Process For Second Generation Lactic Acid Production

Abstract

The present invention provides an improved process for high titer lactic acid production using simultaneous saccharification and co-fermentation process using acid treated lignocellulosic biomass. The process comprises of pretreating lignocellulosic biomass comprising C5 and C6 sugars with dilute acid and superheated steam at a temperature ranging from 150-210° C.; neutralizing the pretreated lignocellulosic biomass of step (i) with NaOH pellets; and adding cellulase enzyme in the range of 1.5 to 10 FPU/g, bacteria, nutrients and buffering agent to the neutralized slurry obtained in step (ii) for hydrolysis and co-fermentation of C5 and C6 sugars at a temperature ranging from 40-45° C. for a period of 120-144 hours to obtain lactic acid. The process of the present invention requires simple nutrients and results in high titer lactic acid production within shortest duration of time.

Claims

1. An improved process for production of lactic acid from a lignocellulosic biomass, the process comprising: i. pretreating lignocellulosic biomass comprising C5 and C6 sugars with dilute acid and superheated steam at a temperature ranging from 150-210° C.; ii. neutralizing the pretreated lignocellulosic biomass of step (i) with NaOH pellets; and iii. adding cellulase enzyme in the range of 1.5 to 10 FPU/g, bacteria, nutrients and buffering agent to the neutralized slurry obtained in step (ii) for hydrolysis and cofermentation of C5 and C6 sugars at a temperature ranging from 40-45° C. for a period of 120-144 hours to obtain lactic acid.

2. The process as claimed in claim 1, wherein the pretreatment of biomass slurry additionally comprises adjusting pH of the slurry to 5.0-5.5 with a pH adjuster.

3. The process as claimed in claim 2, wherein the pH adjuster is selected from aqueous ammonium hydroxide, NaOH, KOH and CaCO.sub.3.

4. The process as claimed in claim 1, wherein the nutrient is yeast extract and diammonium hydrogen phosphate (NH.sub.4).sub.2HPO.sub.4.

5. The process as claimed in claim 1, wherein the buffering agent is calcium carbonate (CaCO.sub.3).

6. The process as claimed in claim 1, wherein the pre-treated biomass slurry is subjected to fermentation without any detoxification.

7. The process as claimed in claim 1, wherein the bacteria is Lactobacillus selected from Lactobacillus lactis 2369 (NCIM) and Lactobacillus delbrueckii 2365 (NCIM).

8. The process as claimed in claim 1, wherein the lignocellulosic biomass is selected from the group consisting of rice straw, wheat straw, sugarcane bagasse, cotton stalk, barley stalk, bamboo or any agriculture residues which contain cellulose or hemicellulose or both.

9. The process as claimed in claim 1, wherein the C5 sugar is xylose and C6 sugar is glucose.

10. The process as claimed in claim 1, wherein the process yields ˜90 g/L lactic acid within 120 hours of fermentation.

11. The process as claimed in claim 1, wherein the fermentation reaction is a batch process.

12. A process for the production of lactic acid from lignocellulosic biomass using a pretreated slurry as claimed in claim 1, wherein the simultaneous saccharification and co-fermentation is performed.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0037] These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings wherein:

[0038] FIG. 1 illustrates the overall steps involved in SSCF process for lactic acid production from lignocellulosic biomass.

DETAILED DESCRIPTION OF THE INVENTION

[0039] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

Definition

[0040] For the purposes of this invention, the following terms will have the meaning as specified therein:

[0041] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0042] The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

[0043] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0044] The term “including” is used to mean “including but not limited to” “including” and “including but not limited to” are used interchangeably.

[0045] Nearly all forms of lignocellulosic biomass, i.e., plant biomass, such as monocots, comprise three primary chemical fractions: hemicellulose, cellulose, and lignin. Cellulose and hemicelluloses in plant cell walls exist in complex structures within the residual material. Hemicellulose is a polymer of short, highly-branched chains of mostly five-carbon pentose sugars (xylose and arabinose), and to a lesser extent six-carbon hexose sugars (galactose, glucose and mannose). Because of its branched structure, hemicellulose is amorphous and relatively easy to hydrolyze into its individual constituent sugars by enzyme or dilute acid treatment. Cellulose is a linear polymer comprising of β (1.fwdarw.4) linked D-glucose in plant cell wall, much like starch with a linear/branched polymer comprising of a (1.fwdarw.4) linked D-glucose, which is the primary substrate of corn grain in dry grain and wet mill ethanol plants. However, unlike starch, the glucose sugars of cellulose are strung together by β-glycosidic linkages which allow cellulose to form closely-associated linear chains. Because of the high degree of hydrogen bonding that can occur between cellulose chains, cellulose forms a rigid crystalline structure that is highly stable and much more resistant to hydrolysis by chemical or enzymatic attack than starch or hemicellulose polymers.

