recovery and reuse of ammonia due to its high volatility. Closer analysis of various pre-treatments with ammonia shows that ammonia is used in the liquid state (ammonia concentration 30-99%) (6-11), supercritical state (12) or as diluted ammonium hydroxide (0.1-28%) ( 13-14). Pre-treatment with the use of percolation in ammonia solution (ARP) (15) and AFEX are the main technologies for the pretreatment of biomass using ammonia. However, most of the pre-treatment methods are mainly pre-treatment of biomass using a liquid pretreatment medium (with various ammonia concentrations, 0.1-99%). Pre-treatment with the use of percolation in ammonia solution (ARP) (15) and AFEX are the main technologies for the pretreatment of biomass using ammonia. However, most of the pre-treatment methods are mainly pre-treatment of biomass using a liquid pretreatment medium (with various ammonia concentrations, 0.1-99%). Pre-treatment with the use of percolation in ammonia solution (ARP) (15) and AFEX are the main technologies for the pretreatment of biomass using ammonia. However, most of the pre-treatment methods are mainly pre-treatment of biomass using a liquid pretreatment medium (with various ammonia concentrations, 0.1-99%).

[0004] Examples of previous ammonia treatment methods are ARP and using dilute ammonium hydroxide. These methods include high temperature treatment (150-180 ° C), long residence time (30-120 min), high pressure liquid recycling, separation of biomass into solid and liquid fractions (by separation of hemicellulose and lignin from cellulose to liquid fraction), low the amount of solids introduced, and the neutralization and / or recovery needed for further processing. Traditionally, the use of gas ammonia gas covers a long residence time (from several hours to several weeks) is expensive and difficult to carry to a higher scale. Such conventional methods for ammonia pre-treatment of lignocellulosic biomass are disclosed, inter alia, in US 2008/0008783 A1, US 2007/0031918 A1 and EP 0 144 930 A2.

[0005] For AFEX biomass is treated with anhydrous liquid ammonia at relatively low temperatures (70-180 ° C), with average residence times (15-45 min), with low humidity (10-200% on dry matter (dwb) )) and higher amounts of ammonia (1: 1-3: 1, ammonia mass / biomass mass). During standard AFEX, due to gravity, liquid ammonia flows to the bottom of the reactor. Some amounts of liquid react with water and form ammonium hydroxide and the remaining liquid is converted into ammonia gas (depending on the thermodynamic gas-liquid state in the reactor). Because the biomass is a poor heat conductor, longer residence times are required (usually 15-45 min) to achieve the desired temperature in the reactor. Mixing and performing a single pre-treatment is a significant problem in the absence of a suitable rotor. Mixing solid suspensions using mixers and screw rotors is energy-independent and not very effective in reducing mass and heat transfer limitations. In other words, only biomass, which is both combined with ammonium hydroxide and suitably preheated (i.e., typically biomass close to the walls or bottom of the reactor), is more pretreated than most of the biomass in the reactor. Another serious economic problem of the AFEZ process is the expensive recovery step, in which ammonia should be recovered after pre-treatment as gas, re-compressed, separated from water and re-used as anhydrous liquid ammonia. AFEX is also difficult to carry out continuously using liquid ammonia under pressure as a chemical used for pre-treatment. The costly release of ammonia at the end of the pre-treatment with AFEX requires a large amount of energy to produce ammonia-water gaseous mixtures, which can lead to commercial use being prevented. The treatments based on supercritical ammonia are much more energy-intensive than AFEX, which makes this option less economical.

BRIEF SUMMARY OF THE INVENTION [0006] The present invention encompasses a method for treating biomass comprising the following steps: providing gaseous ammonia at elevated temperature to a reaction vessel containing biomass; allowing the ammonia gas to react with the water present in the biomass to produce the processed biomass, the contents of the reaction vessel being maintained at a pressure between 100 psi (689 kPa) and 1000 psi (6895 kPa), and without expulsion release at the end of treatment ; removal of the biomass to be treated from the reaction vessel.

[0007] In certain example embodiments of the present invention, the temperature in the reaction vessel may be in the range of 50 ° C to 200 ° C; or the temperature in the reaction vessel may be in the range of 50 ° C to 100 ° C; or the temperature in the reaction vessel may increase after the introduction of ammonia gas into the reaction vessel.

[0008] In other embodiments of the present invention, gaseous ammonia may be introduced into the reaction vessel at a pressure of 100 psi to 1000 psi, 200 psi to 650 psi, or 100 psi to 200 psi.

