PROCESS FOR EFFICIENT HYDROLYSIS OF LIGNOCELLULOSIC FEEDSTOCK IN THE PRESENCE OF ADDITIVES

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority from patent application Ref. no IN202221042375 filed on 25/07/22.

TECHNICAL FIELD

The invention relates to the production of biofuels from lignocellulosic feedstock. More particularly it relates to the efficient enzymatic hydrolysis of lignocellulosic feedstock, preferably softwood, in the presence of additives.

BACKGROUND

Ethanol derived from carbohydrates by fermentation represents one of the best alternatives to replace conventional energy sources and reduce dependence on other fossil fuels. While first-generation bioethanol is produced by enzymatic hydrolysis and fermentation of precious food crops, depletion of which may lead to food crisis, there is great interest in feedstock conversion systems that do not use food crops. Second-generation ethanol has thus been produced sustainably using lignocellulosic feedstock comprising residual non-food parts of crops, such as stems, leaves, bagasse, husks and the like. To be competitive and for acceptance of bioethanol at a commercial scale, the conversion of biomass to ethanol must be cost-effective.

Lignocellulosic feedstock is available in abundance and mainly includes agricultural residues, softwood, forest residues, and hardwood. It is mainly composed of three main polymers, namely, cellulose (35 to 50%), hemicellulose (20 to 30%) and lignin (15 to 25%) organized in a complex entanglement in the lignocellulosic structure . These can be hydrolyzed to produce monomeric sugars, most of which can be fermented. However, obtaining monomeric sugars from cellulose and hemicellulose in high yields from lignocellulosic feedstock is more difficult than deriving sugars from sugar or starch-containing crops, such as sugar cane or maize. In addition, the heterogeneity in feedstock, influence of different process conditions on microorganisms and enzymes makes the lignocellulosic feedstock-to-ethanol process more complex.

Amongst the lignocellulosic feedstock, softwood is a type of wood that is derived from conifers. They are typically lighter in weight and have a lower density than hardwoods. They are known for their straight grain and uniform texture. Softwoods are attractive owing to their higher content of cellulose (40-45%) and lignin (26-34%) and a lower content of pentosans (7-14%) as compared to the hardwoods or deciduous species which typically contain 38-49% cellulose, 23-30% lignin, and 19- 26% pentosans. Lower pentosans are advantageous as more aggressive processing conditions can be employed to remove the lignin without having to compromise for toxic inhibitor production.

While ethanol is produced from softwood in various ways, the common processing steps for producing ethanol from the feedstock typically encompass four steps, namely, (1) pre-treatment to increase the accessibility of lignocellulose, (2) enzymatic hydrolysis or saccharification to release fermentable sugars, (3) microbial fermentation to produce target products and (4) purification of the fermentation broth by distillation.

Pre-treatment involves breaking the natural inert physical barriers and exposing more carbohydrates their downstream hydrolytic process. In general, methods for pre-treatment could involve chemical, physical, biological, or physiochemical steps that brings changes to the biomass structure.

Pre-treatment is followed by cellulose hydrolysis using cellulases. However, the compact structure of cellulose and hemicellulose along with its physical shielding by lignin, makes it more resistant to enzymatic action leading to incomplete or low hydrolysis. Additionally, lignin in the softwood is also known to adsorb to cellulolytic enzymes impeding their action, affecting yields as well as necessitating higher quantities of enzymes. Enzyme consumption is an important consideration in the commercialization of feedstock processing. Cellulase preparations need expensive equipment and have a high operating cost and may pose a major challenge in the commercial downstream processing of the feedstock. Hence there is a need in the art to optimize the process for obtaining maximal yields of fermentable monomeric sugars from lignocellulosic feedstock such as softwoods to make it commercially viable.

A promising key interface between enzyme accessibility and activity may involve application of additives such as surfactants, chelators, proteins, and synthetic polymers that may favourably impact hydrolysis outcome. Their major mode of action may include (1) reducing non-productive enzyme adsorption; (2) increasing the stability and activity of the enzyme and protecting it from denaturing when heated and affected by shear force; (3) lower the surface tension of lignocellulose and increase its cellulose accessibility and (4) facilitate enzyme recycling.

Therefore, the present disclosure relates to using additives to improve the economics of pre-treatment, increase process yields, reduce the quantity of the enzyme required, and improve the enzymatic hydrolysis of softwood to monomeric sugars to obtain ethanol through the process of fermentation.

SUMMARY

This summary is not intended to identify all the essential features of the claimed subject matter, nor is it intended for use in determining or limiting the scope of the claimed subject matter. This summary is provided to introduce concepts related to the use of additives to obtain ethanol through the process of fermentation and the concepts are further described below in the detailed description.

