A process for the preparation of 2, 3 - butanediol (BDO) from BIO-waste

Abstract

The present invention generally relates to the production of 2,3-butanediol in an improved and cost effective manner. The present invention specifically relates to a process of preparation of 2,3-butanediol using waste material i.e., rotten tomatoes without any costly pre-treatment. More specifically, the present invention relates to production of 2,3- butanediol using biobased waste material fermentation media i.e., concentrated tomato juice from waste rotten tomatoes, in presence of bacillus bacterium (specifically novel Bacillus halotolerans EX5-6). The prepared 2,3-butanediol (2,3-BD or BDO) can act as a promising bivalent alcohol with its wide variety of industrial applications such as platform chemical serving as precursor for several industrial products like rubber, solvents, and possess biofuel properties, etc.

Description

[0001] A PROCESS FOR THE PREPARATION OF 2, 3 - BUTANEDIOL (BDO) FROM BIO-WASTE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a process for the preparation of 2, 3 -butanediol in an improved and cost effective manner using a waste material i.e., rotten tomatoes without any costly pre-treatment. More specifically, the present invention relates to production of 2,3-butanediol using bio-based waste material based fermentation media i.e., concentrated tomato juice from waste rotten tomatoes, in presence of Bacillus halotolerans EX5-6 (MCC 0265) using novel process. The prepared 2,3-butanediol (2,3-BD or BDO) can act as promising bivalent alcohol with its wide variety of industrial applications such as platform chemical serving as precursor for several industrial products like rubber, solvents, and possess biofuel properties, etc. The invention shall help attain the 12thsustainable development goal of Responsible Consumption and Production.

[0004] BACKGROUND AND PRIOR ART OF THE INVENTION

[0005] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the present invention, or that any publication specifically or implicitly referenced is prior art.

[0006] 2,3-butanediol (2,3-BD or BDO) is a bivalent alcohol having a wide range of applications in several industries serving as a precursor material, an essential component of drugs, ointments, antifreeze agents, and liquid fuels (biofuels). The 2,3-BD market is growing expeditiously and by the year 2027, it is expected to reach $220 million [Tsigoriyna et al., Fermentation, 7(3), 118, 2021; and Song et al., Journal of Industrial Microbiology and Biotechnology, 46(11), 1583-1601, 2019]. The need for 2,3-BD arises due to a multitude of reasons such as the exhaustive nature of fossil fuels, increasing oil consumption, and environmental issues such as greenhouse gas emissions. Therefore, there is an utmost requirement for renewable energy in the form of biofuels from various renewable waste sources such as lignocellulosic biomass and agro-industrial waste.

[0007] In literature, there are several methods disclosed for preparation of BDO such as chemical methods, microbial methods, etc. Some of the chemical methods cover preparation of BDO from butenes (C4H8) derived from crack gases as raw material or oxidation of butan-2-one (MEK) followed by basic hydrolysis and hydrogenation of acetaldehyde to obtain BDO (refer, Maina, S., et al., Biotechnology advances, 54, 107783, 2022). However, these chemical methods suffer from disadvantages like high energy consumption, expensive catalysts, higher amounts of catalyst requirement, harsh reaction conditions, environmental concern, low conversion yield, high cost and low safety in food and cosmetic application, higher impurities production, lesser selectivity, etc.

[0008] Some of microbial-based methods are also known in the literature for the production of BDO i.e., Klebsiella and other enteric bacterial species. However, these species belong to risk group 2 (pathogenic bacteria) having a potential of opportunistic infections, resulting in an obstacle at industrial level of production due to safety guidelines (Hazeena et al., Bioresource technology, 302, 122873, 2020). Therefore, researchers across the world are exploring GRAS (Generally Regarded As Safe) strains for developing BDO production process with higher yield, productivity, and titer. Bacillus generally are considered as GRAS producers of BDO having flexible metabolic capabilities such as utilizing wide range of substartes (Cellulosic and non-cellulosic), ability to thrive in microaerophilic and aerophilic conditions, and their adaptability of different physical environments (extreme pH and temperatures). In the last decade, various natural and engineered BDO producers belonging to bacillus have been reported such as B. licheniformis, B. subtilis, B. amyloliquefaciens, Paenibacillus spps. and several others, giving a comparable production titer of as high as 147g / l and reaching close to theoretical yield of 0.5 g / g of glucose (Petrov and Petrova, Fermentation, 7(4), 307, 2021). However, these type of microbial methods require higher amounts of costly and synthetic based carbon source materials or combination / mixture thereof (glucose, sucrose, fructose, maltose, etc. upto 25% or more), nitrogen source material(s), additional salts and / or additives to achieve effective synthetic media for BDO production, which ultimately increases the overall cost and makes the process tedious. Moreover, it requires multiple substrates / components to have effective conversion, requires higher temperature of around 55 °C, requires higher alkaline pH, requires higher cost of feedstock and their pretreatment, etc., making it not at all preferable method for BDO production.

