CLIAMS:We Claim:

1. A two step process for production of butanol comprising the steps of:
a) low temperature pretreatment of lignocellulosic biomass; and
b) simultaneous saccharification and extractive co-fermentation of the pretreated lignocellulosic biomass,
wherein the saccharification, the fermentation and the extraction are carried out in a single vessel.
2. The process according to claim 1, wherein the pre-treatment of lignocellulosic biomass comprises of contacting the lignocellulosic biomass with an alkali followed by heating the biomass at low temperature to produce a readily saccharifiable pretreated biomass.
3. The process according to claim 2, wherein the alkali is selected from the group comprising ammonia, sodium hydroxide, calcium hydroxide, potassium hydroxide, ammonia hydroxide and sodium hydroxide in combination with hydrogen peroxide.
4. The process according to claim 2, wherein the low temperature is < 100 °C.
5. The process according to claim 1, wherein the simultaneous saccharification and extractive co-fermentation of the pre-treated lignocellulosic biomass comprises the steps of:
a) saccharifying the pre-treated lignocellulosic biomass with one or more enzymes to form soluble pentose (C5) and hexose (C6) sugars;
b) fermenting the pentose (C5) and hexose (C6) sugars by butanol producing bacteria to form an aqueous fermentation broth; and
c) adding water immiscible organic extractant to the aqueous fermentation broth to form an organic layer over an aqueous layer of the fermentation broth, wherein said addition causes significant amount of butanol to extract into the organic layer;
wherein the saccharification, the fermentation and the extraction are carried out in a single vessel.
6. The process according to claim 5, wherein the one or more enzymes are selected from the group comprising cellulases and hemicellulases.
7. The process according to claim 5, wherein the butanol producing bacteria are Clostridium strains selected from the group comprising Clostridium beijerinckii and Clostridium acetobutylicum.
8. The process according to claim 5, wherein the butanol producing bacteria is micro-aero-tolerant strain of Clostridium beijerinckii.
9. The process according to claim 5, wherein the extractant is selected from a group comprising esters of C12-C22 fatty acids, C12-C22 fatty alcohols, C12-C22 fatty acids, esters of C12-C22 fatty acids, C12-C22 fatty aldehydes, C12-C22 fatty amides and mixtures thereof.
10. The process according to claim 5, wherein the extractant is selected from C12-C22 fatty acid methyl esters.
11. The process according to claim 5, wherein ratio of the extractant to the aqueous fermentation broth ranges from 1:1 to 1:20.
12. The process according to claim 5, wherein ratio of the extractant to the aqueous fermentation broth is 1:2.
13. The process according to claim 1, wherein the simultaneous saccharification and extractive co-fermentation further produces isopropanol and/or acetone.
,TagSPECI:FIELD OF THE INVENTION

[0001] The present disclosure relates to a dual step process for production of butanol from lignocellulosic biomass using Clostridium Beijerinckii.