The presently disclosed subject matter provides an improved process for enzymatic hydrolysis of lignocellulosic feedstock comprising subjecting the lignocellulosic feedstock to impregnation to obtain impregnated slurry; adjusting at least the concentration and pH of the impregnated slurry to obtain a stream; adding additives to the stream to obtain additive -treated stream; subjecting the additive -treated stream to enzymatic hydrolysis to obtain enzyme-hydrolysed stream comprising fermentable sugars; and fermenting the enzyme -hydrolysed stream to obtain fermentation product. The improved process for enzymatic hydrolysis of the lignocellulosic feedstock of the present invention is characterised in that, addition of additives to the stream increases efficiency of enzymatic hydrolysis by at least 7%. BRIEF DESCRIPTION OF DRAWINGS

A description of the drawings is outlined with reference to the accompanying figures. In the figures, the left-most digit (s) of a reference number identifies the Figure in which the reference number first appears. The same numbers are used throughout the drawings to refer to like features and components.

Fig. 1: Flow chart depicting the enzymatic hydrolysis of lignocellulosic feedstock with additives.

DETAILED DESCRIPTION

Reference throughout the specification to “various embodiments,” “some embodiments,” “one embodiment,” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases “in various embodiments,” “in some embodiments,” “in one embodiment,” or “in an embodiment” in places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments.

Before the present process or steps are described, it is to be understood that this disclosure is not limited to any particular process or organism, as there can be multiple possible embodiments that are not expressly illustrated in the present disclosure but may still be practicable within the scope of the present disclosure.

Also, the technical solutions offered by the present disclosure are clearly and completely described below. Examples in which specific reagents or conditions may not have been specified have been conducted under conventional conditions or in a manner recommended by the manufacturer.

Description:

As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. All composition numbers and ranges based on percentages are weight percentages, unless indicated otherwise. All ranges of numbers or conditions are meant to encompass any specific value contained within the range, rounded to any suitable decimal point.

In an embodiment, the presently disclosed subject matter provides an improved process for enzymatic hydrolysis of lignocellulosic feedstock comprising the steps of subjecting the lignocellulosic feedstock to impregnation to obtain impregnated slurry and adjusting the concentration and pH of the treated slurry to obtain a stream. The invention further discloses the addition of additives to the stream to obtain an additive-treated stream and subjecting the additive-treated stream to enzymatic hydrolysis to obtain enzyme-hydrolyzed stream comprising fermentable sugars. The invention also discloses cofermenting the enzyme-hydrolyzed stream to obtain fermentation product, the said hydrolysis is carried out using active dry yeast. The improved process for enzymatic hydrolysis of the lignocellulosic feedstock of the present invention is characterized in that the addition of additives to the stream increases the efficiency of enzymatic hydrolysis at least by 7%.

In one embodiment of the present invention, the process includes several steps. Each step has one or more elements for performing the specific function required in an integrated process for obtaining ethanol and proteins from grains. A person skilled in the art may appreciate different variations and/or combinations of these elements that may be used to perform the objects of the invention disclosed herein.

The term “lignocellulosic feedstock” as used herein refers to plant biomass that comprises cellulose (35-50%), hemicellulose (20-30%), and lignin (15-25%).

Some of the embodiments of the present invention relate to an improved process for the enzymatic hydrolysis of lignocellulosic feedstock. Some embodiments of the present invention enable the processing of lignocellulosic feedstock such as annual grasses, energy crops, or other annually renewable feedstocks. In a preferred embodiment, the lignocellulosic feedstock is selected from softwoods, forest residues, or combinations thereof. The term “softwood” as used herein refers to a type of wood that is derived from conifers and comprise of cellulose (40-45%), hemicellulose (15-25%) and lignin (26-34%). In the instant disclosure, the softwood is preferably selected as lignocellulosic feedstock for the production of ethanol. The softwood also has the potential to produce mainly monomeric hexose sugars, which are much easier to ferment than pentose sugars. Examples of softwoods include pine, spruce, cedar, and fir.

The process of pre-processing the softwood is carried out to obtain a feedstock of desired or predetermined particle size. Pre-processing is optionally carried out depending on the physical form of the softwood using one or more techniques selected from milling, particle size-classifying, and combinations thereof. The term “pre-processing” includes size reduction, before feeding to the digester.

Size reduction is one of the necessary and preliminary steps in the pre-processing in order to obtain adequate yields in the production of ethanol from softwood. It is found to alter important physical and structural configurations of softwood, including surface area-to-volume ratio, shape, number of interparticle bonding, and cellulose crystallinity.

