METHOD FOR THE PREPARATION OF LACTIC ACID

Abstract

A method for preparing a fermentation product including lactic acid, the method including: a) treating lignocellulosic material with caustic magnesium salt in the presence of water to provide treated aqueous lignocellulosic material; b) saccharifying the treated aqueous lignocellulosic material in the presence of a hydrolytic enzyme to provide a saccharified aqueous lignocellulosic material comprising fermentable carbohydrate and a solid lignocellulosic fraction; c) simultaneously with step b), fermenting the saccharified aqueous lignocellulosic material in the presence of both a lactic acid forming microorganism and caustic magnesium salt to provide an aqueous fermentation broth comprising magnesium lactate and a solid lignocellulosic fraction; d) recovering magnesium lactate from the broth, wherein the saccharification and the fermentation are carried out simultaneously.

Claims

1. A method for producing a fermentation product comprising lactic acid from lignocellulosic material, said method comprising: a) providing a lignocellulosic material and treating said material with an alkaline agent in the presence of water to provide a treated aqueous lignocellulosic material, said alkaline agent comprising a caustic magnesium salt; b) saccharifying the treated aqueous lignocellulosic material in the presence of a hydrolytic enzyme to provide a saccharified aqueous lignocellulosic material comprising fermentable carbohydrate and a solid lignocellulosic fraction; c) fermenting the saccharified aqueous lignocellulosic material in the presence of a lactic acid forming microorganism and in the presence of a caustic magnesium salt to provide an aqueous fermentation broth comprising magnesium lactate and a solid lignocellulosic fraction; and, d) recovering lactic acid and/or a lactate salt from said aqueous fermentation broth, wherein said saccharification and said fermentation are carried out simultaneously.

2. The method according to claim 1, wherein the lignocellulosic material provided to step a) has been subjected to one or more of pre-extraction, steam pre-treatment, acid hydrolysis and mechanical comminution.

3. The method according to claim 1, wherein said lignocellulosic material provided to step a) is particulate and has an average particle size of from 0.1 to 250 mm.

4. The method according to claim 1, wherein step a) comprises: i) providing a lignocellulosic material; ii) mixing said lignocellulosic material with said alkaline agent in the presence of water to form a reaction mixture having a solids content; and, iii) heating said reaction mixture such that said solids are held at a temperature of from 130° C. to 250° C. for a time period of from 1 minute to 600 minutes.

5. The method according to claim 4, wherein the concentration of the caustic magnesium salt in the reaction mixture is from 0.1 to 50 wt. % based on the dry weight of the lignocellulosic material (w/w).

6. The method according to claim 4, wherein the total solids concentration of the reaction mixture is from 1 to 70% (w/w).

7. The method according to claim 4, wherein said reaction mixture has a pH of from 8.0 to 14.0.

8. The method according to claim 4, wherein: the reaction mixture has a pH of from 9.0 to 12.0 and, in step iii), said solids are held at a temperature of from 130° C. to 250° C. for a time period of from 1 minute to 240 minutes.

9. The method according to claim 4, wherein: the reaction mixture has a concentration of caustic magnesium salt of from 5 to 25% (w/w); and, said solids of the reaction mixture are held at a temperature of from 140° to 170° C. for a period of from 180 to 600 minutes.

10. The method according to claim 4, wherein: the reaction mixture has a concentration of caustic magnesium salt of from 5 to 25% (w/w); and, said solids of the reaction mixture are held at a temperature of from 170° C. to 230° C. for a period of from 1 to 240 minutes.

11. The process according to claim 1 comprising subjecting the lignocellulosic material to acid treatment prior to treating said material with the alkaline agent of step a), wherein said acid treatment comprises mixing said lignocellulosic material with an acidic aqueous solution having a concentration of 8 wt. % or less of acid selected from the group consisting of inorganic acid, organic acid, amino acid, mineral acid, Bronsted acid, Lewis acid and mixtures thereof, and wherein said mixing takes place at a temperature of from 120° C. to 230° C.

12. The process according to claim 11, wherein the treatment of step a) is performed i) at a pH of from 2 to 10; ii) at a temperature of from 15 to 100° C.; and iii) for a period of from 1 to 30 minutes, to neutralize the acid treated lignocellulosic material.

