A process for the production of lactic acid

Abstract

The present application relates to the technical field of biological fermentation, and particularly relates to a process for producing lactic acid, comprising the following steps: (1) carrying out saccharification on treated lignocellulose raw materials to obtain a saccharification treatment liquid; (2) using a fermentation medium containing the saccharification treatment liquid to ferment and produce lactic acid, and adding growth factors during the fermentation process, wherein the addition amount of the growth factors is 1-3 g / L, and the growth factors comprise vitamin B substances, vitamin D substances, amino acid substances and ammonium sulfate. The present application uses Pediococcus acidilactici to ferment and produce lactic acid, the strain has good adaptability to pretreated lignocellulose biomass systems, can ferment under high solid content conditions, and can improve the lactic acid yield by adding growth factors, and has wide industrialization prospects.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation technology, specifically to a process for producing lactic acid. Background Technology

[0002] Polylactic acid (PLA) is a biodegradable and environmentally friendly new material, hailed as a green fiber. As a substitute for petroleum-derived polymers, it has been widely used in packaging materials, biomedical materials, and other fields. As a monomeric compound of PLA, lactic acid exists in two optical isomers: D- and L-. Poly-D-lactic acid / poly-L-lactic acid, polymerized separately from chiral D-lactic acid / L-lactic acid monomers, exhibits superior heat resistance and mechanical properties compared to poly-D,L-lactic acid polymerized from a mixture of D and L-lactic acids. Microbial fermentation is a very promising method for producing chiral lactic acid. Statistics show that 70%–90% of the world's annual lactic acid production is obtained through microbial fermentation. However, currently, chiral lactic acid is produced using glucose or grain starch, resulting in high costs. Lignocellulose, due to its low price, abundant resources, and renewable nature, is a very promising raw material for producing chiral lactic acid.

[0003] The production of lactic acid from lignocellulose mainly involves straw pretreatment, detoxification, enzymatic hydrolysis, and fermentation. Currently, industrial fermentation processes using cellulose as raw material primarily employ stepwise saccharification fermentation and simultaneous saccharification fermentation. Stepwise saccharification fermentation involves each reaction occurring independently. Its advantage is that each reaction utilizes its optimal conditions without interference. However, its disadvantages include complex processes, increased equipment requirements, and higher investment costs due to the numerous steps. In particular, the high concentration of sugars produced during cellulase hydrolysis inhibits cellulase activity, leading to incomplete enzymatic hydrolysis. Simultaneous saccharification fermentation combines enzymatic hydrolysis and fermentation in the same reactor. This process allows the sugars produced by enzymatic hydrolysis to be rapidly consumed by microorganisms without inhibiting cellulase, thus reducing enzyme usage. However, the optimal temperature for cellulase is generally 50℃, while the optimal temperature for lactic acid fermentation is 42℃. Therefore, high temperatures significantly inhibit the growth and fermentation of lactic acid bacteria. To ensure the survival of the bacteria, the reaction temperature must be appropriately lowered for simultaneous saccharification fermentation, resulting in a decreased enzymatic hydrolysis rate, reduced equipment utilization, and increased costs. The above technical challenges are major obstacles encountered in the fermentation of lactic acid from straw. Summary of the Invention

[0004] To address the aforementioned problems in the existing technology, the present invention provides a process for producing lactic acid, comprising the following steps:

[0005] (1) The treated lignocellulose raw material is saccharified to obtain a saccharification solution;

[0006] (2) Lactic acid is produced by fermentation in a fermentation medium containing saccharification solution. During the fermentation process, growth factors are added. The amount of growth factors added is 1-3 g / L. The growth factors include B vitamins, D vitamins, amino acids and ammonium sulfate.

[0007] In one embodiment, the amount of growth factor added is 1-2.5 g / L, more specifically 1.5-2 g / L.

[0008] In one embodiment, the amount of vitamin B added is 0.05-0.3 g / L, more specifically 0.1-0.2 g / L.

[0009] In one embodiment, the amount of vitamin D added is 0.03-0.2 g / L, more specifically 0.1-0.3 g / L.

[0010] In one embodiment, the amount of the amino acid substance added is 0.1-0.5 g / L, more specifically 0.2-0.4 g / L.

[0011] In one embodiment, the amount of ammonium sulfate added is 1-2 g / L, more specifically 1.5-2 g / L.

[0012] In this invention, the vitamin B substances are selected from one or more of vitamin B1, vitamin B2, vitamin B3, and vitamin B12.

[0013] In this invention, the vitamin D-like substances are selected from vitamin D2 and / or vitamin D3.