In the process to convert a softwood into various components, it is often desirable to impregnate with impregnating agent into the feedstock, prior to downstream processing. In this context, the terms “impregnate” and “impregnation” as used herein refers to the introduction of impregnating agent into the feedstock. In some cases, the impregnating agent potentially dissolves at least some part of the softwood. This can help to break the recalcitrant structure and break the carbohydrate lignin matrix present in the feedstock.

In certain embodiments, the process of impregnation comprises mixing the pre-processed material or the feedstock with water and impregnating agent and adding it to the impregnation chamber using a screw feeder.

In a preferred embodiment, the softwood is subjected to impregnation to obtain impregnated slurry by mixing the pre-processed feedstock or feedstock with at least one reactant selected from but not limited to acid, salt of the acid, base, salt of the base, or a combination thereof. Impregnation of the pre-processed feedstock or feedstock with impregnating agent to obtain impregnated slurry is carried out by treating with sulphuric acid, sulphurous acid, sulphur dioxide, nitric acid, phosphoric acid, hydrochloric acid, acetic acid, formic acid, maleic acid, lactic acid, and combinations thereof.

In a preferred embodiment, impregnation comprises treating processed feedstock with SO2 as an impregnating agent. The SO2 (impregnating agent) is used in gaseous or liquid form. The process of impregnation is carried out in any conventional manner with suitable parameters/conditions known in the art to achieve the objective.

The term "pre -treatment" or “treatments” as used herein refers to a physical and/or chemical treatment that renders a cellulosic component of the feedstock more accessible to an enzyme that converts carbohydrate polymers into fermentable sugars during a subsequent enzymatic hydrolysis process, and/or which renders the physical structure of the feedstock more susceptible to such enzymatic hydrolysis. The conditions for the pre-treatment are suitably chosen in order to achieve the objective.

In some embodiments, the pre-treatment is carried out at any suitable temperature. In an embodiment, it is carried out at a temperature ranging from 100 to 250°C; preferably ranging from 150 to 240°C and more preferably from 180 to 220°C.

The pre-treatment process may be carried out at any suitable pressure. In an embodiment, the pretreatment is carried out at a pressure in the range from equal to or more than 0.5 MPa to equal to or less than 3.5 MPa, more preferably in the range from equal to or more than 0.75 MPa to equal to or less than 3 MPa, more preferably ranging from > 1.0 MPa to <2.5 MPa.

In a preferred embodiment, subjecting the impregnated slurry to further treatment involves heating to a temperature ranging from 180°C to 210°C at a pressure of 1.5 to 1.8 MPa for 5-30 minutes to obtain a treated slurry.

The effectiveness of a treatment depends on a combination of factors such as the temperature, pressure and pH to which the feedstock is exposed and the duration of that exposure. It is usually desirable to use as mild a combination of pre-treatment conditions as possible. In an embodiment, the concentration and pH of the treated slurry are adjusted to obtain a stream. The impregnated slurry subjected to further treatments to obtain treated slurry comprises suspended and dissolved solids, the said suspended solids further comprising cellulosic material. In another related embodiment, the concentration of the treated slurry is adjusted to about 15-25% w/w total solids by diluting it with water or recycled water.

In an embodiment, the pH of the treated slurry is adjusted to range from 5.0 to 5.5 and further maintained at a temperature ranging from 52 to 55°C.

It is found that the addition of an additive to softwood, during a pre-treatment step preceding the enzymatic hydrolysis, can significantly enhance the efficiency of the hydrolysis process. This can lead to improved yields of the sugars which result from the hydrolysis of cellulosic components of the feedstock.

It is believed that the additive binds to lignin present in the feedstock, thus preventing the lignin from binding to the enzyme catalyst during hydrolysis. This in turn results in more of the enzyme available for productive catalysis at the cellulose substrate. The additive tends to remain bound during the downstream process after the pre-treatment process, for example during the subsequent washing of the biomass and during its subsequent hydrolysis.

In an embodiment, additives are added to the stream to obtain an additive-treated stream.

There are many additives such as surfactants, proteins, and synthetic polymers which enhance the enzymatic hydrolysis by blocking the non-productive adsorption sites of lignin. Surfactants such as Triton X-100, Tween 20 and like in the range of 0.03-0.3 g/L showed an increased cellulose conversion rate from 9 to 21 % depending on the lignin content. Also, the addition of protein such as BSA bind to lignin, preventing unproductive binding of cellulases. Further, synthetic polymers containing ethylene oxide such as polyethylene glycol; non-ionic surfactants such as polyol esters, polyoxyethylene esters, poloxamers and inorganic salts such as K2CO3, K3PO4, K2HPO4 , KH2PO4, K2SO4, KC1 are also used as additives for increasing the efficiency of enzymatic hydrolysis. Additives bind to the residual lignin of the substrates through hydrophobic interactions and prevents cellulase adsorption on lignin. Addition of additives modifies lignin surface properties such as change its hydrophobicity, hydrogen bonding ability and surface charges.