[0009] Accordingly, to address such problem of use of tedious and costly synthetic media, there is a one report provided where waste substrates used for the production of BDO. It includes utilizing lignocellulosic hydrolysate, com stover hydrolysate, molasses, bakery waste hydrolysate, and crude glycerol (Petrov and Petrova, Fermentation, 7(4), 307, 2021, and Hazeena et al., Bioresource technology, 302, 122873, 2020). Still, in these substrates and methods, the cost of production remains a hindrance. The cost could be mainly attributed to the pretreatment process required for preparation of lysate and substrate availability.

[0010] Hence, there is a dire need of an alternative waste source material, which requires least chemical treatments and provides effective production of BDO with cheaper substrates and biobased media, thus totally avoiding multicomponent synthetic media.

[0011] Accordingly, keeping in view the drawbacks of the hitherto reported prior art, the inventors of the present invention realized that there exists a dire need to provide a fully bio-based waste material i.e., rotten tomatoes for generating cost-effective and complete media, without the requirement of any pre-treatment steps, which when treated with Bacillus species gives rise to effective production of BDO making it cost effective, simple and viable method for production of BDO. The substrate rotten tomatoes contain carbon and other nutrient sources thereby eliminating the need of glucose, yeast extract and other salts, which increase cost of production. Further, the invention is a green synthesis process, as it does not require any pre-treatment step unlike in other biowastes such as lignocellulose biomass.

[0012] OBJECTIVES OF THE INVENTION

[0013] The main objective of the present invention is therefore to provide a process for the preparation of 2,3-butanediol from bio-waste which obviates the drawbacks of the hitherto reported prior art.

[0014] Another objective of the present invention is to utilize a bio-waste material as a complete medium for the process development and production of 2,3-BDO by Bacillus halotolerans EX5-6 (MCC 0265) in an improved and cost effective manner.

[0015] SUMMARY OF THE INVENTION

[0016] The present invention provides a process for the preparation of 2,3-butanediol [BDO] using bio waste material, the process comprising the steps of: a) preparing a bio based fermentation media from waste material, and b) treating and incubating said bio based fermentation media of step a) in presence of Bacillus halotolerans EX5-6 (MCC 0265) in a bioreactor under specific reaction conditions to obtain the desired BDO; wherein the waste material is waste rotten tomatoes, and other food or bio- waste which are rich in sugars.

[0017] In an embodiment, the present invention further provides a process of preparation of said bio based fermentation media used in above process, the process comprises steps of: i) grounding waste material in a grinder to form a paste / puree, followed by filtration to obtain a filtrate; ii) centrifuging the filtrate of step i) at speed of 8000 to 14000 rpm for 15 to 30 minutes at temperature in the range of 1 to 6°C to obtain diluted media; and iii) concentrating the diluted media of step ii) at a temperature in the range of 40 to 50°C under a vacuum to obtain said bio based fermentation media; wherein the waste material is rotten tomatoes, other food or bio-waste which are rich in sugars, and wherein said obtained media of step iii) is directly used for preparation of BDO without pre-treatment.

[0018] In another embodiment, the bio-waste which are rich in sugars is selected from rotten pumpkin, rotten carrots, rotten beets, waste bagasse, waste sugarcane juice, and so on.

[0019] In still another embodiment, the pre-treatment comprises acid or alkaline hydrolysis or utilization at higher temperatures such as in steam explosion or addition of any external nutrients. The process developed in the present invention avoids such pre-treatment step, which is labor intensive, costly and cover hazardous chemicals. In a further embodiment, the present invention provides a process for the preparation of 2,3-butanediol (BDO) from bio-waste, wherein the said process comprising the steps of:

[0020] (a) grinding the waste material in a grinder to form a paste / puree, followed by filtration to obtain a filtrate;

[0021] (b) centrifuging the filtrate of step (a) at speed of 8000 to 14000 rpm for 15 to 30 minutes at temperature in the range of 1 to 6 degree C to obtain diluted media;

[0022] (c) concentrating the diluted media of step (b) to obtain the desired bio-based fermentation media; or

[0023] (d) optionally, repeating the concentrating step (c) one or more times to obtain nX concentrated media; wherein X refers to ‘n’ times reduction in water concentration of said media, wherein ‘n’ is 2, 3, 4 or 5;

[0024] (e) incubating the bio-based fermentation media of step (c) or step (d) with the bacterial strain of Bacillus halotolerans MCC 0265 in a bioreactor to obtain 2,3-butanediol [BDO],

[0025] In another embodiment, the present invention provides a process wherein the preparation of bio-based fermentation media is done from waste material selected from waste rotten tomatoes, rotten pumpkin, rotten carrots, rotten beets, waste bagasse, waste sugarcane juice.

[0026] In still another embodiment, the present invention provides a process, wherein the bioreactor is selected from flask bioreactor and fed-batch fermenter level bioreactor.

[0027] In yet another embodiment, the present invention provides a process, wherein the biobased fermentation media is directly used for preparation of BDO without pre-treatment.