[0009] Further aspects of the method according to the invention include the condensation of gaseous ammonia with biomass. In addition, the biomass may contain less than 15% of water in relation to the dry matter; from 15% water to 233% water against dry matter: or gaseous ammonia reacts with water in biomass.

[0010] In another aspect of the present method, the time for reacting gaseous ammonia with biomass can be from 2 hours to 36 hours, from 2 hours to 12 hours, from about 1 to 120 minutes, from 1 minute to 20 minutes.

[0011] Also in the present method according to the invention, the biomass can be treated uniformly using ammonia gas; the method can be continuous, half-loaded; and the reaction vessel may be a fixed bed reactor, a fluidized bed reactor or a semi-fluid bed reactor.

[0012] In certain embodiments of the present invention, the carrier may be introduced into the reaction vessel after the ammonia gas has been introduced into the reaction vessel. The carrier may be combined with gaseous ammonia and may be an inert gas, it may be oxidising (e.g., air) and may be a vapor. It is also possible to combine the inert gas and steam with gaseous ammonia before introducing the gaseous ammonia into the reaction vessel.

[0013] In a further embodiment, the present method may comprise reintroducing at least a portion of the gaseous ammonia as the gas used for the treatment method.

[0014] The present invention also includes a method for treating biomass comprising: impregnating biomass with ammonia; introducing biomass into the reaction vessel; providing a gas carrier; supplying a gas carrier to the reaction vessel; leaving the gas carrier during the reaction with the biomass in the reaction tank; removal of biomass from the reaction vessel.

BRIEF DESCRIPTION OF THE DRAWINGS [0015] The above summary, as well as the following detailed description of the invention, will be better understood after reading it with reference to the accompanying drawings. For the purpose of illustrating the invention, some of the currently preferred embodiments are shown in the drawings and tables.

Figure 1 shows a comparison of the conventional AFEX (I) method and the pre-treatment with ammonia gas (GAP) (II). In Figure 1, liquid ammonia is added to the reaction vessel for conventional AFEX treatment, whereas in the case of GAP the ammonia injection tank is heated to convert the liquid ammonia to a gaseous state (at P1 pressure) and gaseous ammonia is added to the biomass in the reaction vessel (yes that the final pressure in the reaction vessel is P2).

Figure 2 shows the glucose yield from pre-treated corn enzyme hydrolysis using AFEX (control) and the GAP method with two different residence times as a function of the introduced ammonia.

Figure 3 shows the percent glucose yield (% glucan conversion) of corn straw as a function of different GAP conditions, the effect of ammonia on biomass during GAP treatment and the pretreatment effect seen during enzymatic hydrolysis (I) and pressure during treatment (II). In (I) and (II) the biomass relative to the amount of ammonia is shown on the x-axis, analyzed at various pressures P1 and temperatures (gaseous ammonia before adding to the reaction vessel containing corn straw). Also on (I) the y-axis represents the total glucose output as a function of the different GAP conditions.

Figure 4 shows the role of explosive removal as compared to slow ammonia release during the AFEX and GAP pretreatment process on the glucose yield obtained from treated maize straw.

Figure 5 shows the potential use of fluidization during the GAP process using gaseous ammonia with or without suitable hot carrier gases.

Figure 6 shows glucose and xylose yields for maize straw without treatment, using AFEX at high humidity (60% on dry weight) and low humidity (5% on dry weight).

Figures 7A-7D are photographs of a transmission electron microscope of corn cell walls subjected to and untreated using ammonia; Figure 7A, untreated; Figure 7B processed under AFEX at low humidity; Figures 7C and 7D are different fragments and magnification of samples treated with AFEX at low humidity. Maize non-treated corn has characteristic multilayer cell walls compared to nanoporous cell walls when processed using AFEX. After AFEX you can see signs of surface deposits with a wavy, amorphous appearance on the outer layers of the cell wall.

Figure 8 shows the ammonia recovery system and process flow diagram using conventional AFEX methods (Fig. 8A) and GAP (Fig. 8B).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0016] The details of one or more embodiments of the present invention are defined in the following description. Preferred embodiments of the invention may be more readily understood with reference to the following detailed description of specific embodiments and examples provided herein.