In some embodiments, the additives are selected from polyhydroxy alcohol, glycols, non-ionic surfactants, inorganic salts, and combinations thereof. In a related embodiment, the additives are added in a ratio ranging from 0.25 to 15.0% w/w of the total solids at 50 to 55°C over 5-60 minutes.

The process according to the instant invention comprises a subsequent enzyme hydrolysis process, in which the additive-treated stream is subjected to enzymatic hydrolysis of one or more of its cellulosic components to convert it into a sugar.

In an embodiment, the additive-treated stream is treated with enzymes to obtain enzyme -hydrolysed stream comprising fermentable sugars.

In particular, enzymatic hydrolysis comprises contacting the additive-treated stream with cellulolytic enzyme cocktail including but not limited to endoglucanases, exoglucanases, hemicellulases, and mixtures thereof.

In a specific embodiment of the invention, the enzymes are used at a dose from 0.1 to 25%, preferably from 0.5 to 10% and more preferably from 1 to 6 %.

In another embodiment, the enzymatic hydrolysis is carried out a temperature ranging from 10 to 90°C, preferably ranging from 20 to 70°C and more preferably ranging from 40 to 60°C.

In a related embodiment, the enzymatic hydrolysis is carried out at a pH ranging from 2-7, preferably ranging from 3 to 7 and more preferably from 4 to 6.

The enzymatic hydrolysis process is carried out for any suitable duration. It is carried out for a period of 20 to 90 hrs, preferably from 30 to 80 hours and more preferably from 60 to 75 hours. In a preferred embodiment, the enzymatic hydrolysis is carried out by cellulolytic enzyme cocktail at a dose ranging from 1 to 6% for 72 hours at a pH ranging from 4.8 to 5.2 and temperature ranging from 50 to 55°C. The enzymatic hydrolysis results in one or more sugars such as glucose which can advantageously be used in a fermentation process to produce one or more alcohols.

In another embodiment, the said enzyme hydrolyzed stream comprises glucose ranging from 4 to 8%, xylose ranging from 0.5 to 1.5%, galactose between 0.2% to 0.5%, and mannose between 2.0 to 3%.

The fermentation of the enzyme -hydrolyzed stream is carried out in any conventional manner. In an embodiment, the fermentation of the enzyme-hydrolysed stream is carried out to obtain the fermentation product. It may in particular be catalysed by a microorganism, more particularly yeast or bacteria such as the Saccharomyces, Lactobacillus, Actinobacillus. Pichia or Candida species.

In an embodiment, the co-fermentation of the enzyme -hydrolyzed stream to obtain fermentation products is carried out using whole cells (yeast or bacteria). In a preferred embodiment, cofermentation of the enzyme-hydrolysed stream to obtain fermentation product is carried out using active dry yeast.

Fermentation products may be any product obtainable from fermentation including ethanol, isopropanol, acetone, n-butanol, isobutanol, 1,4-butanediol, succinic acid, lactic acid, and like. In a preferred embodiment, the fermentation product includes ethanol.

A process according to an embodiment of the invention may comprise one or more additional processing steps after the fermentation step. For instance, the process may comprise separating out the biomass or feedstock, particularly residue from the fermentation broth, for instance by centrifugation, and/or recovering the desired fermentation product, for instance by distillation, and/or purifying the recovered fermentation product. The fermentation product yield (e.g., ethanol yield) is the yield of the final product produced in fermentation.

The term "bioethanol" or “ethanol” is used interchangeably herein with "ethanol" and refers to ethanol generated from the conversion of plant matter. According to an aspect, the invention provides the efficient enzymatic/enzyme -catalysed hydrolysis of softwood in the presence of additives for the generation of fermentable sugar from softwood, for one or more of the following purposes: i. improving the efficiency of the process, or of a processing step forming part of the process, in particular, the hydrolysis of a cellulosic component of the softwood; and ii. improving the yield of the fermentable sugars.

An improvement in the yield of the fermentable sugar may be manifested by a higher yield in any one or more fermentable sugars, at a specific point in time and/or over a specific period of time and/or on completion of the process or of a hydrolysis step which forms part of the process.

