CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority from Iranian Patent Application Ser. No. 139950140003005451, filed on Sep. 16, 2020, and entitled “Modified high-efficiency bioprocess for bio-ethanol production from wheat milling lignocellulosic by-products” which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present disclosure generally relates to a method for converting wheat milling lignocellulosic by-products to high added value chemicals; particularly to a method for converting wheat bran to bioethanol, protein, and lignin derivatives, and more particularly to a method for extracting biochemical products using simultaneously fermentation and cell wall degrading enzymes (CWDEs).

BACKGROUND

[0002] Energy is one of the significant parameters for the economic growth and sustainable development of any country. The limited supply and ever-rising cost of fossil fuel and environmental issues have forced the world to find renewable, cleaner, and cheaper alternatives to fossil fuel to meet their energy demands. Therefore, the use of inexpensive and available resources such as biomass, municipal waste, agricultural residues, low-value food, or food- related products is rapidly increasing to obtain biofuels, chemicals, and other products with high added value.

[0003] Biofuels (Biodiesel and Bio-ethanol) obtained from the biochemical process have developed as one of the most suitable renewable alternatives to fossil fuel. They are less polluting than fossil fuel, reduce greenhouse gas emissions, and are essential components of decarbonization in the transport sector. Accordingly, biofuels are the preference in nearly every country, and an increasing proportion of policies are promoting the establishment of those on a large scale.

[0004] Nations' demand for inexpensive and low-cost resources to develop biofuels results in lignocellulosic biomass usage; it is known as abundantly available raw material on the Earth for biofuels, mainly bio-ethanol. It should be noted that the first generation bio-ethanol manufactured by the fermentation of plant sugars competes with other food products obtaining from these sources; while the advanced biofuel or 2nd generation does not have this limitation. [0005] Despite the cheap and abundant lignocellulosic materials, second -generation bio- ethanol production costs are still high. As a result, biorefineries were developed to decrease the production cost of biofuels by converting the biomass into a spectrum of bio-based products such as chemicals, feed, and biofuels.

[0006] The wheat milling process for wheat flour production has different by-products, the major of which are wheat bran and wheat germ having a high economic value followed by other valuable by-products. Wheat bran is a lignocellulosic source for biofuels production and is known as a potential plant protein source. It is well known that most of the proteins that exist in wheat gran are in the Aleurone layer. Proteins deposited in the Aleurone layer are surrounded by cell walls comprising of complex insoluble carbohydrates and lignin, resulting in difficult protein digestion in the human digestive system. In addition, concentrated phytic acid and protein in the wheat bran form a complex, making the solubility and bioaccessibility of bran protein limited.

[0007] Recently, bio -processing of wheat bran using carbohydrazides and proteases increases the protein solubility up to 55%. Additionally, activating the endogenous enzymes of bran improves the protein solubility by approximately 75%. Accordingly, both biological processing using hydrolytic enzymes and lactic acid fermentation have been known as the most effective method in improving the digestion of bran proteins.

[0008] The capital required to set up a bio -ethanol production plant depends on the production capacity, process type, raw material selection criteria, and various output products. Thereby, there is a need for an industrial process to produce bio-ethanol from a lignocellulosic feedstock, especially wheat bran, wherein proteins and lignin derivatives are simultaneously produced. It is believed that the disclosed process overcomes the above mentioned drawbacks in using wheat bran, providing a cost effective solution for producing bio-ethanol with proteins and lignin derivatives on an industrial scale.

SUMMARY

[0009] This summary is intended to provide an overview of the subject matter of the present disclosure, and is not intended to identify essential elements or key elements of the subject matter, nor is it intended to be used to determine the scope of the claimed implementations. Its sole purpose is to present some concepts of one or more aspects in a simplified form as a prelude to the more detailed description that is presented later. The proper scope of the present disclosure may be ascertained from the claims set forth below in view of the detailed description below.

[00010] In one general aspect, the present disclosure relates to an inexpensive, highly efficient, and industrial process that extracts high potential value-added products from wheat bran. The invented method includes sets of experiments carried out step by step in a cascade sequence to create products, namely, bio-ethanol, proteins, and lignin derivatives. The invention disclosed a method for converting the low-cost and high amount of wheat bran to different chemicals using an inexpensive, easy -to-do, and highly efficient process. It is further disclosed that high quality proteins, bio-ethanol, and lignin derivatives are extracted from wheat bran using the invention method. [00011] Another aspect of the present disclosure may direct to a high-efficiency bio-process technology of wheat milling by-products for producing biochemical products such as protein, bio-ethanol, and lignin biopolymers.

[00012] In one or more exemplary embodiments, the invention disclosed a bio-process comprising of both fermentation and cell wall degrading enzymes (CWDEs) for extracting biochemical from wheat bran. As a result, the accessibility and digestion of wheat bran proteins and the breakdown of its hydrocarbon simultaneously increase.