BACKGROUND OF THE INVENTION

[0002] Fossil fuels have served the mankind for ages. However, the recent awakening to the realization of dismal scenario of fossil fuel availability, perils of fossil fuel resource exhaustion, and stringent environmental legislation governing worldwide, has led to search for alternative energy sources. Therefore, several alternative fuels are being investigated, and developed which can either completely be used in pure form and replace the petroleum derived fuels (gasoline and diesel) or can be blended with petroleum fuels to certain ratio.
[0003] Butanol was found to be of the best choice among the alternative fuels as it is superior replacement for gasoline. According to the data from U.S. Environmental Protection Agency, hydrocarbon, carbon monoxide, and nitrogen oxide releases can be greatly reduced by use of biobutanol. Another advantage is that biobutanol has higher energy content than ethanol, almost 20% more by density. Due to its similarities to conventional gasoline, it is able to blend much better than ethanol with gasoline. It has even shown promise when using 100% biobutanol in a conventional gasoline engine. Besides these, biobutanol experiences a lower chance of separation and corrosion than ethanol. Biobutanol also resists water absorption, allowing it to be transported in pipes and carriers used by gasoline. A very exciting advantage of biobutanol is that vehicles require no modifications to use it. This means that with effective pumping systems, it can be implemented immediately. Currently, funds are quickly rising for biobutanol production and the only requirement is a cheap and fast modification to the ethanol plants which already exist. As yield efficiencies rise, the cost of biobutanol will continue to drop from its already reasonable price.
[0004] Butanol or butyl alcohol (sometimes also called biobutanol when produced biologically), is a primary alcohol with a 4 carbon structure and the molecular formula of C4H10O. It is primarily used as a solvent for a wide variety of chemical and textile processes, as an intermediate in chemical synthesis, and as a fuel. It is also used as paint thinner and a solvent in other coating applications where it is used as a relatively slow evaporating latent solvent in lacquers and ambient-cured enamels. It finds other uses such as a component of hydraulic and brake fluids. It is also used as a base for perfumes, but on its own has a highly alcoholic aroma.
[0005] Today, there is a paramount interest in producing fuels like butanol and ethanol using microorganisms by fermentation due to environmental aspects as this is a renewable mode of production of solvents. Butanol is a superior fuel and has more calorific value than ethanol.
[0006] Butanol is an important industrial solvent and potentially a better fuel extender than ethanol. The market demand is expected to increase dramatically if green butanol can be produced economically from low cost biomass.
[0007] Biobutanol production via anaerobic bacteria fermentation has been observed since 1861, when it was witnessed by Pasteur. During anaerobic bacteria fermentation processes, butanol is a single product among many. Another result is the production of acetone, which was first witnessed in 1905 by Schardinger. By the beginning of the 20th century, interest in butanol had risen sharply. This was due to butanol’s involvement in the solution to a material shortage. A shortage of natural rubber had struck society and efforts were undertaken to make a synthetic rubber. It was found that butadiene or isoprene rubber could be synthesized from butanol or isoamyl alcohol, another fermentation product. This discovery stimulated great interests in anaerobic fermentative processes for compound production.
[0008] Between 1912 and 1914, Chaim Weizmann, a chemist, performed one of the first microorganism screenings to study microbiology in hopes to better understand the fermentation process. One species he isolated, Clostridium acetobutylicum, was able to yield more acetone and butanol than previous species while feeding on a larger range of biomass.
[0009] Acetone butanol ethanol (ABE) fermentation is a process that uses bacterial fermentation to produce acetone, n-Butanol, and ethanol from starch. Clostridium acetobutylicum is the most well-known strain, although Clostridium beijerinckii has also been used for this process with good results. However, cost issues, the relatively low-yield and sluggish fermentations, as well as problems caused by end product inhibition and phage infections, meant that ABE butanol could not compete on a commercial scale with butanol produced synthetically and almost all ABE production ceased as the petrochemical industry evolved. Biobutanol can be produced from cereal crops, sugar cane, sugar beet, etc, and also from cellulosic raw materials.
[00010] M. M. Shah et al in “Simultaneous saccharification and extractive fermentation for acetone/butanol production from pretreated hardwood”, Applied Biochemistry and Biotechnology, vol.34-35, Issue 1, pp 557-568, discloses simultaneous saccharification and extractive fermentation (SSEF), and production of acetone/butanol from pretreated hardwood by Clostridium acetobutylicum and cellulase enzymes. However, the SSEF takes place in an integrated bioreactor-extractor wherein the products of fermentation were extracted from the broth through a semipermeable membrane. Further there is no disclosure of performing SSEF in a single vessel.
[00011] United States publication US20100196980 provides a method of production of a fermentation product from a lignocellulose containing material comprising pretreatment, hydrolysis and fermentation, wherein hydrolysis and fermentation step are carried out simultaneously. However US20100196980 fails to disclose the simultaneous Saccharification and Extractive Co-Fermentation in a single vessel. Further US20100196980 discloses the method of production of ethanol and not butanol as described in various embodiments and examples.
[00012] PCT Publication WO2010087737 discloses saccharifying the pretreated plant material with enzymes, fermenting the sugars by butanol, acetone, ethanol producing bacteria, removing organic solvents and fermentation gases, and recovering end product, wherein the saccharification and the fermentation are carried out in a single phase. However WO2010087737 fails to disclose the simultaneous Saccharification and Extractive Co-Fermentation in a single vessel. There is no disclosure of extractive co-fermentation through phase extraction. Furthermore, this method is for production of butanol, acetone and ethanol.
European patent publication EP 1954798 discloses method of production of ethanol comprising non-pressurised pre-treatment of mono- and/or polysaccharide containing waste fractions, enzymatic hydrolysis and fermentation wherein yeast is preferably used for fermentation. There is no disclosure of butanol production in any manner whatsoever.
[00013] United States Publication US20110097773 discloses a method of recovering butanol from a fermentation medium by extracting butanol into a water-immiscible extractant.
[00014] There are many drawbacks and disadvantages associated with each of these processes for production of butanol. One of the main drawbacks associated with the current processes is the use of multiple process steps and/or vessels which is a cost intensive method of production of butanol. Further there are problems including butanol toxicity and inhibition of the fermenting microorganism resulting in low butanol yield. Thus, there is a need in the art to develop an economically viable method for butanol production. The new method must also combat the effect of butanol toxicity. The new process must be cost-effective by eliminating the need of high temperature pretreatment, detoxification and purification of solvents. The new process must reduce operating time by removal of separate reactors and some operations, for example concentration and sterilization of soluble sugars before fermentation. The present invention satisfies the existing needs, as well as others, and generally overcomes the deficiencies found in the prior art.