13. The method according to claim 1, wherein the alkaline agent of step a) comprises or consists of a caustic magnesium salt selected from MgO, Mg(OH).sub.2, MgCO.sub.3, Mg(HCO.sub.3).sub.2 and mixtures thereof.

14. The method according to claim 13, wherein the caustic magnesium salt of step a) comprises at least one of MgO and Mg(OH).sub.2.

15. The method according to claim 1, wherein the caustic magnesium salt of step c) is selected from at least one of MgO, Mg(OH).sub.2, MgCO.sub.3 and Mg(HCO.sub.3).sub.2.

16. A method as defined in claim 1 for preparing lactic acid, said method comprising the steps of: a) treating a lignocellulosic material with an alkaline agent in the presence of water to provide a treated aqueous lignocellulosic material, said alkaline agent comprising a caustic magnesium salt; b) saccharifying the treated aqueous lignocellulosic material in the presence of a hydrolytic enzyme to provide a saccharified aqueous lignocellulosic material comprising fermentable carbohydrate and a solid lignocellulosic fraction; c) fermenting the saccharified aqueous lignocellulosic material in the presence of a lactic acid forming microorganism and in the presence of a caustic magnesium salt to provide an aqueous fermentation broth comprising magnesium lactate and a solid lignocellulosic fraction; d) recovering magnesium lactate from said aqueous fermentation broth; e) providing a feed comprising hydrogen chloride, said feed being either an aqueous solution comprising hydrogen chloride or a gas feed comprising gaseous hydrogen chloride; and, f) acidifying the magnesium lactate to lactic acid by bringing said magnesium lactate into contact with said feed comprising hydrogen chloride, thereby forming a liquid effluent comprising lactic acid and magnesium chloride, wherein said saccharification and said fermentation are carried out simultaneously.

17. The method according to claim 16, further comprising: g) separation of lignin from the liquid effluent product of step f).

18. The method according to claim 16, further comprising: g) separation of lignin from the liquid effluent product of step f); and, h) separation of the lactic acid and magnesium chloride present in the liquid effluent product of step g) to obtain a lactic acid product stream and a magnesium chloride solution or suspension.

19. The method according to claim 18, wherein the separation step h) comprises a lactic acid extraction step.

20. The method according to claim 18, wherein the separated magnesium chloride solution or suspension is subjected to a temperature of at least 300° C., thereby decomposing magnesium chloride into magnesium oxide and hydrogen chloride and thus obtaining a solid comprising magnesium oxide and a gas comprising gaseous hydrogen chloride.

21. The method according to claim 20, wherein said magnesium oxide derived from said step of thermally decomposing magnesium chloride is used directly in at least one of step a) and step c) or, alternatively, is used as a precursor for a caustic magnesium salt used in at least one of step a) and step c).

22. The method according to claim 21, wherein heat conserved in the solid magnesium oxide from the thermal decomposition of magnesium chloride is transferred to the lignocellulosic material of step a) and/or the fermentation broth of step c).

23. The method according to claim 20, wherein said feed comprising hydrogen chloride as used in step e) is at least partially derived from gaseous hydrogen chloride obtained from the thermal decomposition of the magnesium chloride.

24. The process according to claim 16 wherein the magnesium lactate as recovered in step d) is at least partially in crystalline form.

25. The process according to claim 16 wherein said feed comprising hydrogen chloride is an acidic aqueous solution in which the concentration of hydrogen chloride is at least 5 wt. %, based on the total weight of the acidic solution.

26. Method according to claim 19, wherein the extracted lactic acid is subjected to a purification step.

Description

EXAMPLES

Raw Materials and Their Analysis:

[0115] Bagasse was provided by Purac Thailand Ltd. (Rayong, Thailand).

[0116] Ground wheat straw was obtained from a local supplier. For Examples 1 to 9 below, the wheat straw was milled using a Retch Cutting Mill (SM100) and then screened to attain a median particle size in the range from 500 microns to 1 mm. For Examples 10 to 12 below, the wheat straw was milled using hammer mill (Apex Commuting Mill) with a screen size of 1.5 mm.

[0117] Using a Mettler Toledo Advanced Moisture Analyzer, the dry weight contents of selected 0.5-2.0 g milled wheat straw samples shed—were measured.