[0046] Therefore, a pretreatment process is typically used to alter and open up the cell wall matrix, to hydrolyze the hemicelluloses, and to reduce crystallinity. Pretreatment disrupts the non-easily digestible portion of lignocellulosic biomass, e.g., cellulose and lignin, thus improving its digestibility. After pretreatment, much of the biomass becomes easily digestible while a portion remains non-easily digestible. Ultimately, the pretreatment process makes the cellulose and/or hemicellulose more accessible (during a subsequent hydrolysis process) for conversion of the carbohydrate polymer into fermentable sugars.

[0047] “Cellulase enzyme” used herein is a mixed form of enzyme which is mostly composed of exo-hydrolase, endo-hydrolase and beta-glucosidase and other auxiliary enzymes. Cellulase breaks down the cellulose molecule into monosaccharide and shorter polysaccharides or oligosaccharides.

[0048] “Pretreated biomass” or “Pretreatment of biomass” used herein clears away physical and chemical barriers that make native biomass recalcitrant and exposes cellulose for better enzymatic hydrolysis. In most of the pretreatment, chemical (acid or alkali) and physical (high temperature or steam or pressure) parameters are used individually or in mixed manner to remove barriers for enzymatic hydrolysis and improve the enzymatic digestibility.

[0049] “Detoxification” used herein is the process where the inhibitors (toxic compound such hydroxymethyl furfural, furfural, acetic acids, formic acids etc.) produced during the pretreatment process are removed or neutralized from pre-treated biomass by chemical, physical or biological process.

[0050] The present invention relates to bioconversion of acid-treated biomass for high-titer lactic acid production with lesser nutrient requirements, operation with high solid loading and low enzyme loading resulting in a cost-effective lactic acid production.

[0051] The objective of the present invention is to produce high titer lactic acid from waste lignocellulosic biomass in one pot simultaneous saccharification and co-fermentation process (SSCF). Particularly, the present invention provides a process for production of lactic acid from lignocellulosic biomass using a dilute acid pretreatment, wherein the pretreatment method solubilizes maximum of hemicelluloses and improves the digestibility of cellulose rich pretreated slurry using commercial cellulases. The dilute acid pretreated lignocellulosic biomass comprises soluble C5 and solid C6 sugars for enzymatic hydrolysis. This is followed by co-fermentation of C5 and C6 sugars present in the pretreated biomass with a cellulase enzyme, bacteria and nutrient to obtain lactic acid in a single unit system with a fixed temperature regime for effective lactic acid production.

[0052] Thus, according to one aspect of the present invention, there is provided an improved process for production of lactic acid from a lignocellulosic biomass, the process comprising:

i. pretreating lignocellulosic biomass comprising C5 and C6 sugars with dilute acid and superheated steam at a temperature ranging from 150-210° C.
ii. neutralizing the pretreated lignocellulosic biomass of step (i) with NaOH pellets; and
iii. adding cellulase enzyme in the range of 1.5 to 10 FPU/g, bacteria, nutrients and buffering agent to the neutralized slurry obtained in step (ii) for hydrolysis and cofermentation of C5 and C6 sugars at a temperature ranging from 40-45° C. for a period of 120-144 hours to obtain lactic acid.

[0053] In an embodiment of the present invention, there is provided a process for the production of lactic acid, wherein the pretreatment of biomass slurry additionally comprises adjusting pH of the slurry to 5-5.5 with a pH adjuster.

[0054] In another embodiment of the present invention, there is provided a process for production of lactic acid, wherein the pH adjuster used for adjusting pH of the slurry is selected from aqueous ammonium hydroxide, NaOH, KOH and CaCO.sub.3.

[0055] In yet another embodiment of the present invention, there is provided a process for production of lactic acid, wherein the nutrient is yeast extract and diammonium hydrogen phosphate (NH.sub.4).sub.2HPO.sub.4.

[0056] In still another embodiment of the present invention, there is provided a process for production of lactic acid, wherein the buffering agent is calcium carbonate (CaCO.sub.3).

[0057] In an embodiment of the present invention, there is provided a process for production of lactic acid, wherein the pre-treated biomass slurry is subjected to fermentation without any detoxification.

[0058] The production of lactic acid is commonly carried out by fermentation of a strain of the bacterial genus Lactobacillus and more particularly by the species Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus pentosus and Lactobacillus lactis as examples. In a preferred embodiment of the present invention, the Lactobacillus is selected from Lactobacillus lactis 2369 (NCIM) and Lactobacillus delbrueckii 2365 (NCIM). The lactobacillus species used in the present invention is capable of utilizing both glucose and xylose fraction of the pretreated biomass and results in high-titer lactic acid production.