[0028] In still another embodiment, the present invention provides a process, wherein the specific reaction conditions for the preparation of BDO in a flask bioreactor comprising:

[0029] (i) temperature in the range of 25 to 37 °C;

[0030] (ii) no external pressure; (iii) stirring speed in range of 150-200 rpm in a rotatory shaker;

[0031] (iv) pH in range of 5.0 to 7.0;

[0032] (v) inoculum concentration of bacterium in range of 8%- 10%; and

[0033] (vi) time period of incubation in range of 20 to 30 hrs.

[0034] In yet another embodiment, the present invention provides a process, wherein the specific reaction conditions for the preparation of BDO in a fed-batch fermenter level bioreactor comprising:

[0035] (i) fermentation vessel volume in the range of 2 to 5 L;

[0036] (ii) working volume in range of 800 to 1600 ml;

[0037] (iii) media comprising 2X to 5X concentration of tomato juice;

[0038] (iv) sparging in range of 2 to 2.5 1 per minute (LPM);

[0039] (v) inoculum concentration of bacterium in the range of 8 to 10%;

[0040] (vi) stirring speed in range of 300 to 350 RPM;

[0041] (vii) pH in range of 5.0 to 7.0;

[0042] (viii) temperature in range of 25 to 35 degree C; and

[0043] (ix) time period in range of 32 to 40 h.

[0044] In still another embodiment, the present invention provides a process, wherein the flask bioreactor is having volume in the range of 200 to 500 ml, with media volume in the range of 25 to 100 ml.

[0045] In still another embodiment, the present invention provides a process, wherein the filtration is done by manual coarse filtration using muslin cloth. In yet another embodiment, the present invention provides a process, wherein the concentration in step (iii) or (iv) is done using rotatory evaporator at a temperature in the range of 40 to 50 degree and 80 to 120 rpm under 30 bar vacuum.

[0046] In still another embodiment, the present invention provides a process, wherein the biobased fermentation media is free of additive or synthetic component; and wherein the process provides acetoin as an additional product.

[0047] BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0048] Figure 1 shows yield and productivity of BDO when 2X concentrate of biobased media is used (TJ means tomato juice media without concentration, TJ-2X means tomato juice media with 2X concentration) where bacterium used is Bacillus halotolerans EX5-6. 2,3- BDO production at flask level. The production titer calculated using equation y=17.58x- 1.2114. GC analysis with conditions column FFAP (30mx0.25mmx0.25pm), Detector and injector temperature of 300°C, sample volume 1 ul was carried out.

[0049] Figure 2 shows yield and productivity of BDO when X concentrate of biobased media is used with or without additional components or nutrients [(TJM= Tomato juice concentrate (2X)+ Yeast extract and salts (no glucose)] where bacterium used is Bacillus halotolerans EX5-6. 2,3-BDO production at flask level. The production titer calculated using equation y=17.58x-1.2114. GC analysis with conditions column FFAP (30mx0.25mmx0.25pm), Detector and injector temperature of 300°C, sample volume 1 ul was carried out.

[0050] Figure 3a shows graph with standard peak for 2,3- butanediol upon GC analysis with conditions column FFAP (30mx0.25mmx0.25pm), Detector and injector temperature of 300°C, sample volume 1 pL.

[0051] Figure 3b shows graph with standard peak for Acetoin at 17.33 minutes and water 19.20 minutes from HPLC analysis using Aminex HPX-87H column with flow rate of 0.6ml / min of 5mM H2SO4.

[0052] Figure 3c shows graph with standard peak for Glucose at 9.1 minutes and water 19.2 minutes from HPLC analysis using Aminex HPX-87H column with flow rate of 0.6ml / min of 5mM H2SO4. Figure 4 shows graph with 2,3-BDO production in a Fed-batch process. The production titer calculated using equation y=17.58x-1.2114. GC analysis with conditions column FFAP (30mx0.25mmx0.25pm), Detector and injector temperature of 300°C, sample volume 1 pL was carried out.

[0053] DETAILS OF BIOLOGICAL RESOURCES USED IN THE INVENTION

[0054] The bacterial strain used for the purposes of the present invention is Bacillus halotolerans EX5-6 (MCC 0265). The bacterial strain MCC 0265 was isolated from the polycyclic aromatic hydrocarbon (PAH) enriched sediment of Pawana River, Pune, Maharashtra, India (18.57305, 73.83121) on Nutrient Agar medium (Himedia, India). Rotten tomatoes were collected from Pashan market, Pune, India (18.53718, 73.79206).

[0055] The isolated bacterial strain Bacillus halotolerans EX5-6 has been deposited with the IDA NCCS, Pune vide Accession number MCC 0265.

[0056] DETAILED DESCRIPTION OF THE INVENTION

[0057] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0058] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0059] Reference throughout this specification to “one embodiment” or “an embodiment” or “another embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this 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.