Unless defined otherwise, all technical and scientific expressions used herein have the meaning as commonly understood by those skilled in the art to which this invention belongs. Preferred materials, devices and methods are described herein, although any methods, devices and materials similar or equivalent to those described herein can be used to apply or test the invention.

[0018] In this specification and the appended claims, singular forms also include the plural, unless the context clearly dictates otherwise.

[0019] As used herein, the term "ammonia" means the nitrogen and hydrogen compound of formula NH3.

[0020] As used herein, the term "biomass" means an organic material such as wood, plants and organic waste that can be transformed into a fuel.

[0021] As used herein, the term "gaseous" means a state of matter distinguished from a solid and a liquid state by density, viscosity and / or depressurization.

[0022] The inventors have developed a method known as "ammonia gas treatment" (GAP) in which hot ammonia gas (ammonia gas) is used to treat biomass in a reaction vessel such as a reactor or other tank capable of maintaining biomass under pressure. For example, in the GAP process, the contents of the reactor may be maintained at a pressure ranging from 100 psi to 1000 psi, from 200 psi to 500 psi, or from 100 psi to 200 psi. In one example, water is used to pre-wet the biomass, hot ammonia gas is supplied to the biomass under pressure. Gaseous ammonia is supplied to the reaction vessel at pressures ranging from 0 psi to 1000 psi, 200 psi to 500 psi, or 100 psi to 200 psi. Then the hot ammonia gas is condensed on biomass and reacts with water. When using this method, the desired temperature (from 50-200 ° C) is obtained immediately due to the exothermic reaction between water and ammonia. The formation of ammonium hydroxide occurs wherever water is associated with biomass. During the process, the biomass is uniformly pretreated using ammonia (i.e., most of the biomass achieves approximately the same parameters) and requires a short pre-treatment period (e.g.

from 1 to 120 min or from 1 to 20 minutes). The short pretreatment time also reduces the formation of potentially inhibitor degradation products that may adversely affect further biological processes. In certain embodiments of the present invention, longer pretreatment times may be used, e.g. from 2 to 36 hours or from 2 to 12 hours. With this method no further expansion release occurs at the end of the pre-treatment, which allows significant energy savings during the reintroduction of ammonia into the circulation [0023] The method of the present invention can be easily adapted to a continuous process using an ammonia gas stream (a). re-circulated, (b) a mixture of ammonia introduced into circulation and steam, (c) ammonia introduced similarly to a combined cycle with an inert gas or other carrier gas, or (d) a recycled ammonia gas / vapor mixture combined with an inert / carrier gas in a fluidized bed, semifluidized bed reactor or fixed bed reactor. It is predicted that only a small portion of ammonia (0.5 to 3% on a mass basis of ammonia / biomass) will react during the present process (due to the reaction of ammonia with various components of the cell wall) and the remaining part of ammonia (i.e. from 50% to 99 , 5%, from 75% to 99.5% or from 97% to 99.5%, in mass ratio of ammonia / biomass) can be re-circulated in the gaseous state. or (d) a re-circulated ammonia gas / vapor mixture combined with an inert / carrier gas in a fluidized bed, semifluidized bed reactor or a fixed bed reactor. It is predicted that only a small portion of ammonia (0.5 to 3% on a mass basis of ammonia / biomass) will react during the present process (due to the reaction of ammonia with various components of the cell wall) and the remaining part of ammonia (i.e. from 50% to 99 , 5%, from 75% to 99.5% or from 97% to 99.5%, in mass ratio of ammonia / biomass) can be re-circulated in the gaseous state. or (d) a re-circulated ammonia gas / vapor mixture combined with an inert / carrier gas in a fluidized bed, semifluidized bed reactor or a fixed bed reactor. It is predicted that only a small portion of ammonia (0.5 to 3% on a mass basis of ammonia / biomass) will react during the present process (due to the reaction of ammonia with various components of the cell wall) and the remaining part of ammonia (i.e. from 50% to 99 , 5%, from 75% to 99.5% or from 97% to 99.5%, in mass ratio of ammonia / biomass) can be re-circulated in the gaseous state.