[0118] The wheat straw and bagasse were analyzed for carbohydrate, acid soluble lignin, acid insoluble lignin and ash contents in accordance with the procedure given in: Determination of Structural Carbohydrates and Lignin in Biomass: Laboratory Analytical Procedure (LAP), National Renewable Energy Authority (August 2012) http://www.nrel.gov/docs/gen/fy13/42618.pdf; and, Determination of Ash in Biomass: Laboratory Analytical Procedure (LAP), National Renewable Energy Authority (January 2008) http://www.nrel.gov/docs/gen/fy08/42622.pdf. Where applicable, current practice relating to said procedures may be found at htt://www.nrel.gov/biomass/analytical_procedures.html.

[0119] Where applicable, glucose was determined using the Megazyme D-Glucose assay kit (glucose oxidase/peroxidase; GOPOD) employing a pulsed amperometric detector (Roche/Hitachi GOD-PAD) and spectrophotometry (Hitachi U-2800, 540 nm). Xylose may be determined using the Megazyme D-xylose kit and spectrophotometry (Hitachi U-2800, 340 nm).

[0120] The theoretical maximum yield of glucose from wheat straw was determined to be 37.1 wt. %, based on the total dry weight.

Examples 1-9

[0121] Pre-Treatment:

[0122] Weighed 12.45 g portions of the milled wheat straw or bagasse were separately slurried in 150 ml demineralised water, following which the solid magnesium oxide or, where applicable, solid calcium oxide or sodium hydroxide were added thereto; no further basic compounds were added. For completeness, it is noted that in Example 9 below, the NaOH was added to the milled wheat straw after the solid magnesium oxide, thereby raising the pH of that sample.

[0123] The pre-treatment of said samples was carried out in a double wall stainless steel, stirred reactor (Buchi Autoclave). The reactor was rated for 60 Bar and was equipped with a pressure safety spring. Heating was carried out using hot oil up to 190° C.

[0124] The properties of each separate portion and the different conditions of temperature and residence time to which they were subjected are shown in Table 1 herein-below. All samples were continuously stirred for the requisite residence time in the reactor. Further all samples that were treated with caustic magnesium or calcium oxide had an initial pH of from 8.5-9.4 prior to any heating step.

[0125] Preparation for Enzymatic Hydrolysis:

[0126] The thus pre-treated solids were subjected to a first solid/liquid separation step using a Buchner filter under reduced pressure (approx. 200 mBar). The liquid fraction was collected for analysis. The solids were collected, dispersed in water and neutralized to a pH of from 6-7 with lactic acid.

[0127] Next the solids were subjected to a two-stage second solid/liquid separation step using a filtering centrifuge (Hermle Sieva 2) equipped with a 5 micron filter cloth. In a first stage, the centrifuge is initiated at 5000 rpm before being increased to 10000 rpm; the filtrate is collected and added to the centrifuge again; samples of the then derived filtrate and filter cake are collected for analysis. In a second stage, the centrifuge is re-initiated with the addition of 1 litre of demineralized water thereto. The separated solid fraction was then collected; its dry matter content was measured before being subjected to enzymatic hydrolysis.

[0128] Enzymatic Hydrolysis:

[0129] At a dry matter content of 10% (w/w), the pre-treated solids were hydrolyzed in 50 mL polypropylene tubes with a cellulase enzyme mix CMAX4 available from Dyadic. A potassium phosphate buffer (pH, 6.4) was employed and, furthermore, sodium azide (0.02%, w/w) was present to prevent microbial infection of the hydrolyzate. The added amount of enzyme was varied in the experiments—as indicated in Table 1—with enzyme loading of 20 mg/g dry weight being more usual, noting that this loading should ensure satisfactory release of carbohydrates (NREL, 2011).

[0130] The hydrolyzation reactions were incubated at 52° C. at 300 rpm. After 24 hours, 48 hours and 72 hours, duplicate 0.2 ml samples were taken and filtered using a micro plate; the duplicate supernatants were then analyzed.

[0131] The concentrations of glucose are given in Table 1 below.