OBJECTS OF THE INVENTION

[00015] An object of the present disclosure is to provide a dual step cost-effective process for production of butanol from lignocellulosic biomass.
[00016] It is an object of the present disclosure to compress the multiple process steps of saccharification, fermentation and extraction of butanol to a single process step of “Simultaneous Saccharification and Extractive Co-Fermentation’ (also referred to as SSECF hereinafter) that lowers the cost of production.
[00017] It is another object of the present disclosure to provide a dual-step process for production of butanol from lignocellulosic biomass wherein saccharification, fermentation and extraction are carried out in a single vessel.
[00018] It is another object of the present disclosure to provide a high-yielding and long-term stable process for production of butanol from lignocellulosic biomass.
[00019] It is another object of the present disclosure to provide a dual step process for production of butanol from lignocellulosic biomass which requires low maintenance.
[00020] It is a yet another object of the present disclosure to provide a continuous and industrially scalable butanol production process with improved butanol productivity.
[00021] It is still another object of the present disclosure to provide an efficient process for the production of high yield of butanol from pretreated lignocellulosic biomass using a micro-aero-tolerant strain of Clostridium beijerinckii.
[00022] It is the object of the present disclosure to provide optimal fermentation conditions for enhanced production of butanol from pre-treated lignocellulosic biomass using Clostridium beijerinckii.
[00023] Other objects of the present disclosure will be apparent from the description of the invention herein below.