[00013] The method further includes the enzymes that degrade the wheat bran cell wall, which leads to an increase in the number of fermentable sugars and available carbohydrates of the wheat bran. They also improve the microbial growth of bran yeast microorganisms, accelerating the acidification of bran bio-process, leading to activating the endogenous enzyme of bran, contributing to an increase in protein hydrolysis and dissolution. The synergy in increasing the acidity and improving the digestion of extracted protein is the reason for enhancing the dissociation of wheat bran in the first step. In one or more exemplary embodiments, the present invention disclosed a formulation on enzyme composition, hydrolysis time, and temperature to optimize the extraction of fermentable sugars and carbohydrates of wheat bran.

[00014] Indeed, this invention proposes a multi-step process to obtain different chemicals from wheat bran. In one or more exemplary embodiments of the present invention, after hydrolyzing bran to simple sugars in the first step, the method further includes a second step wherein other embedded chemicals in wheat bran are extracted using aerobic and anaerobic fermentation. Bio-ethanol and lignin derivatives are some kind of the valuable byproducts which can produce in the second step.

[00015] This Summary is provided to introduce a selection of concepts in a simplified form; these concepts are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

[00016] The novel features which are believed to be characteristic of the present disclosure, as to its structure, organization, use and method of operation, together with further objectives and advantages thereof, will be better understood from the following drawings in which a presently preferred embodiment of the present disclosure will now be illustrated by way of example. It is expressly understood, however, that the drawings are for the purpose of illustration and description only and are not intended as a definition of the limits of the present disclosure. Embodiments of the present disclosure will now be described by way of example in association with the accompanying drawings in which:

[00017] FIG. 1A illustrates a flow chart of a method for the production of bioethanol, protein, and lignin from lignocellulosic biomass, consistent with a preferred exemplary embodiments of the present disclosure;

[00018] FIG. IB illustrates a detail flow chart of the pre-processing step of a method for the production of bioethanol, protein, and lignin from lignocellulosic biomass, consistent with a preferred exemplary embodiments of the present disclosure.

DETAILED DESCRIPTION

[00019] In the following detailed description, numerous specific details are set forth by way of examples to provide a thorough understanding of the relevant teachings related to the exemplary embodiments. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well known methods, procedures, and components have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings. [00020] The following detailed description is presented to enable a person skilled in the art to make and use the methods and devices disclosed in exemplary embodiments of the present disclosure. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of the present disclosure. However, it will be apparent to one skilled in the art that these specific details are not required to practice the disclosed exemplary embodiments. Descriptions of specific exemplary embodiments are provided only as representative examples. Various modifications to the exemplary implementations will be plain to one skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the scope of the present disclosure. The present disclosure is not intended to be limited to the implementations shown, but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.

[00021] It must be noted that, as used in this specification, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[00022] As used herein, the terms “comprising,” “including,” “constituting,” “containing,” “consisting of,” and grammatical equivalents thereof are inclusive or open-ended terms that do not exclude additional, unrecited elements or method steps.

[00023] Reference herein to “one embodiment,” “an embodiment,” “some embodiments,” “one or more embodiments,” “one exemplary embodiment,” “an exemplary embodiment,” “some exemplary embodiments,” and “one or more exemplary embodiments” indicate that a particular feature, structure or characteristic described in connection or association with the embodiment may be included in at least one of such embodiments. However, the appearance of such phrases in various places in the present disclosure do not necessarily refer to a same embodiment or embodiments.

[00024] The word “exemplary” is used herein to mean “serving as an example, instance, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments. Likewise, the term “embodiments of the invention” does not require that all embodiments of the invention include the discussed feature, advantage or mode of operation.

[00025] It should also be noted that in some alternative implementations, the functions/acts noted may occur out of the order noted in the figures. For example, two operations shown in succession may in fact be executed substantially concurrently or may sometimes be executed in the reverse order, depending upon the functionality/acts involved.

[00026] The terms used in this specification may generally have their ordinary meanings in the art, within the context of the disclosure, and in the specific context where each term is used. Certain terms that are used to describe the disclosure are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the disclosure. It will be appreciated that a term or a phrase may be said in more than one way.

[00027] Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein. Nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and is not intended to further limit the scope and meaning of the disclosure or of any exemplified term. Likewise, the disclosure may not be limited to various embodiments given in the present specification.

[00028] In a general embodiment of the invention, the present invention discloses a modified bioprocess to treat wheat bran for producing protein, bio-ethanol, and lignin biopolymer. Wheat bran as a lignocellulosic byproduct of wheat milling is an inexpensive and abundant source of biomass. The present invention discloses a bioconversion process of wheat bran into different high value-added products. Referring to FIGs. 1, in a general aspect of the invention, the modified bio-process for treating the bran (100) comprises some main steps. The first step, the most important part of the invention, is an integrated pre-processing of wheat bran, lignocellulosic waste, or biomass (102). In this step, the fermentation of 2nd generation sugars and converting them to cellulosic ethanol is performed by controlling the viscosity and enzymatic hydrolysis of the mixture (104). In other words, the preprocessing increases the bioaccessibility of the bran resources for later molecular dissociation using primary bran decomposing.