[0024] In one embodiment, the hot ammonia gas is used for pretreatment of biomass (from 15% to 233% humidity, e.g. water content, based on dry matter (dwb)) in the reactor with continuous circulation of the ammonia gas gas mixture (e.g. ammonia gas / steam). In another embodiment, the hot ammonia-water gas mixture is used for pretreating pre-wet or dry biomass (less than 15% moisture, based on dry matter), which ammonia-water gaseous mixture is fed continuously into the reactor and the ammonia gas mixture. it is brought back into the reactor. In a further embodiment, the hot ammonia gas is introduced into the reactor in combination with a hot inert / carrier gas (e.g., nitrogen, air) for pre-wet pre-treatment (from 15% to 233% moisture, based on dry matter) or dry biomass, which hot ammonia gas is continuously introduced into the reactor with recycled ammonia-water-inert gas mixture. The oxidizing gas such as air or oxygen can alternatively be combined with gaseous ammonia. In another embodiment, a mixture of hot ammonia-water gas is introduced into the reactor in combination with hot inert / carrier gas (e.g., steam, nitrogen, air) for pre-wet pre-treatment (from 15% humidity to 233% humidity, based on dry matter) or dry biomass (15% moisture or less on dry matter), which mixture is hot ammonia - the vapor is fed continuously into the reactor with the recycled ammonia-vapor-inert gas mixture. It is envisaged that the recycle step (recirculation step), as set forth in this invention, will reduce the amount of ammonia required for the pretreatment of the biomass. Since only a small amount of ammonia gas reacts with biomass (from 0.5 to 3% w / w ammonia / biomass), hot inert carrier gas is expected to provide a suitable heat transfer medium and mass replacing the ammonia used in the present methods (AFEX) .

[0025] Further in other embodiments, the carrier gas [both oxidising (e.g., oxygen or air) or non-oxidizing (e.g., nitrogen or vapor)] is used with gaseous ammonia during the pre-treatment or pre-treatment process (to remove residual ammonia) from biomass).

[0026] In the present process according to the invention, the effective amount of biomass to ammonia is from 1: 0.01 to 1: 5, from 1: 0.2 to 1: 2 or from 1: 0.2 to 1: 1. Then, in the present method of the invention, the glucan conversion rates are the same or higher (10-15%) than the conversion rates for the standard AFEX. For example, as depicted in Figure 2, 15 minutes of reaction with GAP, a relatively equivalent conversion rate is obtained as for 45 minutes of reaction time with AFEX. At 30 minutes of reaction time with GAP, however, the glucan conversion is expected to increase by 10-15% compared to the AFEX process. In general, the conversion factor depends on other factors such as the relevant cellulases and hemicellulases, the type and combination of enzymes and the amount of enzymes used for enzymatic hydrolysis.

[0027] The present invention also includes impregnating biomass using liquid or gaseous ammonia and water (using concentrated / diluted sodium hydroxide) to achieve lower amounts of ammonia introduced (from 0.01 to 0.3 kg of ammonia per kg of biomass) and then continuous introduction of biomass. into the reactor where it is pretreated using a hot inert carrier gas (containing little or no ammonia). In one embodiment, this method is performed in a fixed biomass reactor using ammonia / vapor / inert gas / carrier gas mixtures purged through the reactor.

The gas stream is recirculated continuously using compressors and heaters for recirculation through a fixed biomass reactor.

[0028] Figure 1 shows a schematic sketch of comparison (I) of a standard AFEX apparatus 10 and 30 for carrying out the GAP method according to the invention (II). In contrast to AFEX, where liquid ammonia (pressure 100-200 psi) is introduced into the reactor through the bottom valve of the ammonia supply tank 12; in GAP, ammonia in the supply tank 32 is preheated (to the supply tank) and fed through the upper delivery valve valve 32. This allows the hot ammoniac gas to condense on the GAP in biomass (unlike the standard AFEX), thus causing rapid (e.g., immediate) temperature increase in the reactor. In the case of the AFEX method, it usually takes 15-45 minutes to achieve the desired pre-treatment temperature in the reactor (e.g. 100 degrees Celsius), after which the temperature is maintained for another 5-45 min. For the AFEX method, the typical biomass pre-treatment time is between 20-90 min. For comparison, in the case of GAP (depending on the temperature and pressure P1 of hot ammonia gas entering the reactor), the desired pretreatment temperature in the reactor can be quickly reached (e.g. from 50 ° C to 200 ° C or from 50 ° C to 100 ° C) , with a total residence time from 1 minute to 120 minutes.

[0029] The inventors have also developed novel reactor configurations for continuous topping up of a column reactor using hot ammonia and / or inert carrier gas mixtures that were reused and fed into the reactor in a gaseous state (without compressing gaseous ammonia to liquid ammonia or mixtures of ammonium hydroxide).