TABLE-US-00001 TABLE 1 Enzyme Alkaline dosage Pre- Treatment Residence Agent (mg Glucose Washed Temp. Time Alkaline Dosage protein/g concentration Example Substrate (Y/N) (° C.) (min.) Agent (% w/w) dry solids) (g/L, at 72 h) 1 Milled Y 190 60 MgO 12 5 23.3 Wheat straw 2 Milled Y 190 120 MgO 12 5 14.9 Wheat straw 3 Milled Y 190 60 MgO 8 5 18.1 Wheat straw 4 Milled Y 190 120 MgO 8 5 13.0 Wheat straw 5 Milled Y 190 60 MgO 10 20 40.6 Wheat straw 6 Milled N 190 60 MgO 10 20 37.5 Wheat straw 7 Milled Y 190 30 MgO 10 20 37.2 Wheat straw 8 Milled Y 85 480 CaO 8 20 22.9 (Comparative) Wheat straw 9 Bagasse Y 190 20 MgO + NaOH 10 20 13.7 (pH = 11)

[0132] It is clear from Table 1 that the elevated temperature pre-treatment of the wheat straw with caustic magnesium salt facilitates the subsequent hydrolysis of this biomass, as evidenced by the attained concentrations of glucose.

Example 10

[0133] This Example is intended to demonstrate the scaling up of the pre-treatment process to accommodate larger amounts of lignocellulosic material.

[0134] Pre-Treatment:

[0135] Weighed 1.6 kg portions of the milled wheat straw were separately slurried in 13.4 litres of demineralised water, following which the solid magnesium oxide was added thereto; no further basic compounds were added.

[0136] The pre-treatment of said samples was carried out in a double wall stainless steel, jacketed reactor equipped with an anchor propeller (50 litre Buchi Autoclave). The reactor was rated at greater than 20 Bar and was equipped with a pressure safety spring. Heating to 190° C. was carried out using both direct injection of pressurized steam and hot oil circulated through the jacket. The reaction mixture was stirred continuously during its residence time in the reactor.

[0137] After the reaction was performed, the reactor was cooled using oil circulated in the jacket followed by a rapid cooling effected by releasing the pressure of the reactor. The contents of the reactor were then collected.

[0138] The properties of the collected material and the conditions of temperature and residence time to which that material had been subjected are shown in Table 2 herein-below. Further the samples that were treated with caustic magnesium oxide in this manner had an initial pH of from 8.5-9.4 prior to any heating step.

[0139] Preparation for Enzymatic Hydrolysis:

[0140] The thus pre-treated solids were subjected to a first solid/liquid separation step under gravity using a 1 mm screen, the liquid fraction being collected in a 120 liter vessel. The solids were collected, dispersed in water in a further 120 liter vessel and neutralized to a pH of from 6-7 with lactic acid (50 wt. % aqueous solution).

[0141] The slurry formed was again filtered under gravity as described above (1 mm screen) except that 26 liters of demineralized water was sprinkled evenly over the filter cake. The filter cake was divided into six portions (SPs) each of which was pressed at an applied pressure of 250 Bar using a bench press filter (Fischer MachineFabriek). Each separated solid fraction was then collected; the cakes were subsequently broken up, homogenized and distributed over two containers (SP1, SP2). The dry matter content of each container was measured and found to be from 38-42% (w/w).

[0142] Enzymatic Hydrolysis:

[0143] The pre-treated solids were hydrolyzed in polypropylene tubes with a cellulase enzyme mix CMAX4 available from Dyadic; the pre-treated solids were added so as to be in an amount of 10 wt. %, by dry weight (c. 1 g dry weight). A potassium phosphate buffer (pH, 6.4) was employed and, furthermore, sodium azide (0.02%, w/w) was present to prevent microbial infection of the hydrolyzate. The enzyme loading was 20 mg/g dry weight.

[0144] The hydrolyzation reactions were incubated at 52° C. at 300 rpm. After 24 hours and 48 hours, duplicate 0.2 ml samples were taken and filtered using a micro plate; the duplicate supernatants were then analyzed.

[0145] The concentration of glucose are given in Table 2 below.

TABLE-US-00002 TABLE 2 Enzyme dosage Alkaline (mg Treatment Residence Agent protein/g Glucose Temp. Time Alkaline Dosage dry concentration Example Substrate (° C.) (min.) Agent (% w/w) solids) (g/L, at 48 h) 10 Milled 190 59 MgO 10 20 53.2 Wheat straw

Example 11

[0146] Pre-Treatment:

[0147] Weighed 1.6 kg portions of the milled wheat straw were pre-treated in an identical manner to that described for Example 10 above. The properties of the collected material and the conditions of temperature and residence time to which that material had been subjected are thus shown in Table 2 herein-above. Further, as previously the samples that were treated with caustic magnesium oxide in this manner had an initial pH of from 8.5-9.4 prior to any reactor heating step.