SUMMARY OF THE INVENTION

[00024] The present disclosure provides a two step process for production of butanol comprising:
a) low temperature pretreatment of lignocellulosic biomass; and
b) simultaneous saccharification and extractive co-fermentation (also referred to as SSECF hereinafter) of the pretreated lignocellulosic biomass,
wherein the saccharification, the fermentation and the extraction are carried out in a single vessel.
[00025] In one embodiment of the present disclosure, pre-treatment of lignocellulosic biomass comprises of contacting the lignocellulosic biomass with an alkali followed by heating the biomass at low temperature to produce a readily saccharifiable pretreated biomass, also referred to as alkali treated biomass herein.
[00026] In one embodiment of the present disclosure, the alkali is selected from the group comprising ammonia, sodium hydroxide, calcium hydroxide, potassium hydroxide, ammonium hydroxide, sodium hydroxide in combination with hydrogen peroxide and the like.
[00027] In one embodiment of the present disclosure, the pretreated lignocellulosic biomass comprises of cellulose and hemicelluloses.
[00028] In another embodiment of the present disclosure, there is provided a process of simultaneous saccharification and extractive co-fermentation of pre-treated lignocellulosic biomass comprising the steps of:
a) saccharifying the pre-treated lignocellulosic biomass with one or more enzymes to form soluble pentose (C5) and hexose (C6) sugars;
b) fermenting the pentose (C5) and hexose (C6) sugars by butanol producing bacteria to form an aqueous fermentation broth; and
c) adding water immiscible organic extractant to the aqueous fermentation broth to form an organic layer over an aqueous layer of the fermentation broth, wherein said addition causes significant amount of butanol to extract into the organic layer;
wherein the saccharification, the fermentation and the extraction are carried out in a single vessel.
[00029] In one embodiment of the present disclosure, the simultaneous saccharification and extractive co-fermentation further produces isopropanol and/or acetone along with butanol.
[00030] In one embodiment of the present disclosure, the one or more enzymes used for the saccharification of lignocellulosic biomass are cellulases and hemicellulases. The enzymes degrade or convert cellulose and hemicelluloses into soluble pentose (C5) and hexose (C6) sugars.
[00031] In one embodiment of the present disclosure, the butanol producing bacteria are Clostridium strains selected from the group comprising Clostridium beijerinckii and Clostridium acetobutylicum. In one preferred embodiment, the Clostridium strain used is Clostridium beijerinckii.
[00032] In one embodiment of the present disclosure, the extractant used for the extraction or removal of butanol is selected from a group comprising esters of C12-C22 fatty acids, C12-C22 fatty alcohols, C12-C22 fatty acids, esters of C12-C22 fatty acids, C12-C22 fatty aldehydes, C12-C22 fatty amides and mixtures thereof. In one of the preferred embodiments, the extractant is fatty acid methyl esters (FAME).

BRIEF DESCRIPTION OF THE DRAWINGS

[00033] These and other features, aspects and advantages of the invention will become better understood when the description is read with reference to the accompanying drawings, wherein:
[00034] Figure 1 illustrates effect of temperature and ammonia concentration on the biomass recovery during pretreatment process.