[0030] There are several variations of reactors that can be used to carry out the GAP method. For example, a semi-batch or continuous reactor with a fluidized or semi-fluidic biomass fed continuously into the reactor, where the biomass is combined with hot ammonia and / or inert carrier gas. In one embodiment, the hot gas can be recovered and recycled. In another embodiment, the batch solid-batch reactor is continuously purged using ammonia and / or an inert carrier gas; and the hot gas can be recovered and re-introduced into the reactor (see Figure 5). Figure 5 shows a potential process flow chart showing how the GAP can be performed by using biomass fluidization using gaseous ammonia and other carrier gases.

[0031] In the present GAP pretreatment process, the pre-treatment is homogeneous as much as possible; and unimportant problems regarding mass transfer, negligible problems regarding residence time, low ammonia / water use and complex water-ammonia separation procedures were avoided.

[0032] Next, as shown in Table 1, the GAP method using the fluidization method has several advantages over AFEX.

<td>AFEX</td><td>Fluidized GAP method</td>

<td>Liquid phase reaction</td><td>The reaction of the gas phase</td>

<td>Mixing using rotors (heterogeneous mixing)</td><td>Mixing carried out by gas to fluidization (homogeneous mixing)</td>

<td>Use of water (40-100%) (difficulty in ammonia separation after pre-treatment)</td><td>Minimizing the use of water (&lt;10%)</td>

<td>Pre-heated liquid ammonia (costly recovery and liquefaction)</td><td>Pre-heated gaseous ammonia (rec entering into circulation without liquefaction)</td>

<td>Poor mixing (more required ammonia-water)</td><td>Effective mixing (more efficient use of ammonia-water)</td>

<td>Long residence time (15-45 min)</td><td>Short residence time (1-15 min)</td>

<td>Hotspots due to inhomogeneous heating</td><td>Homogeneous heating and better control reaction kinetics</td>

[0033] Figures 8A and 8B show a block diagram of the flow of biomass and ammonia / inert gas for the recovery process for each of the AFEX and GAP methods, respectively. In the case of the GAP method shown in Figure 8B, the method includes a GAP reactor, where biomass is introduced with or without water followed by the injection of hot ammonia gas (NH3) / nitrogen (N2) into the GAP reactor using a heater and a compressor. Most of the hot ammonia gas and nitrogen is recovered after the GAP process and heated again for further use in the pretreatment process. The ammonia / nitrogen residues present in the pretreated biomass are recovered using a condenser and used for a subsequent pre-treatment.

[0034] As described herein, there are several preconditioning conditions (ammonia gas pre-treatment temperature, ammonia pressure P1 before introduction, ammonia pressure P2 after introduction, GAP reactor reaction time, water content in biomass and amount of ammonia) that can affect the way GAP (see various ranges shown in Figure 8B).

[0035] The preconditioning conditions are also dependent on each other, i.e. changing one condition may affect the other. For example, the reaction time depends on the temperature and pressure of the ammonia. The higher the pressure and temperature of the ammonia gas that is fed into the GAP reactor, the shorter the reaction time in the reactor (and the pressure P2 in the reactor will also be higher). Conversely, the lower the pressure P1 and the temperature of the gaseous ammonia fed in the reactor, the longer the reaction time in the reactor (and also the pressure in the reactor would be lower). Also, the diffusion coefficient of ammonia by biomass particles is higher in the case of increasing pressure, which means that the reagent can have much faster access to reactive bonds and thus can reduce the overall reaction time. In theory, when the ammonia pressure is doubled, the reaction time should be reduced by almost half, since the majority of reactions in biomass are pseudo-first order reactions. Achieving the set temperature can be achieved much more quickly at increased pressure, because hot gas ammonia also has the task of transferring heat through the solid phase to the interior of the biomass where the reaction takes place. For reaction temperatures close to room temperature (ie 25-40 ° C), reaction times can be extended up to 24 hours (depending on the amount of ammonia) to achieve nearly 90% conversion, while at 100 ° C the total residence time can be reduced to 15 minutes ( depending on the amount of ammonia). In addition to the pressure and temperature of the ammonia gas, the size of the biomass particles may also affect the reaction time. The smaller the particle size, the faster the set temperature and pressure inside the molecule is obtained, which means that complete conversion should take place more quickly. The pressure of P2 in the reactor can also be reduced by lowering the amount of ammonia introduced into the reactor or by lowering the moisture content of the biomass.