[0060] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be constructed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0061] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

[0062] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”

[0063] Also, use of "(s)" as part of a term, includes reference to the term singly or in plurality, for example, the term pharmaceutically acceptable salt(s) indicates a single salt or more than one salt of the compound of formula (I). The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0064] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description that follows, and the embodiments described herein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.

[0065] It should also be appreciated that the present disclosure can be implemented in numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.

[0066] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0067] Unless otherwise indicated, the following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention herein and the appended claims. These definitions should not be interpreted in the literal sense as they are not intended to be general definitions and are relevant only for this application. The term "or", as used herein, is generally employed in its sense including "and / or" unless the content clearly dictates otherwise.

[0068] The BDO described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. The BDO of the present invention that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically inactive starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. L-, D-, and meso isomers of the BDO of the present invention are described and may be isolated as a mixture of isomers or as separated isomeric forms. In some embodiments, the compound has the mixture of all forms of BDO i.e., L, D and meso. In some embodiment, BDO has (L)-configuration. In some embodiment, BDO has (D)-configuration. In some embodiment, BDO has (meso)-configuration.

[0069] Generally, D and L enantiomers refer to the configurational stereochemistry of the molecule. L isomers have the hydroxy group attached to the left side of the asymmetric carbon furthest from the carbonyl, while D isomers have the hydroxy group on the right side. Meso isomers are the ones which have presence of two or more than two chiral centres along with presence of c / i / Sn symmetry element / s in one or more conformers. For present invention, the BDO covers such isomers as below:

[0070] The term, “BDO or 2,3 -butanediol,” used herein is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of structures depicted.

[0071] In some embodiments, the BDO as synthesized, or salts thereof, are substantially isolated. By “substantially isolated” is meant that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compounds of the invention. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the BDO of the invention, or salt thereof. Methods for isolating compounds and their salts are routine in the art.

[0072] The expressions, “ambient temperature” and “room temperature” or “rt” as used herein, are understood in the art, and refer generally to a temperature, e.g. a reaction temperature, that is about the temperature of the room in which the reaction is carried out, for example, a temperature from about 25° C. to about 35° C.

[0073] Generally, the tomato is the one of the most important vegetable and is a key component of diet worldwide and is well-implicated for their role in lowering blood cholesterol. China is the largest producer of tomato, followed by India, contributing 11% of world’s production of tomato. As per the World Processing Tomato Council (WPTC), out of 130 million tons of tomato processed every year, around 8 million tons represent waste worldwide [Trombino et al., Molecules, 26(16), 5062, 2021]. Tomato has a lower shelf life and thus more prone to rotting due to fungal attacks and improper storage facilities. Most of the tomatoes are freshly consumed and around 2% are used to produced sauce and paste, however, more than 31% go to waste during tomato harvest in the peak season. Chemically, tomato consists of various nutrients and bioactive compounds including fructose and glucose as the major carbon sources. Compared to abundant lignocellulosic biomass, tomato contains non-cellulosic sugars that can be utilized by microbes without the need of any pretreatment process. In view of this, in general embodiment of the present invention, the inventors have provided a novel and improved process for production of BDO utilizing rotten tomatoes, and developed a cost efficient and simple process for the production of 2,3-butanediol.

[0074] In an aspect, the bioreactor is selected from flask bioreactor and fermenter level bioreactor.

[0075] In another aspect, the bio-based fermentation media is directly used for preparation of BDO without pre-treatment.

[0076] In still another aspect, the specific reaction conditions for the flask bioreactor comprise one or more of: 1) temperature in the range of 25 to 37 °C,

[0077] 2) no external pressure,

[0078] 3) stirring speed in range of 150-200 rpm in a rotatory shaker,

[0079] 4) pH in range of 5.0 to 7.0,

[0080] 5) inoculum concentration of bacterium in range of 8%- 10%, and

[0081] 6) time period of incubation in range of 20 to 30 hrs.

[0082] In yet another aspect, the specific reaction conditions for the fed batch fermentation conditions comprise one or more of:

[0083] 1) ferment vessel volume in the range of 2 to 5 L,

[0084] 2) working volume in range of 800 to 1600 mL,

[0085] 3) media comprises 2X-5X concentration of tomato juice,

[0086] 4) sparging is in range of 2 to 2.5 Litres per minute (LPM),

[0087] 5) inoculum concentration of bacterium in range of 8%- 10%,

[0088] 6) stirring speed in range of 300-350 RPM,

[0089] 7) pH is in range of 5.0-7.0,

[0090] 8) temperature kept in range of 25 to 35 °C, and

[0091] 9) time period in range of 32 to 40 h.

[0092] In still another aspect, the flask bioreactor is having volume in the range of 200 to 500 mL, with media volume in the range of 25 to 100 ml.

[0093] In yet another aspect, the flask level bioreactor is having volume 250 mL, with media volume of 50 ml. In still another aspect, the concentration of step iii) or iv) is done using rotatory evaporator at a temperature in the range of 40 to 50°C and 80-120 rpm under 30 bar vacuum.

[0094] In yet another aspect, the bio-based fermentation media is free of additive or synthetic component.