[0059] In an embodiment of the present invention, there is provided a process for production of lactic acid, wherein the lignocellulosic biomass is selected from the group consisting of rice straw, wheat straw, sugarcane bagasse, cotton stalk, barley stalk, bamboo or any agriculture residues which contain cellulose or hemicellulose or both.

[0060] In another embodiment of the present invention, there is provided a process for production of lactic acid, wherein C5 sugar is xylose and C6 sugar is glucose.

[0061] In an embodiment of the present invention, there is provided a process for production of lactic acid, wherein the process yields ˜90 g/L lactic acid within 120 hours of fermentation.

[0062] In another embodiment of the present invention, the biomass loading of 10 to 25% (w/w) was used for lactic acid production. In a preferred embodiment of the present invention, high solid loading of 25% (w/w) is used for high-titer lactic acid production.

[0063] In an embodiment of the present invention, there is provided a process for production of lactic acid, wherein the fermentation reaction is a batch process.

[0064] In an aspect of the present invention, there is provided a process for production of lactic acid from lignocellulosic biomass using a pretreated slurry wherein simultaneous saccharification and co-fermentation is performed.

[0065] Thus, the process of the present invention produces high titer lactic acid from waste lignocellulosic biomass in one pot simultaneous saccharification and co-fermentation process (SSCF). The process claimed in the present invention requires simpler nutrients for microbial growth and fermentation can be performed in an open with no sterilization. Further, high solid loading of 25% (w/w) and low enzyme dosage (1.5 to 10 FPU/g biomass) is required for lactic acid fermentation. In addition, the results obtained in the fermentation process as indicated in Table 2-4 of examples demonstrates that the strains Lactobacillus lactis and Lactobacillus delbrueckii used in the process results in high-titer lactic acid production from pretreated biomass via SSCF process.

[0066] Although the subject matter has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible.

EXAMPLES

[0067] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.

Example 1: Preparation of Pretreated Lignocellulosic Biomass

[0068] For the preparation of lignocellulosic biomass, rice straw (Oryza sativa), agricultural waste material was collected from a field near Mathura, Uttar Pradesh (27.28° N 77.41° E) during harvesting period. The rice straw was air dried and shredded to 10 mm size using a knife cutting mill and was further, preserved using sealed poly bags maintained at room temperature (25° C.) for experimentation. Table 1 indicates chemical composition of native biomass and pretreated biomass. The dilute acid pretreatment of shredded rice straw was conducted using in-house designed (250 kg/day) continuous pilot-scale reactor system using dilute sulfuric acid (1.8%). The plant consists of a size reduction mill, high-pressure reactor, screw feeder, hydraulic press, slurry tank and feed hoper. Milled rice straw (10 mm) was presoaked in an acid solution (1.8%) for a duration of 30 minutes. Further, acid-soaked biomass was then pressed to reduce the excess water ˜50% and soaked biomass was fed through feed hopper at a flow rate of 10 Kgh.sup.−1. Rice straw was pretreated at the optimized temperature at ˜162° C. and 5.2 bar for a residence time of 10 minutes using superheated steam. After dilute acid pretreatment, the pretreated slurry was collected and cooled down to room temperature and the chemical composition were determined by the NREL method (NREL/TP-510-42621).

TABLE-US-00001 TABLE 1 Chemical Compositional analysis of native and pretreated rice straw Biomass Extractive Glucan Xylan Lignin Ash Native biomass 18 38.94 17.57 17.73 9.33 Pretreated biomass 0 42.51 17.22 28.53 14.95 Water extractives; Ethanol extractives; Glucan and Xylan were calculated using NREL Lap equations

Example 2: Simultaneous Hydrolysis and Co-Fermentation Process (SSCF) for Lactic Acid Production by Lactobacillus lactis 2369

[0069] For the lactic acid production, first dilute acid pretreated rice straw was neutralized with NaOH pellets to pH 4.8-5.0. The pretreated neutralized slurry was added with commercial cellulase enzyme using 5 FPU/g of cellulase per gram of substrate, yeast extract (1%), (NH.sub.4).sub.2PO.sub.4 (0.1%) and CaCO.sub.3 (˜0.45 g/g total sugar), 10% (v/v) of Lactobacillus lactis 2369 NCIM culture and fermentation was performed in a single step. The simultaneous hydrolysis and co-fermentation process was performed in batch mode by maintaining the reactor temperature at 42° C. for a duration of 120-144 hours with a fixed rpm of 200. Table 2 below indicates the lactic acid production and acetic acid production by using dilute acid pretreated rice straw. It was observed that the final lactic acid production was 108 g/L. All aqueous samples were analyzed by HPLC to determine lactic acid and by-product concentrations using Waters HPLC equipment (Water, Switzerland) equipped with HPX 87H and HPX 87 P column (Bio-Rad Labs, USA) and Waters Refractive index (RI 2414) and Waters UV-vis spectrophotometer (2489) measuring at a wavelength 210 nm. A racemic mixture of lactic acid (D/L), with D-lactic acid as a major fraction was obtained, as evident from D/L-lactic colorimetric assay kit.