[0148] Preparation for Simultaneous Saccharification and Fermentation:

[0149] The thus pre-treated solids were subjected to a first solid/liquid separation step under gravity using a 1 mm screen, the liquid fraction being collected in a 120 liter vessel. The filter cake was divided into 11 portions (SSFPs) each of which was pressed at an applied pressure of 250 Bar using a bench press filter (Fischer MachineFabriek). Each separated solid fraction was then collected; the cakes were subsequently broken up, homogenized and distributed over two containers (SSF1, SSF2). An averaged dry matter content of each container was measured as: 40.26% w/w, SSF1; and, 42.95% w/w, SSF2.

[0150] Simultaneous Saccharification and Fermentation

[0151] As used in this Example, Bacillus coagulans DSM2314 is a publically available, non-GMO strain. A work stock of Bacillus coagulans DSM2314 was taken from a −80° C. freezer and pre-cultured in a sterile medium containing 7.7 g/l dextrose monohydrate, 2 g/l DAP, 3.5 g/l DAS, 1 g/l CaCl.sub.2.5H.sub.2O, and 10 g/l yeast extract paste (50% Dry Solids).

[0152] Seed Fermentation:

[0153] To generate a seed fermentation, a standard Minifors unit was loaded with 1 litre of said sterile fermentation medium. To this medium was added 50 ml of the above inoculum. The medium was stirred at 200 rpm and maintained at a temperature of 52° C. for approximately 20 hours. The pH of the medium was also maintained at 6.4 during this period by the addition of magnesium hydroxide (aqueous solution) thereto.

[0154] Simultaneous Saccharification and Fermentation:

[0155] the simultaneous saccharification and fermentation was performed in a further minifors unit adapted to include a high torque, helical stirrer and an external 1 rpm pump for the addition of sludge to said unit.

[0156] In this Example, a two hour pre-saccharification step was employed, whereby the substrate (SSF1) was first loaded into the adapted minifors unit in the absence of the inoculum at a substrate loading of 5% (w/w) dry matter. The pre-saccharification was performed with cellulase enzyme mix CMAX4 (Dyadic) at an enzyme loading of 20 mg/g dry weight; at a start volume of 1300 ml, the pre-saccharification medium was stirred at 200 rpm and maintained at a temperature of 52° C. and a pH of 6-7.

[0157] After two hours, 100 ml of the inoculum was introduced and the reactor maintained at the above conditions of stirring, temperature and pH for a further 3 hours, again using magnesium hydroxide to control the pH.

[0158] Following this, the stirrer was stopped and first 50 g dosage of substrate (SSF1) introduced; the stirrer was then re-started to 200 rpm and the temperature of the reactor moderated to 52° C., if required. The pH was monitored and further 50 g dosages of substrate added in the same manner when the pH of the medium fell below 6.4. Such substrate addition was continued until the content of the reactor was approximately 20% (w/w) dry matter.

[0159] 25 ml samples of the supernatant were taken regularly, on average every 3 hours and used to determine the concentration of glucose, xylose and lactic acid, lactic acid concentration being determined using HPLC. Measurements were stopped at 24.8 hours total time (pre-saccharification plus SSF time).

TABLE-US-00003 TABLE 3 Final Solid *Initial *Initial Total Lactic loading Glucose Xylose Fermentation/ acid pre- concentration concentration SSF time concentraation Ex. saccharification (g/L) (g/L) (hours) (g/L) 11 5% wt 17.8 7.3 24.8 99.6 *Concentration measured at the end of pre-saccharification

[0160] FIG. 1 appended hereto illustrates the change in concentration of glucose, xylose and lactic acid over time during the simultaneous saccharifcation and fermentation.

[0161] It will be apparent to those skilled in the art, upon consideration of the specification, that various modifications can be made in the disclosed embodiments without departing from the scope of the invention. It is therefore intended that the embodiments and examples be considered illustrative only, with the true scope of the invention being indicated by the following claims.