[0095] In still another aspect, the process provides acetoin as an additional product.

[0096] In yet another aspect, the bio-waste which are rich in sugars is selected from rotten pumpkin, rotten carrots, rotten beets, waste bagasse, waste sugarcane juice, and so on.

[0097] In still another aspect, the pre-treatment comprises acid or alkaline hydrolysis or utilization at higher temperatures such as in steam explosion (160 °C to 260 °C (Ziegler- Devin et al., 2021. Frontiers in Chemistry, 9, 705358)) causing rupture of the biomass fibers rigid structure, or addition of any external nutrients.

[0098] In another aspect, the bacillus bacterium is selected from Bacillus halotolerans. Similarly, other Bacillus species such as B.licheniformis and B.subtilis, which are well reported for 2,3-BDO production (Petrov and Petrova 2021) can also be utilized for the process of BDO production as disclosed above in this application.

[0099] In still another aspect, in the fed batch fermentation conditions, the ferment vessel volume is specifically 3L, the working volume is specifically is in range of 1200 to 1300 mL, and the temperature specifically is kept at 28 °C.

[0100] In a specific aspect, X refers to the multiple (times) concentration, where the 2X means doubling the concentration of media containing sugars and other contents of rotten tomato juice by reducing half the quantity of water. Similarly, 3X indicates removing three times of water from the original volume by rota evaporation. E.g. 1 kg of tomatoes approximately 500 to 700 ml of juice / media when X is 1, if X is 2, then the amount of juice / media is 250 to 350 ml, if X is 3, then the amount of juice / media is 125 to 175 ml, and so on.

[0101] In still another aspect, the filtration of step i) is done by manual coarse filtration using muslin cloth. This filtration step is not limited to muslin cloth, and one can use any type of filtration medium or machine can be used which is known in the art to effectively obtain filtrate.

[0102] In yet another aspect, the concentration of step iii) or iv) is done using rotatory evaporator at a temperature in the range of 40 to 50°C and 80-120 rpm under 30 bar vacuum. This concentration step is not limited to said temperature, rpm and vacuum, and one can use any type of concentration method or machine thereof known in the art to effectively obtain said media.

[0103] In still another aspect, no additional components or additives are used or added in the media obtained for production of BDO. E.g. if additional component like nitrogen source is externally added in said biobased media, it reduces the production and yield of BDO (refer, figure 2)

[0104] In yet another aspect he process produces acetoin as a by-product, which can also be converted into 2,3-BDO, further enhancing the overall efficiency and yield.

[0105] In still another aspect, the biobased media from waste rotten tomatoes provided 16.62 g / 1 of 2,3-BDO with productivity of 0.7g / l / h in batch fermentation at flask level. It is noted that this yield and productivity of BDO can be increased when the process is done in fed batch mode, refer figure 4.

[0106] The present invention proves to be very effective in terms of use of waste materials without addition of any external supplement or synthetic, to obtain BDO effectively. Moreover, inventors have successfully carried out FED-BATCH experimentation as per process of present invention to increase the production titre to 27.66 g / L compared to initial 16.6 g / L produced at 250 ml flask level.

[0107] The fed-batch mode of fermentation was carried out in 3L fermenter vessel in BioFlo / CelliGen 115 fermenter bioreactor (New Brunswick Eppendorf). The working media, which was 2X Tomato concentrate with a total volume was 1200 ml including 8% inoculum volume. The pH of media was 5.0 to 5.4. The temperature was kept constant at 28 °C. The agitation speed and sparging LPM (Liters per minute) was 300 and 2, respectively. Procedure: The fermentation media comprises of 2X tomato Juice with no external nutrient supplementation. The media was autoclaved at 10 psi for 10 minutes. 8% inoculum grown in seed medium (2% Glucose (GRM 016, Himedia), 0.5% each of peptone (RM667, Himedia), yeast extract (RM0277, Himedia), beef extract (0176000500, Loba Chemie), sodium chloride (MB023, Himedia), pH of 7.0) (OD at 600nm- 8.0) was inoculated in fermentation medium. After 16hrs of incubation, 6X concentrated Tomato Juice (200 ml) was fed to the fermentation media at IX concentration and monitored for production of BDO for 44hrs. We observed highest titer of 27.66 g / L at 32ndhrs with productivity of 0.86 (Fig. 5), higher than the flask level of 16.62g / L and 0.7g / L / h.

[0108] The inventors of present invention have also developed the optimized synthetic media for production of BDO using said bacillus bacterium where the synthetic media comprises 150g / L of Glucose (GRM 016, Himedia), 25 g / L of Yeast Extract (RM0277, Himedia), 3g / L K2HPO4 (26735, Fisher Scientific), 0.2g / L of MgSO4.7H2O (RM684, Himedia), 5g / L of NaCL (MB023, Himedia), and pH-5.0. This synthetic media provided 73.19 g / L and 0.5g / g of BDO titre and yield, respectively.