DETAILED DESCRIPTION OF THE INVENTION

[00035] The present disclosure relates to a two step process for producing butanol from lignocellulosic biomass comprising the steps of:
a) low temperature pretreatment of lignocellulosic biomass; and
b) simultaneous saccharification and extractive co-fermentation (also referred to as SSECF hereinafter) of the pretreated lignocellulosic biomass,
wherein the saccharification, the fermentation and the extraction are carried out in a single vessel.
[00036] In one embodiment of the present disclosure, pre-treatment of lignocellulosic biomass comprises of contacting the lignocellulosic biomass with an alkali followed by heating the biomass at low temperature to produce a readily saccharifiable pretreated biomass, also referred to as alkali treated biomass herein. In one embodiment of the present disclosure, the low temperature in above step is <100°C.
[00037] In one embodiment of the present disclosure, the alkali is selected from the group comprising ammonia, sodium hydroxide, calcium hydroxide, potassium hydroxide, ammonia hydroxide, and sodium hydroxide in combination with hydrogen peroxide, and the like
[00038] Biomass used in the present disclosure is lignocellulosic, which contains polysaccharides such as cellulose and hemicellulose, and lignin. Polysaccharides of biomass may also be called glucans and xylans. The types of biomass broadly refer, but not limited to, bioenergy crops, agricultural residues, industrial solid waste, municipal solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. It may also include, but not limited to, corn stover, corn cobs, corn husks, wheat straw, barley straw, oat straw, canola straw, hay, rice straw, sugar cane bagasse, sorghum bagasse or stover, soybean stover, components obtained from milling of grains, waste paper, grasses, switchgrass, miscanthus, cord grass, reed canary grass, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers and animal manure. Biomass may include other crop residues, forestry wastes such as aspen wood, other hardwoods, softwood and sawdust; and post-consumer waste paper products; and fiber process residues such as corn fiber, beet pulp, pulp mill fines and rejects; as well as other sufficiently abundant lignocellulosic material.
[00039] Lignocellulosic biomass that is particularly useful for the present disclosure comprises biomass that has relatively high cellulose and hemicellulose content, is relatively dense, and/or is relatively easy to collect, transport, store and/or handle. The lignocellulosic biomass, as used in the present disclosure, may be used either directly as obtained from the source or pre-processed, for example, by applying mechanical energy to reduce the moisture or size of the biomass. Coarse size reduced material of size greater than 0.1 mm may be produced by using suitable methods of size reduction. These methods may include, but not limited to, knife milling, crushing, shredding, chopping, disc refining, and coarse hammer milling. Such size reduction process may be done before or after the pre-treatment with ammonia. Drying the pretreated biomass may be performed by conventional means such as using a drying oven, rotary dryer, flash dryer, or superheated steam dryer. Further, even air drying may be sufficient to reach the desired biomass moisture content that is less than about 15%, preferably between about 7 to 10%.
[00040] Lignocellulosic biomass is typically treated prior to saccharification to prepare it for hydrolysis. This pretreatment improves the hydrolysis, or release of sugars, during saccharification. Sugar release, primarily glucose and xylose, from the polysaccharides of biomass is difficult due to the presence of lignin that constitutes a physical barrier and also a surface for non-productive binding of saccharification enzymes. In addition, the crystallinity and tight packing of cellulose microfibrils restricts access of the enzymes.
[00041] In accordance with one exemplary embodiment of the present disclosure, the pre-treatment step comprises soaking or contacting raw biomass of suitable particle size in liquor ammonia at temperatures lower than 100 °C, wherein ammonia concentration is not more than 25% (v/v), to produce ‘treated biomass’. In one preferred embodiment, the biomass pretreatment step comprises soaking or contacting biomass in ammonia for around 4h to 72h. The alkaline pretreatment process as described herein is performed in one single reactor. The solids loading of the biomass may be varied from 5 – 25 % (w/v).
[00042] In accordance with the present disclosure, the treated biomass may be directly used for the subsequent process (SSECF) without any detoxification or intermediate washing steps.
[00043] In one embodiment of the present disclosure, a process of simultaneous saccharification and extractive co-fermentation of the pre-treated lignocellulosic biomass comprises the steps of:
a) saccharifying the pre-treated lignocellulosic biomass with one or more enzymes to form soluble pentose (C5) and hexose (C6) sugars;
b) fermenting the pentose (C5) and hexose (C6) sugars by butanol producing bacteria to form an aqueous fermentation broth; and
c) adding water immiscible organic extractant to the aqueous fermentation broth to form an organic layer over an aqueous layer of the fermentation broth, wherein said addition causes significant amount of butanol to extract into the organic layer;
wherein the saccharification, the fermentation and the extraction are carried out in a single vessel.
[00044] In one embodiment of the present disclosure, a process of simultaneous saccharification and extractive co-fermentation takes place at a temperature ranging from 30 to 35 °C
[00045] In accordance with the present disclosure, the saccharification, the fermentation and the extraction are performed simultaneously in a single reaction vessel. Thus the number of reaction vessels required is reduced thereby resulting in capital cost savings.
[00046] Hydrolysis usually means cleavage of chemical bonds by addition of water. Where a carbohydrate is broken into its component sugar molecules by hydrolysis (e.g. sucrose being broken down into glucose and fructose), this is termed as saccharification. Before a pre-treated lignocellulose biomass is fermented, it is hydrolyzed to break down cellulose and hemicellulose into fermentable sugars. In a preferred embodiment of the present disclosure, the pre-treated biomass is hydrolyzed/saccharified, preferably enzymatically, before/during fermentation.
[00047] In a preferred embodiment of the present disclosure, hydrolysis is carried out enzymatically. The pre-treated lignocellulose biomass may be hydrolyzed by one or more cellulolytic enzymes, such as cellulases or hemicellulases, or combinations thereof.
[00048] In addition to cellulose, hemicellulose and lignin, the lignocellulose containing feedstock may contain other constituents and thus hydrolysis and/or fermentation may be performed in presence of additional enzymes like proteases, amylases and lipases.