[0036] Next, based on various preconditioning conditions, the inventors have found that the addition of water together with ammonia during the pre-treatment process gives rise to two competing reactions; namely hydrolysis (using a hydroxyl ion) and ammonolysis (using ammonia). Degradation products formed by hydroxyl ions are mainly acids, which are strong inhibitors for microorganisms and further fermentation processes. On the other hand, the ammonium reaction leads to the formation of amides, which are much less inhibitory for microbes (unpublished data from Ming W. Lau and Bruce E. Dale). In a typical AFEX method, 0.5-2 kg of water per kg of biomass is used. Because ammonia is soluble in water, it is costly to distil ammonia from pre-treated water for re-use in a continuous biorefining process.

[0037] Using the GAP method, the inventors expect that a biomass containing from 5 to 15% moisture relative to dry matter (the desirable moisture content of field dried biomass without external water supplementation during pretreatment) can be pretreated using hot ammonia gas and percent conversion of glucans. is similar to those obtained with ammonia pre-treatment with high humidity (15% or more on a dry basis) as shown in Figure 6.

[0038] The GAP method can be used in lignocellulose biorefineries. More precisely, the modern biorefinery uses about 2,000 tonnes / day of lignocellulosic biomass ("biomass") for the production of biofuels and biochemicals (2). For this pre-treatment, the costs of treatment and greenhouse gas (GHG) emissions are considered as a few of the limitations for such a biorefinery. With the GAP method, both the pre-treatment and GHG emission costs can be reduced and the technology is more suitable for biorefineries (see, e.g., Table 2 below).

[0039] The GAP method can be used in the edible oilseed and expeller industry. Oil seeds are usually extracted in two steps: (i) a mechanical expeller / extraction press to reduce the oil content to 20-25% (by weight), then (ii) extraction with hexane to remove residual oil (16). The extracted cake is then roasted (or desolvated) by steam stripping / boiling to remove residual solvent and pre-conditioned (i.e., to detoxify the anti-nutritive components from the oilseeds) for animal ingestion and / or protein extraction. Pre-conditioning processes are generally dependent on the type of oilseeds, but usually require boiling of biomass (with appropriate moisture content) with steam at 90-110 ° C for 15-30 minutes. The GAP method can be used together with typical steam roasting methods for pretreatment of biomass before subsequent biological treatment for the production of biofuels and chemical agents (e.g. ethanol and biodiesel). Part of the fiber in the cake can be fermented to ethanol and also reacted with the oil extracted from the biodiesel seeds.

[0040] One of the main advantages of the AFEX and GAP methods is that, unlike other thermochemical treatments (e.g., dissolved acid, organosolv), the pretreatment temperature is relatively low (e.g., 50-150 ° C for GAP compared to 150-220 ° C for acid treatment). Low temperatures allow for the reduction of protein degradation and improve the degradation of important amino acids such as lysine. Ammonium-based treatments are currently used to detoxify oilseeds such as peanuts to remove toxic aflatoxins (17). The inventors performed pre-treatment of the extracted cakes using the standard AFEX method and hydrolysis using cellulase enzymes. The AFEX method gives a significant increase in the coefficient and yield of the maximum sugars obtained compared to the untreated sample (data not shown). GAP or AFEX processing methods can be used for pre-treatment of cakes for biomass conversion applications (see, e.g., Balan, V., et al, 2009, Journal of the American Oil Chemists Society, 86, 157-165).

[0041] The GAP method can be used to extract proteins from animal feed. In the Pro-Xan (18) method, proteins are extracted from alfalfa by grinding in a percussive mill to break up cell walls, followed by extraction of juices by a screw press and steam injection for protein coagulation. Then soluble substances are added to the squeezed mass and sold as animal feed. In this method, ammonia is used to kill various microorganisms and increase the pH (which allows the extraction of proteins). Again, GAP can be used at slightly elevated temperatures (instead of at room temperature) from 30 ° C to 50-100 ° C. This will further improve the extraction of proteins and at the same time lead to the pretreatment of biomass, which can be used in biorefineries for the production of biofuels and biochemicals.

[0042] The inventors performed both in vivo and in vitro digestion studies of biomass treated with AFEX and observed that it is very well digested. Based on digestion studies, animals need significantly less expensive feed to achieve adequate milk growth and production if these feeds are pre-treated with ammonia.