[0109] In a nutshell, the present invention addresses the problems such as high cost of substrates, product recovery cost, and microbial type (group 2 or GRAS) which have hindered its industrial implementation by providing a cheaper feedstock requiring lesser pretreatment unlike other lignocellulosic biomass. Specifically, the invention covers Bacillus halotolerans EX5-6, isolated from Pawana River sediment (Pune, India), which was screened for 2, 3 -butanediol production. At the 250ml flask level batch fermentation, upon optimization, inventors obtained 2,3-BDO concentration of 73.19 g / 1 with Yield and Productivity of 0.5 g / g and 1.02 g / l / h, respectively, using optimized synthetic media. This is a first report of BDO production from Bacillus halotolerans strain. Further, in order to address the cost challenges of feedstock, for the first time, inventors utilized tomato juice as a substrate for BDO production. India is a second largest producer of tomatoes, however, due to its lower shelf life, storage facilities issues, and fungal attacks, most of these end up getting wasted and dumped. Inventors developed a process wherein rotten tomatoes were concentrated to a various concentrations 2X, 3X, and 4X. In comparison to optimized synthetic media containing costly substrates, 16.62 g / 1 of 2,3-BDO was obtained from concentrated 2X rotten tomatoes with productivity of 0.7g / l / h in batch fermentation at flask level and 27.66 g / L with productivity of 0.86g / L / h from Fed-batch process which is high as compared to many other reports utilizing different lignocellulosic and other wastes. Tomato waste served as a complete medium as the addition of optimized medium constituents had no increasing effect on the BDO production thereby substituting the synthetic medium.

[0110] EXAMPLES

[0111] The following examples are given by way of illustration only and therefore should not be construed to the scope of the present invention in any manner.

[0112] Biological and other materials used in present invention:

[0113] The bacterium Bacillus halotolerans (EX5-6) used in the present invention was isolated from the polycyclic aromatic hydrocarbon (PAH) enriched sediment of Pawana River, Pune, Maharashtra, India (18.57305, 73.83121) on Nutrient Agar medium (Himedia, India).

[0114] Rotten tomatoes were collected from Pashan market, Pune, India (18.53718, 73.79206).

[0115] The screening for BDO production was done using synthetic fermentation media with composition of Glucose 50g / L (GRM 016, Himedia), K2HPO4 3g / L (26735, Fisher Scientific), Yeast Extract lOg / L (RM0277, Himedia), Peptone lOg / L (RM667, Himedia), Sodium chloride 5g / L (MB023, Himedia), MgSO4.7H2O 0.2 g / L (RM684, Himedia), and pH 7.0.

[0116] Example 1: Bacterial screening for BDO production:

[0117] The culture with said Bacillus halotolerans (EX5-6) bacterium was grown overnight in Nutrient Broth No. 3 (M1902, Himedia, India) at 37°C for 12 hours and was inoculated in the fermentation media for 24hrs. After incubation period, the broth was analysed for the production of 2,3-butanediol by HPLC (UHPLC, Dionex ultimate 3000, ThermoFisher). The column used was Aminex HPX-87H (1250140, BioRad, USA), 5mM H2SO4 was used as a solvent with column flow of 0.6ml / min, oven temperature of 60°C, and run time of 30 mins with detection using Refractive Index (RI) detector. Standard 2,3-butanediol was procured from Merck Sigma (USA). The further detection of 2,3-BD was carried out on Gas Chromatography using the following conditions: Column name- FFAP (30mx0.25mmx0.25pm), Detector and injector temperature of 300°C, sample volume 1 ul, Total run time of 15minutes, Instrument (Trace 1300 Gas Chromatograph, Thermo Scientific) Example 2: Molecular characterization of the bacterial isolate

[0118] The DNA was extracted from bacteria using DNeasy Powersoil Pro kit (Qiagen, 47014).

[0119] The 16S rRNA gene was amplified using 27F (AGAGTTTGATCMTGGCTCAG - SEQ ID: 1) and 1492R (GGTTACCTTGTTACGACTT - SEQ ID: 2) universal primers and sequenced using 3500 xl genetic analyser (Thermofisher, USA). The NCBI accession number for 16S rRNA sequence is OR263467. The phylogenetic analysis was carried out using MEGA vl 1 software using Neighbour Joining Method.

[0120] The IDA accession number is MCC 0265, Processing Reference Number is D_JUL_23_187. The culture is submitted in IDA facility of National Centre for Microbial Resource at National Center for Cell Sciences, Pune, India on date of 31stJuly, 2023.