[00049] Enzymatic hydrolysis is preferably carried out in a suitable aqueous environment under conditions which can readily be determined by one skilled in the art. In a preferred embodiment of the present disclosure, hydrolysis is carried out at suitable, preferably optimal, conditions for the enzyme(s) in question. Suitable process time, temperature and pH conditions can readily be determined by one skilled in the art.
[00050] Preferably, enzymatic hydrolysis of SSECF in accordance with the present disclosure is performed by adding a mixture of cellulolytic enzymes consisting of a consortium of cellulases and hemicellulases capable of hydrolyzing both cellulosic and hemicellulosic components of lignocellulosic biomass respectively. One skilled in the art would recognize how to optimize amounts of enzymes to be added for the hydrolysis of polymers. Addition of suitable amounts of the enzymes results in a release of hexose (C6) and pentose (C5) sugars into a surrounding aqueous medium used to suspend the solid pretreated biomass.
[00051] Fermentation can be carried out in a batch, fed-batch, or continuous reactor. Fed-batch fermentation may be fixed volume or variable volume fed-batch. In one embodiment, fed-batch fermentation is employed. The volume and rate of fed-batch fermentation depends on, for example, the fermenting organism, the identity and concentration of fermentable sugars, and the desired fermentation product. Such fermentation rates and volumes can readily be determined by one of ordinary skill in the art.
[00052] The butanol producing bacteria may be cultured in a suitable fermentation medium to produce butanol. Consideration must be given to appropriate fermentation medium, pH, temperature, and requirements for anaerobic/micro-aero tolerant conditions.
[00053] According to one embodiment of the present disclosure, butanol producing bacteria ferments pentose (C5 sugars) and hexose (C6) sugars for the production of butanol. Desirably, the present disclosure uses both the C5 and C6 sugars for the fermentation process, which may reduce complexity and costs of processing and related equipment.
[00054] The C5 and C6 sugar stream as produced by a saccharification step during SSECF can be converted into a hydrocarbon or an oxygenated hydrocarbon using fermentation processes, preferably butanol. The residue can be used for a secondary step of conversion (power and/or hydrocarbon or/and oxygenated hydrocarbons). In one embodiment of the present disclosure, the fermentation may further produce small amounts of acetone or isopropanol along with butanol.
[00055] Pentose broadly refers to five (5) carbon member sugars or saccharides (monomers), corresponding disaccharides (dimers), corresponding trisaccharides (trimers), corresponding tetrasaccharides (tetramers), modified pentose, derivatives of pentose, acetylated groups and/or the like. Pentose includes xylose, ribose, arabinose, ribulose, xylulose, lyxose, any other isomer of five carbon sugars, and/or the like. Desirably, at least a portion of pentose may be separated or derived from hemicellulose. Pentose may include or form complexes of relatively simple sugars, such as a disaccharide and/or a trisaccharide. According to one embodiment, pentose refers to sugar bound in polymer form that can be liberated or separated, such as with mild to moderate processing to break down the hemicellulose into simpler segments or monosaccharide units.
[00056] Hexose broadly refers to six (6) carbon member sugars or saccharides (monomers), corresponding disaccharides (dimers), corresponding trisaccharides (trimers), corresponding tetrasaccharides (tetramers), and/or the like. Hexose includes glucose, glacatose, sucrose, fructose, allose, altrose, gulose, idose, mannose, sorbose, talose, tagatose, modified hexose, derivatives of hexose, any other isomer of six carbon sugars, and/or the like. Hexose may include and/or form complexes of relatively simple sugars, such as a disaccharide including sucrose, lactose, and maltoseand/or a trisaccharide.
[00057] Butanol producing bacteria include species of Clostridium, including Clostridium beijerinckii and Clostridium acetobutylicum, as well as other bacteria known in the art. In one preferred embodiment of the present disclosure, the process uses a wild type micro-aero-tolerant strain, Clostridium beijerinckii, for fermentation. Clostridium strains present in the medium utilize the released C6 and C5 sugars and produce solvents, majorly butanol along with acetone or isopropanol depending on the strain used for the process. It was observed that the strains initially show a lag phase varying from 10-12 h followed by a high rate of solvent production. One skilled in the art would know that butanol fermentations are anaerobic production processes. Iso-propanol and/or acetone are also produced during the anaerobic production of butanol.
[00058] An extractant useful in the process described herein is water-immiscible. A suitable organic extractant composition should meet the criteria for an ideal solvent for a two-phase extractive fermentation for the production of butanol. Specifically, the extractant should be (i) biocompatible with the microorganisms, (ii) substantially immiscible with the aqueous fermentation medium, (iii) low cost and nonhazardous. Further, for improved process operation and economics, the extractant should have (iv) low tendency to form emulsions with the fermentation medium,(v) low viscosity (μ), (vi) low density (ρ) relative to the aqueous fermentation medium, and (vii) a boiling point suitable for downstream separation of the extractant and the butanol. The extractant should be biocompatible with the microorganism, that is, nontoxic to the microorganism or toxic only to such an extent that the microorganism is impaired to an acceptable level, so that the microorganism continues to produce the butanol product into the fermentation medium.
[00059] In accordance with the present disclosure, the extractant used is selected from a group comprising esters of C12-C22 fatty acids, C12-C22 fatty alcohols, C12-C22 fatty acids, esters of C12-C22 fatty acids, C12-C22 fatty aldehydes, C12-C22 fatty amides and mixtures thereof. In one of the preferred embodiments, the extractant is fatty acid methyl esters (FAME). The extractant is added to an aqueous fermentation broth to form a water-immiscible organic layer over an aqueous layer of the fermentation broth, which causes significant amount of butanol to extract into the organic layer through a water-organic layer interface. The water immiscible extractant of the present disclosure is not inhibitory to microbial growth. This organic layer may also act as a barrier to entry of air in the aqueous fermentation broth and may thus be a cost effective mechanism for maintaining anaerobic fermentation environment. In one embodiment of the present disclosure, the ratio of organic layer to aqueous broth ranges from of 1:1 to 1:20. In one preferred embodiment of the present disclosure, the ratio of organic layer to aqueous broth is 1:2. Thus, in accordance with the present disclosure, the organic layer can be added to a system without any intermediate membrane separating it from the aqueous fermentation broth.
[00060] The present disclosure is illustrated with working examples, which is intended to illustrate the working of the disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure.