In a preferred embodiment, the improved process for enzymatic hydrolysis of the softwood is characterized by the addition of additives to the stream increase the efficiency of the enzymatic hydrolysis by at least 7%.

Furthermore, a process for obtaining fermentable sugars from softwood, or any other process for the treatment of softwood which involves subjecting the softwood to enzymatic hydrolysis, may be marketed with an indication that it benefits from an improvement due to the use of an additive in the process, in particular a greater efficiency.

Examples

The following examples have been included to provide illustrations of the presently disclosed subject matter. Considering the present disclosure and the general level of skill in the art, those of skill will appreciate that the following examples are intended to be exemplary only and that numerous changes,

modifications, and alterations can be employed without departing from the spirit and scope of the presently disclosed subject matter.

Source of material:

The examples have been carried out using Norway Spruce sawdust sourced from Nordic countries. Cellulase enzymes have been procured from Novozymes. Active dry yeast is obtained from Leaf technologies, from Chile.

Example 1: Process for enzymatic hydrolysis of softwood

Softwood sawdust from Norway Spruce (60kg) and Pine (60 kg) were used as lignocellulosic feedstock to determine the effect of additives on enzymatic hydrolysis. The initial analysis of the Norway Spruce and Pine revealed composition comprising total solids, dissolved solids, lignin, ash and protein (Table 1).

Table 1: Initial composition of Norway Spruce and Pine The softwood was subjected to impregnation using SO2 (liquid or gaseous or combination thereof) with an inlet flow of 60kg/h delivering about 0.36kg/h to obtain an impregnated slurry. Prior to impregnation, the softwood may be optionally subjected to pre-processing by size reducing steps to enhance impregnation efficiency. The impregnated slurry was passed through a continuous pre-treatment system maintained at a temperature of 195°C and pressure of 1.8 MPa for 10 minutes to obtain 130kg/h of pre -treated slurry in an hour.

Further, 5.3kg of the treated slurry having pH 1.5 was diluted with 1.7 kg of water to adjust the concentration of the slurry comprising 19.67% and 16.81% total solids in Norway Spruce and Pine respectively (Table 2). Also, pH 1.5 was raised to pH 5.5 by treating with 81g of ammonia solution to obtain a stream.

Table 2: Analysis of SO2 pretreated slurry

Effect of different additives on the enzymatic hydrolysis of Norway Spruce as a feedstock was determined using additives including PEG 1500, PEG 4000, Tween 80, Tween 20 and Triton X 100 alone or in combination at 0.25-2% w/w and a 3% enzyme dose (Cellic SE 1.0. enzyme activity of 2343 BHU/g) to obtain additive treated stream which was maintained at 50-52°C and pH 5.0 to 5.2 for 72h with continuous stirring at 300rpm.

Significant difference was observed in the enzyme efficiency, ranging from 67.72% to 75.73%, by varying additives alone or in combination. Additive treated stream with Tween 80 at a dose of 0.5- 2%%w/w exhibited the highest percentage of 75.73% followed by PEG 4000 with 74.20% of enzyme efficiency (Table 3) when compared with control (63.87%). The use of additives alone proved more effective when compared to the use of additives in combination. Table 3: Effect of additives on enzyme hydrolysis of SO2 pre -treated Norway Spruce

The effect of varying enzyme dose on efficiency of enzymatic hydrolysis was determined using additive treated stream of Norway Spruce comprising (18.18% w/w) and Pine (16.8% w/w) total solids. The additive started streams were treated with varied doses of cellulolytic enzyme cocktail (Cellic SE 1.0, 2343BHU/g of enzyme activity) ranging from 1.5 to 15 % w/w (based on total solids) at a temperature of 45 to 55°C and pH ranging from 4.5 to 5.5 for about 72-120 hours to obtain enzyme-hydrolysed stream.

The percentage of glucan to glucose conversion efficiency during enzymatic hydrolysis of the enzyme-hydrolysed streams increased with the increase in the enzyme dose from 1.5% to 15%. An enzyme dose of 15% w/w exhibited the maximum conversion percentage (Table 3). Enzyme- hydrolysed stream with PEG 4000 at a dose of 15% exhibited a maximum conversion of glucan to glucose at 86.10% and 86.5% in Norway Spruce and Pine respectively.

Table 3: Effect of different enzyme doses on SO2 pre -treated Spruce and Pine 0.7kg from the additive-treated enzyme hydrolyzed stream of Norway Spruce and Pine were further treated with active dry yeast of 1 kg/KL ethanol and 0.1% w/w of urea at a temperature of 32°C for 48 hours to obtain fermentation broth rich in ethanol.