[0121] Results: Identification and genomic characterization

[0122] The 16S rRNA sequence (1425 bp) was analyzed for closest type strain similarity using EZtaxon. The top-hit strain was Bacillus halotolerans ATCC 25096 with 99.86% similarity and 95.2% completeness. Further, phylogenic investigation using Neighbour

[0123] Joining method with bootstrap of 1000, suggested close phylogeny to Bacillus halotolerans. Further details of said bacterium are provided below in table 1:

[0124] Table 1:

[0125] B. haiotaierans EX5-6 Total Sequence Length (bp) :4226035 Num ber of Sequences :4 Longest Sequences (bp) :4143713 NSO (bp) : 4143713

[0126] Gap Ratio (%) :O.OOOOOO GCcontent (%) :43.5 Num ber of CDSs :4251 Average Protein Length :289.4 Coding Ratio (%):87.3 Num ber of rRNAs :17 Num ber of tRNAs :72 Num ber of CRI SPRs zO AN I value >98

[0127] The DNA of the bacterial isolate was extracted using DNeasy PowerSoil Pro kit (Qiagen), followed by Library Preparation using ligation sequencing kit SQK-LSK-109 and Native Barcoding NBD 112 (Oxford Nanopore) for Nanopore sequencing on MK1C using R9 flow cell. The data was basecalled, trimmed, and demultiplexed using Guppy. The genome assembly was carried out using FLYE assembler v2.9 followed by polishing using Medaka tool. The resultant assembled, fasta file was uploaded on DFAST tool (https: / / dfast.ddbj.nig.ac.jp / ).

[0128] Example 3: Optimization of 2,3-BD production in synthetic medium

[0129] The optimization of media components and physical parameters such as pH and temperature was done using one factor at a time (OFAT) approach. The concentration of C-source, Yeast Extract, and Peptone was optimized. For all the optimization, seed medium consisted of Yeast Extract 5g / L, Peptone 5g / L, NaCl 5g / L, Beef extract 5g / L, Glucose 20g / l, and pH 7.0. A colony was inoculated in to seed medium at 37°C till O.D. 600 reached to 8.0. Further 10 % of inoculum (inoculum concentration was further optimized) was inoculated into fermentation medium to produce 2,3-BD.

[0130] Results: Optimization of 2,3-BD production using Bacillus halotolerans EX5-6

[0131] We utilized OFAT strategy to optimize the media and physical parameters for BDO production. Higher production was obtained at 28°C and at pH 5. Glucose and yeast extract was found to be the favourable carbon and nitrogen source, respectively. Further, concentration optimization suggested 15% and 2.5% as the best glucose and yeast extract concentrations for 2,3 -butanediol production by Bacillus halotolerans EX5-6. Upon final validation, we could obtain 73.19 g / E of BDO with yield and productivity of 0.5g / g glucose and 1.02 g / E / h from synthetic medium, respectively.

[0132] This example confirms that the bacterium Bacillus halotolerans (EX5-6) is effectively producing BDO.

[0133] Example 4: Rotten Tomatoes processing for BDO production

[0134] Rotten tomatoes were grounded using a home grinder. The resultant juice was filtered using a muslin cloth. The juice was collected whereas the peel and seeds were autoclaved and discarded. The juice was further concentrated using a rota evaporator (Heidolph, Germany) to increase the sugar concentration to 2X, 3X, and 4X times. Here “X” refers to the multiple (times) concentration. So the 2X means doubling the concentration of sugars and other contents of juice by reducing half the quantity of water. Similarly, 3X indicates removing three times of water from the original volume by rota evaporation. These concentrate were then used for BDO production either alone or with supplementation of optimised media components.

[0135] Analytical analysis

[0136] HPLC and Gas chromatography were employed for the detection of metabolites. HPLC conditions included: Column- Aminex HPX-87H (1250140, BioRad), Solvent- 5mM H2SO4, Flow rate- 0.6ml / min, Detector- RI, runtime- 30 minutes, HPLC (UHPLC, Ultimate 3000, ThermoScientific). The GC conditions were Column name: FFAP (30mx0.25mmx0.25pm), Detector and injector temperature of 300°C, sample volume 1 ul, and following oven conditions, Gas Chromatography (ThermoScientific, Trace 1300). The production titer and yield of 2,3-BDO, Glucose, and Acetoin was calculated using standard equation generated from HPLC and GC analysis of the standards: 1. (2R,3R)-(-)- 2,3-Butanediol (237639-5G, Sigma), Acetoin (40127-U, Sigma), Glucose (GRM 016, Himedia). The standard detection peak is in the Fig. 4.

[0137] Standard Equation:

[0138] For HPLC:

[0139] 1. Glucose: y = 2.5158x + 0.3534, R2=0.99

[0140] 2. 2,3- butanediol: y = 1.9168x + 2.1252, R2=0.99

[0141] 3. Acetoin: y = 1.7698x - 0.1496, R2=0.99

[0142] For GC:

[0143] 1. 2,3- butanediol: y = 17.58x - 1.2114, R2=0.99

[0144] Results: Rotten Tomato concentrates were used for BDO production with the above optimized conditions (pH 5.0, 180 rpm, Temperature 28°C). Inventors at 2X concentration of said tomato based media obtained 16.62 g / L of BDO at a productivity rate of 0.69 g / L / h (refer, Figure 1). Additionally, inventors obtained acetoin as another major product with a titre of 2.39 g / L. Since Acetoin can be easily converted to 2,3 -BD, many research studies combine its yield with BDO. Therefore, the combined BDO titre stands at 19.01 g / L from 2X tomato juice. There was a decrease in the production of BDO upon supplementation of Tomato concentrate with optimized media constituents suggesting Tomato concentrate as the complete nutrient substitute for BDO production (refer, figure 2 and 3). It could replace all the medium components and thus probably will result in cheaper production process (Fig. 4).