EXAMPLES

Example 1: Pretreatment of the Lignocellulosic Biomass
[00061] Based on the merits of ammonia based pretreatment found in literature and results of a few trial experiments, ammonia based pretreatment was chosen as the pretreatment step and thus explored. The main parameters that were studied and optimized were temperature, ammonia concentration and reaction time, as shown in Table 1. Figure 1 shows the effect of temperature and ammonia concentration on the biomass recovery during pretreatment. At 70 °C, pretreatment using 15 % (v/v) ammonia showed a better biomass recovery (81 %) compared to 25 % (v/v) ammonia.

Table 1: Optimized pretreatment conditions and corresponding sugar recoveries obtained
Temp (oC) Time
(h) Ammonia Conc.
(%) Biomass recovered
(%) Glucose
(g/L) Xylose
(g/L) Sugar recovered/g pretreated biomass (g) Overall sugar recovery per g raw biomass (g) Extent of hydrolysis
(%)*
70 72 15 81 18 6 0.57 0.46 70
*Enzymatic hydrolysis at 50 0C for 4 hours

Example 2: Extractive Fermentation
[00062] Different volumes (0, 15 & 30mL) of organic solvent consisting of FAME were added to 30 mL of fermentation broth (T6 medium containing 60 g/L glucose) kept in different anaerobic bottles. Fermentation was carried out for 72 h at 35 °C and both the phases were analyzed for butanol at the end, as shown in Table 2. Around 0.2-0.3% (w/v) of butanol concentration was found to get distributed in biodiesel phase. This resulted in lower butanol concentrations (much below the inhibitory levels) in the aqueous fermentation broth which in turn resulted in slight increase of overall butanol production.