TABLE-US-00002 TABLE 2 SSCF process of lactic acid production using Lactobacillus lactis #Initial #Initial Final Final Glucose xylose Fermentation Lactic Acetic concentration concentration time acid acid Titer Substrate (g/L) (g/L) (h) Titer(g/L) (g/L) Dilute acid 10.3 33.6 120 108.1 11.5 pretreated rice straw #Initial sugar concentration is measured in the feed slurry before adding the seed culture

Example 3: Simultaneous Hydrolysis and Co-Fermentation Process (SSCF) for Lactic Acid Production by Lactobacillus delbrueckii 2365

[0070] The bacterial strain Lactobacillus delbrueckii is evaluated to demonstrate lactic acid production from C5 and C6 sugars of the pretreated lignocellulosic biomass. In this process, at first dilute acid pretreated rice straw was neutralized with NaOH pellets to pH 4.8-5.0 as described in Example 2. Neutralized (20˜25 wt. % on dry weight), commercial cellulase (5 FPU/g), yeast extract (1%), (NH.sub.4).sub.2PO.sub.4 (0.1%) and CaCO.sub.3 (˜0.45 g/g total sugar) and 10% (v/v) of Lactobacillus delbrueckii NCIM culture was added in single go. Simultaneous hydrolysis and co-fermentation process were performed in batch mode by maintaining the reactor temperature at 42° C. Table 3 below indicates the lactic acid production and acetic acid production by using strain Lactobacillus delbrueckii 2365. It was observed that lactic acid concentration of 97 g/L is achieved within 120 hours of reaction. The strain produces a homogenous mixture of L-Lactic acid (L), as evident from D/L-lactic colorimetric assay kit.

TABLE-US-00003 TABLE 3 SSCF process of lactic acid production using Lactobacillus delbrueckii #Initial #Initial Final Final Glucose xylose Fermentation Lactic Acetic concentration concentration time acid acid Titer Substrate (g/L) (g/L) (h) Titer(g/L) (g/L) Dilute acid 10.3 33.6 120 97.4 9.7 pretreated rice straw #Initial sugars concentration is measured in the feed slurry before adding the seed culture

Example 4 Simultaneous Hydrolysis and Co-Fermentation Process (SSCF) for Lactic Acid Production Via Co-Culture of Lactobacillus delbrueckii 2365 and Lactobacillus lactis 2369

[0071] In this process, a co-culture of Lactobacillus delbrueckii and Lactobacillus lactis is demonstrated for lactic acid production from C5 and C6 sugars of the pretreated lignocellulosic biomass with a mixing ration of 1:1. In this process, at first dilute acid pretreated rice straw was neutralized with NaOH pellets to pH 4.8-5.0. Neutralized (20˜25 wt. % on dry weight), commercial cellulase (5 FPU/g), yeast extract (1%), (NH.sub.4).sub.2PO.sub.4 (0.1%) and CaCO.sub.3 (˜0.45 g/g total sugar), 5% (v/v) of Lactobacillus delbrueckii and 5% (v/v) of Lactobacillus lactis were added in single go. Simultaneous hydrolysis and co-fermentation process were performed in batch mode by maintaining the reactor temperature at 42° C. The result obtained (depicted in Table 4 below) indicates that by using the co-culture of Lactobacillus lactis and Lactobacillus delbrueckii, the lactic acid concentration of 82 g/L was observed in 120 hours of reaction. The strains produces a racemic mixture of Lactic acid (D/L), as evident from D/L-lactic colorimetric assay kit.

TABLE-US-00004 TABLE 4 SSCF process of lactic acid production using co-culture of Lactobacillus lactis and Lactobacillus delbrueckii #Initial #Initial Final Final Glucose xylose Fermentation Lactic Acetic concentration concentration time acid acid Titer Substrate (g/L) (g/L) (h) Titer(g/L) (g/L) Dilute acid 10.3 33.6 120 82.8 9.7 pretreated rice straw #Initial sugars concentration is measured in the feed slurry before adding the seed culture