[0043] In the general description of the invention, it will be easier to understand it by reference to the following Examples presented for illustrative purposes only.

EXAMPLES [0044] Example 1: Pre-treatment of lignocellulosic biomass using ammonia gas.

[0045] Anhydrous ammonia gas was introduced into a stainless steel cylinder and pre-heated to obtain 450-900 psi. In parallel, the biomass of adequate humidity (60%) was maintained in a preheated (up to 140 ° C and 160 ° C) stainless steel reaction vessel and a vacuum was introduced to remove the air and create a negative pressure to introduce ammonia. Pre-heated ammonia gas was introduced into the reaction vessel. The unreacted ammonia in the tank was measured and the present ammonia introduced into the pre-treatment reactor during the process was calculated. A sudden increase in biomass temperature (from the initial temperature of 30 ° C to about 100-200 ° C) was observed depending on the pressure / temperature of the pre-heated ammonia gas. The reaction had different residence times and then the pressure was released slowly.

[0046] Example 2: Enzymatic hydrolysis of pre-treated maize straw using the AFEX (control) and GAP methods with different amounts of ammonia and various times of residence.

[0047] The treated biomass was dried overnight in the extract and the pretreatment efficiency was determined using biomass digestion using commercial enzymes (15 FPU Spezyme CP from Genencor and 64 pNPGU beta-glucosidase from Novozyme per gm of glucans) at 50 ° C for 72 h Hydrolysates were analyzed for glucose using a YSI glucose analyzer. Figure 2 shows 5 and 15 minute reaction times showing the GAP process, 45 minute reaction time using the AFEX method and different ratios of biomass to ammonia. The data in Figure 2 shows equal or better pre-treatment efficiency using GAP with significantly shorter reaction times than for AFEX.

[0048] Example 3: Conversion of biomass glucans as a function of different GAP conditions.

[0049] The biomass moisture was determined to be 60% and the biomass to ammonia concentration ranged from 1: 1.2 to 1: 0.2 (the amount of biomass to ammonia, w / w) to further understand the effect of the ammonia concentration required in the GAP process. In addition, the feed pressure of P1 ammonia (before feed) and the reactor temperature were changed. The results are shown in Figure 3. Figure 3 clearly shows that up to 1: 0.8 conversions are comparable to the standard AFEX method (60% humidity, 1: 1 amount of biomass to ammonia, 45 min total residence time). With further reduction of the amount of biomass to ammonia (to 1: 0.2) only 10-15% decrease in glucose yield was observed compared to the control. This means that there is almost the same percentage of glucose conversion for corn straw processed by GAP, as for corn straw treated with AFEX with much lower ammonia and pressure of the reaction vessel. In (II) the y-axis shows the reactor pressure as a function of the GAP conditions and shows that the pressure P2 in the reactor decreases with the amount of ammonia. In reducing the amount of ammonia from the biomass, the pressure in the reaction vessel also decreases (see Figure 3 (II)) to between 50-150 psi.

[0050] Despite the fact that the glucose yield decreases by 10%, the pressure P2 in the reactor tank also falls below 100 psi. For this reason, the operating and capital costs for GAP at lower pressure (and lower amounts of ammonia) will be significantly lower compared to AFEX and other ammonia based pre-treatments. In the GAP process, with the correct selection of an enzyme cocktail (containing the appropriate cellulases and hemicellulases), the inventors expect a further increase in conversion and reduction of processing costs by further reducing the amount of biomass to ammonia (biomass to ammonia 1: 0.05 - 1: 0.2 relative to dry matter ) during the GAP method.

[0051] Example 4: Effect of pressure release during a pre-treatment process.