[00029] Referring to FIG. IB, in one or more exemplary embodiments, the first step or preprocessing of wheat bran (102) consists of one or a plurality of sub steps, of which they can be included or excluded regarding the quality of the bran. The exemplary first step may include, but is not limited to, a plurality of sub steps consistent with exemplary aspects and embodiments described herein. In one embodiment, wheat bran in a sub-step can be soaked to remove the impurities and dirties (1022). Wheat bran can be kept in a vertical storage tank in contact with a fluid for a while. It should be mentioned that the type of fluid, storage tank, and time of soaking are not limited to the mentioned example. In other words, the other methods of bran cleaning, which skilled persons in the relevant art know, are also in the scope of the present invention. In another exemplary embodiments, the wheat bran can also be disinfected (1024), grinded or milled (1026). It should be noted that herein there are some examples of sub step, but they are not limited to the mentioned activities.

[00030] In the second step, the mixture's viscosity reduces, and cellulosic compounds decompose using enzymatic hydrolysis (104). Finally, in the next step comprises fermentation and extraction of lignin.

[00031] As can be seen in FIG. IB, after filtration of cleaned wheat bran, they are exposed to the saturated vapor to break the bonds between lignin, cellulose, and hemicellulose, contributing to activate the bran cell wall degrading enzymes (CWDEs) (1028). This present invention reduces inhibitors formation and their effects on the following steps, and also benefits the advantages of water- and vapor-based processes. Additionally, this disclosed invention does not use chemicals; providing the highly efficient separation of cellulose and hemicellulose structures leads to operation costs decrease. The synergism in increasing the acidity and improving the digestibility of the extracted protein improved the pretreating the cellulose structure in the bran and its decomposition in the preprocessing step. At the same time, it brings the highest protein extraction efficiency with high digestibility. The preprocessing step is effective in obtaining bioaccessibility in the bran structure and improves the following separation process. It should be noted that in this step, the decomposition process has not yet begun, and the first step is only a preprocessing stage.

[00032] In one or more exemplary embodiments, the second step comprises the viscosity reduction and enzymatic hydrolysis of cellulose and hemicellulose. In the second step, the enzymes and endogenous biochemical decomposition are simultaneously used. The cell wall degrading enzymes (CWDEs) catalytically increase the fermentable sugars, the carbohydrate content of wheat bran, and the extraction of digestible proteins. The second step uses decomposition and liquefying of the pre-processed feed simultaneously to ensure a continuous process and transfer the material to the fermentation stage. The performance of the generated enzymes combined with the proper design provides a highly efficient decomposition for the liquid mixtures within a short stay time and high dry content.

[00033] In one or more exemplary embodiment, after the first pre-processing step (102), a mixture of hydrolytic enzymes comprises of xylanase, hemicellulose and amylase with ratio equal to 2: 1: 1 are used to process the preprocessed wheat bran (104). The process performs in a pre-processed substrate of bran lactic acid and phosphoric acid buffer at 32 °C, and pH level between 5-6 (106). Consequently, the microbial growth of endogenous yeast microorganisms is accelerated, leading to an increase in the acidification rate of the biological process happening in the operating medium, contributing to promoting the hydrolysis and dissolution of the protein in 24 hours. Consequently, the microbial growth of endogenous yeast microorganisms is accelerated, leading to an increase in the acidification rate of the biological process happening in the operating medium, contributing to promoting the hydrolysis and dissolution of the protein. This synergism facilitates the pre-concentration process of extractable proteins and enhances the decomposition of cellulose into sugar. At the same time, it provides high efficient digestible protein extraction of up to 90%.

[00034] Referring to FIG. 1A, in one or more exemplary embodiment of the disclosed invention, the protein separation stage (108) in the second step carried out at temperature between 30-40 °C and atmospheric pressure. Then the residual fractions are processed for further sugar extraction and cellulosic breaking down. Further processing of the enzymatic medium is performed at 60°C, at pH level of 5 and for 48 hours. At the end of this step, both the extraction of protein and the enzymatic decomposition of cellulose decrease the medium viscosity, leading to an increase in the solid content of the solution, followed by high efficient fermentation for bio-ethanol production.

[00035] In one or more exemplary embodiment, the next steps comprise of fermentation, bio- ethanol extraction (HO) and lignin derivatives production (112). These steps use the combination of bacterial strains and yeast to ferment sugar-containing fractions and decompose the micromolecular- and oligo- saccharide compounds. These steps in one or more exemplary embodiments can be performed in an ammonium phosphate buffer, at a pH level between 5-6, in the presence of glucoamylase enzyme and one or more nutrients, for 24 hours. One or more nutrients can be selected from the group consisting of malt, inofolic acid, and a mixture thereof. It should be noted that a molecular sieve continuously extracts the bio-ethanol to prevent the growth of fermentation inhibitors. [00036] In one or more exemplary embodiments of the invention, after the first 24 hours of the third step, the residual fraction is processed for alcohol distillation. After extracting the remaining alcohol, taking out the lignin derivatives could be performed. In the last part of the third step, the extracted lignin is converted to ligno sulfonate using the sulfonation method within concentrated sulfuric acid at 80 °C . The ligno sulfonates are separated using the ultrafiltration method.