Table 2: In situ product separation with FAME as extractant
Aqueous Phase Organic Phase Total Butanol production
(ppm)
Strain Biodiesel (mL) Acetone (ppm) Isopropanol (ppm) Butanol (ppm) Butanol (ppm)
C-01 0 (Control) 763 1235 5948 - 5948
15 - 1102 4640 3300 6290
30 599 871 3504 2430 5934
B-17 0 (Control) 2013 - 5785 - 5742
15 1775 - 4814 3012 6321
30 1388 - 3413 2381 5794

[00063] In another set of experiments, three different strategies of organic layer addition were tried with 30 mL of fermentation broth (T6 medium containing 60 g/L glucose) using strain Clostridium beijerinckii strain B-17: Strategy I: 15 mL of FAME was added to 30 mL of T6 media at the beginning of experiment; Strategy II: 5 mL of FAME was added to 30 mL of T6 media at 24 h, 48 h and 72 h of fermentation. Strategy III: 15 mL of FAME was added to 30 mL of T6 media at the beginning of experiment. Thereafter, 5 mL of biodiesel was removed and 5 mL of fresh biodiesel was added at 24 h & 48 h.

Table 3: Extractive fermentation using FAME as the extractant
Aqueous Media (30 mL) FAME (15 mL)
Strategy Ethanol (ppm) Acetone (ppm) IPA (ppm) Butanol (ppm) Ethanol (ppm) Acetone (ppm) IPA (ppm) Butanol (ppm) Total Solvents (g/L)
Control 78 495 1969 6763 NA 9.31
I 95 387 1320 3905 - 345 368 5982 9.06
II 78 318 2052 4891 - 285 448 6741 11.1
III 91 399 1520 4245 130 315 344 5768 10.5

Biodiesel (5mL sampled out) at 24 h - 323 160 1572
Biodiesel (5mL sampled out) at 48 h - 358 209 3383
Example 3: Simultaneous Saccharification and Extractive Co-Fermentation (SSECF)
[00064] In the results shown here, the SSECF step was done in one single batch reactor system. As fermentations were found to take almost 48 h to complete, low enzyme loadings (5 - 10 U/gds) were explored. The solid substrate loading of the pretreated biomass was 10 % (w/v). The temperature range explored was 30-35 0C. Acetate buffer (50 mM, pH 5.5) was used for all SSECF experiments. The ratio of organic layer: aqueous broth was kept at 1:2. Butanol was found to be partitioned between the aqueous and organic layers, as shown in Table 4. The three processes, namely hydrolysis, co-fermentation (glucose and xylose utilization) and extraction, were thus integrated into one single step where all the processes occur simultaneously.

Table 4: Solvent production in an SSECF process
Aqueous Media (40 mL) Organic Phase (20 mL)
Strategy Acetone (ppm) Butanol (ppm) Acetone (ppm) Butanol (ppm) Total Solvents (g/L)
Control 4085 11676 - - 15.7
SSECF 3933 8086 1483 9845 17.7

 
ADVANTAGES OF THE INVENTION

[00065] The present disclosure provides a dual step cost-effective process for production of butanol from lignocellulosic biomass.
[00066] The present disclosure compress the multiple process steps of saccharification, fermentation and extraction of butanol to a single process step of “Simultaneous Saccharification and Extractive Co-Fermentation’ (herein also referred to as SSECF) that lowers the cost of production.
[00067] The present disclosure provides a dual-step process for production of butanol from lignocellulosic biomass wherein saccharification, fermentation and extraction are carried out in a single vessel.
[00068] The present disclosure provides a high-yielding and long-term stable process for production of butanol from lignocellulosic biomass.
[00069] The present disclosure provides a dual step process for production of butanol from lignocellulosic biomass which requires low maintenance.
[00070] The present disclosure provides a continuous and industrially scalable butanol production process with improved butanol productivity.
[00071] The present disclosure provides an efficient process for the production of high yield of butanol from pretreated lignocellulosic biomass using a micro-aero-tolerant strain of Clostridium beijerinckii.
[00072] The present disclosure provides an optimal fermentation conditions for enhanced production of butanol from pre-treated lignocellulosic biomass using Clostridium beijerinckii.
[00073] Other advantages of the present invention will be apparent from the description of the invention.