[0145] Moreover, it is submitted that the production of BDO using said bio waste material rotten tomatoes based media provides higher yield and productivity than those reported in literature i.e., OHair, J. et al., Waste Management, 120, 248-265, (2021). Vis-a-vis comparison is provided below in table 2:

[0146] Table 2. State-of-the-art for 2,3 BDO production from tomato waste ADVANTAGES OF THE INVENTION

[0147] • The present invention provides first report of production of 2,3-butanediol by utilizing tomato waste interestingly using Bacillus halotolerans bacterium.

[0148] • Waste management.

[0149] • Green synthesis, as the process does not require any pre-treatment step unlike in other biowastes such as lignocellulose biomass.

[0150] • Our waste serve as a complete nutrient media and does not require any external supplementation of nutrients.

[0151] • Very cost effective, as our substrate rotten tomatoes contains carbon and other nutrient sources thereby eliminating the need of glucose, yeast extract and other salts, which increase cost of production.

[0152] • The incubation is at room temperature and thus eliminates the requirement of incubator, thereby further reducing the costs.

[0153] • The benefits of using present process for producing BDO includes waste management, and green synthesis because the process does not require any pretreatment step unlike in other biowastes such as lignocellulose biomass, serving as a complete nutrient media and does not require any external supplementation of nutrients.

[0154] • Cost effective because of the substrate rotten tomatoes contains carbon and other nutrient sources thereby eliminating the need of glucose, yeast extract and other salts, which increase cost of production.

Claims

WE CLAIM:

1. A process for the preparation of 2, 3 -butanediol (BDO) from bio-waste, wherein the said process comprising the steps of:(a) grinding the waste material in a grinder to form a paste / puree, followed by filtration to obtain a filtrate;(b) centrifuging the filtrate of step (a) at speed of 8000 to 14000 rpm for 15 to 30 minutes at temperature in the range of 1 to 6 degree C to obtain diluted media;(c) concentrating the diluted media of step (b) to obtain the desired bio-based fermentation media; or(d) optionally, repeating the concentrating step (c) one or more times to obtain nX concentrated media; wherein X refers to ‘n’ times reduction in water concentration of said media, wherein ‘n’ is 2, 3, 4 or 5;(e) incubating the bio-based fermentation media of step (c) or step (d) with the bacterial strain of Bacillus halotolerans MCC 0265 in a bioreactor to obtain 2,3-butanediol [BDO].

2. The process as claimed in claim 1, wherein the preparation of bio-based fermentation media is done from waste material selected from waste rotten tomatoes, rotten pumpkin, rotten carrots, rotten beets, waste bagasse, waste sugarcane juice.

3. The process as claimed in claim 1, wherein the bioreactor is selected from flask bioreactor and fed-batch fermenter level bioreactor.

4. The process as claimed in claim 1, wherein the bio-based fermentation media is directly used for preparation of BDO without pre-treatment.

5. The process as claimed in claim 1, wherein the specific reaction conditions for the preparation of BDO in a flask bioreactor comprising:(i) temperature in the range of 25 to 37 °C;(ii) no external pressure;(iii) stirring speed in range of 150-200 rpm in a rotatory shaker;(iv) pH in range of 5.0 to 7.0;(v) inoculum concentration of bacterium in range of 8% - 10%; and(vi) time period of incubation in range of 20 to 30 hrs.

6. The process as claimed in claim 1, wherein the specific reaction conditions for the preparation of BDO in a fed-batch fermenter level bioreactor comprising:(i) fermentation vessel volume in the range of 2 to 5 L;(ii) working volume in range of 800 to 1600 ml;(iii) media comprising 2X to 5X concentration of tomato juice;(iv) sparging in range of 2 to 2.5 1 per minute (LPM);(v) inoculum concentration of bacterium in the range of 8 to 10%;(vi) stirring speed in range of 300 to 350 RPM;(vii) pH in range of 5.0 to 7.0;(viii) temperature in range of 25 to 35 degree C; and(ix) time period in range of 32 to 40 h.

7. The process as claimed in claim 1, wherein the flask bioreactor is having volume in the range of 200 to 500 ml, with media volume in the range of 25 to 100 ml.

8. The process as claimed in claim 1, wherein the filtration is done by manual coarse filtration using muslin cloth.

9. The process as claimed in claim 1, wherein the concentration in step (iii) or (iv) is done using rotatory evaporator at a temperature in the range of 40 to 50 degree and 80 to 120 rpm under 30 bar vacuum.

10. The process as claimed in claim 1, wherein the bio-based fermentation media is free of additive or synthetic component; and wherein the process provides acetoin as an additional product.

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