[0052] Two independent pre-treatments were performed using the AFEX and GAP methods using a biomass to ammonia of 1: 1. In the first set of experiments, the pressure was released in an explosive manner and in the second set of experiments the pressure was released slowly after the process. In explosive release, the pressure drops suddenly (less than 1 second) from reaction pressure (200-400 psi) to atmospheric pressure (15 psi). In slowing down the slow pressure drops gradually to atmospheric pressure (over 2 minutes to the pressure drop). The obtained raw material was collected on a tray and dried under a hood overnight. The next day, the work-up was tested for ease of digestion using commercial enzymes at 50 ° C for 72 h as described above (see Figure 4). In Figure 4, the y-axis represents the% glucose yield (% glucan conversion) for the biomass samples treated in a different manner. The inventors observed a marginal decrease in the conversion of the pre-treatment method carried out with slow release compared to the explosion, and this fall was within the error range. This means that an explosion or sudden expansion release during pre-treatment is unnecessary or not very important. It is therefore possible to carry out a continuous pre-treatment of the biomass fed into the reactor under constant pressure into which hot ammonia gas (and water) and / or inert / carrier gas mixtures are introduced. The inventors observed a marginal decrease in the conversion of the pre-treatment method carried out with slow release compared to the explosion, and this fall was within the error range. This means that an explosion or sudden expansion release during pre-treatment is unnecessary or not very important. It is therefore possible to carry out a continuous pre-treatment of the biomass fed into the reactor under constant pressure into which hot ammonia gas (and water) and / or inert / carrier gas mixtures are introduced. The inventors observed a marginal decrease in the conversion of the pre-treatment method carried out with slow release compared to the explosion, and this fall was within the error range. This means that an explosion or sudden expansion release during pre-treatment is unnecessary or not very important. It is therefore possible to carry out a continuous pre-treatment of the biomass fed into the reactor under constant pressure into which hot ammonia gas (and water) and / or inert / carrier gas mixtures are introduced.

[0053] Example 5: Hydrolysis of maize straw untreated and processed using AFEX.

[0054] Figure 6 shows the hydrolysis of maize straw untreated and processed using AFEX. Standard AFEX was carried out at 90 ° C, the amount of biomass to ammonia 1: 1 at 60% humidity relative to the dry weight at 5 minutes of residence time; and AFEX at low humidity were carried out at 90 ° C, the amount of biomass to ammonia 1: 1 at 5% humidity against dry matter and 5 minutes residence time after 24 hours incubation at 50 ° C at 200 rpm.

[0055] To prove that biomass with low humidity (5% moisture) yields comparable pretreatment results as high humidity biomass (60% moisture vs. dry weight), the inventors performed pre-treatment for these conditions and enzymatic hydrolysis using 15 FPU cellulases and 64 pNGPU beta-glucosidase. The results of the transformation are shown in Figure 6. The y-axis shows the yields of glucose and xylose after enzymatic hydrolysis for different preconditioning conditions.

[0056] Furthermore, electron tomography images have shown that the pretreated low humidity biomass has a higher porosity in the cell wall than when using a higher moisture content (Figure 7A and 7B). The increased porosity allows better access for enzymes for more efficient hydrolysis of pretreated biomass. A slightly lower conversion for the lower AFEX sample to be treated may result from the lack of appropriate hemicellulase during enzymatic hydrolysis and poor heat / mass transfer during pretreatment with AFEX.

[0057] The inventors expect to obtain better results than compared to standard AFEX conditions with proper control of the above factors, but instead use GAP-based fluidization. The advantage of low-humidity treatments based on ammonia, especially during GAP, is the simpler recovery of ammonia from water. Because of this, the more water circulated, the more expensive it is to recover (and re-circulate) the ammonia from the system.

[0058] Example 6: Comparison of raw material savings and GHG emissions For energy assessment, raw material saving and greenhouse gas (GHG) emissions for the GAP method compared to the AFEX method, the inventors performed a calculation based on the Aspen model and the results are presented in the Table 2. The results show significant savings in heat, electricity and water together with a 3-fold reduction in GHG emissions for the GAP method.

<td></td><td colspan="3">Process data</td><td colspan="3">GHG</td>

<td></td><td>Unit</td><td>GAP</td><td>AFEX</td><td>Unit</td><td>GAP</td><td>AFEX</td>

<td>corn straw</td><td>mt</td><td>1</td><td>1</td><td></td><td></td><td></td>

<td>Ammonia</td><td>^kg</td><td>8.8</td><td>8.8</td><td>^kg</td><td>24</td><td>25</td>

<td>Water</td><td>^kg</td><td>0.0</td><td>896</td><td>^kg</td><td>0</td><td>1</td>

<td>Electric current</td><td>MJ</td><td>19</td><td>33</td><td>^kg</td><td>4</td><td>7</td>

<td>Heat</td><td>MJ</td><td>449</td><td>2521</td><td>^kg</td><td>35</td><td>194</td>

<td>biomass *</td><td>mt</td><td>1.0</td><td>1.0</td><td></td><td></td><td></td>

<td>Together</td><td></td><td></td><td></td><td></td><td>63</td><td>226</td>

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