[0014] In this invention, the amino acid is selected from one or more of lysine, methionine, leucine, and phenylalanine.

[0015] In one embodiment, the vitamin B substances include vitamin B1 and vitamin B12, and further, the amount of vitamin B1 added is 0.03-0.08 g / L, and the amount of vitamin B12 added is 0.15-0.2 g / L.

[0016] In one embodiment, the vitamin D-like substance includes vitamin D2.

[0017] In one embodiment, the amino acid substances include methionine, leucine, and phenylalanine. Further, the amount of methionine added is 0.1-0.3 g / L, the amount of leucine added is 0.05-0.1 g / L, and the amount of phenylalanine added is 0.1-0.2 g / L.

[0018] In one embodiment, the solid content of the treated lignocellulosic raw material is 30% to 70%, more specifically 40% to 50%. For example, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50% (w / w). The solid content represents the mass percentage of solid materials, including but not limited to cellulose and hemicellulose, in the pretreated material obtained by pretreating the lignocellulosic raw material.

[0019] In this invention, the lignocellulose raw materials include, but are not limited to, at least one of straw, rice straw, rice husk, bagasse, wood, and sawdust.

[0020] In one embodiment, saccharification is performed using cellulase, wherein the amount of cellulase solution relative to the lignocellulose raw material is 10-50 U / g, or more specifically 10-20 U / g.

[0021] In one embodiment, during the saccharification process, the temperature is controlled at 40-60°C, and further at 45-55°C.

[0022] In one embodiment, during the saccharification process, the pH value is controlled at 4.5-6.5, and more specifically at 5-6.

[0023] In one embodiment, during the fermentation process, the temperature is controlled at 45-55°C, further at 45-52°C, and further at 48-51°C.

[0024] In one embodiment, during fermentation, the pH is controlled at 4.5-6.5, and more specifically 5-6. This invention allows for further saccharification using cellulase during fermentation, thus achieving simultaneous saccharification and fermentation.

[0025] In this invention, the fermentation medium may contain 5-15 g / L peptone, 5-15 g / L yeast extract, 1-3 g / L diammonium hydrogen citrate, 1-10 g / L anhydrous sodium acetate, 1-3 g / L dipotassium hydrogen phosphate, 0.1-0.3 g / L magnesium sulfate heptahydrate, and 0.1-0.5 g / L manganese sulfate monohydrate.

[0026] In one embodiment, the seed culture is inoculated into the fermentation medium at an inoculation amount of 5-25%, preferably 10-20%, where the percentage is a volume percentage.

[0027] In one embodiment, the bacterial cells in the seed liquid are selected from Pediococcus lactis, which can grow and ferment to produce lactic acid at 45-55°C.

[0028] As one embodiment of the present invention, the *Pediococcus acidilactici* is Cath501; it is currently deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, China, accession number CCTCC M 20211364, deposit date November 4, 2021.

[0029] In one embodiment, the processing of lignocellulose raw materials may include a pretreatment step. Those skilled in the art understand that the purpose of pretreatment is primarily to alter the inherent dense structure of natural lignocellulose, disrupt the chemical and physical bonds between cellulose, lignin, and hemicellulose, reduce the crystallinity of cellulose, or remove lignin, increasing the porosity of the raw material to enhance effective contact between cellulase systems and cellulose. This facilitates subsequent saccharification and fermentation. This invention does not particularly limit the method of pretreatment; any method that achieves the aforementioned effects is acceptable. Pretreatment methods include dilute acid pretreatment, steam explosion pretreatment, ammonia cellulose explosion pretreatment, alkali pretreatment, and ionic liquid pretreatment, etc.

[0030] In one specific embodiment of the present invention, the alkali pretreatment includes: crushing the lignocellulose raw material, adding alkali solution, and treating it at 100-130℃ for 10-30 minutes.

[0031] In one embodiment, the concentration of the alkaline solution is 1-5%, and the alkaline solution includes one or more of NaOH, KOH, Ca(OH)2, and ammonia water.

[0032] In one embodiment, the mass ratio of lignocellulose raw material to alkaline solid-liquid mixture is controlled to be 1:(5-10).

[0033] In this invention, the lactic acid yield prepared by the above process is above 1.5 g / L / h, further above 1.7 g / L / h, further above 1.9 g / L / h, even further above 2.0 g / L / h, and even further above 2.3 g / L / h.

[0034] In addition, the present invention provides a method for preparing lactic acid, comprising:

[0035] (1) Lactic acid fermentation broth was prepared using the above process;

[0036] (2) The obtained lactic acid fermentation broth is subjected to extraction and purification treatment to obtain lactic acid. The extraction and purification treatment can be, for example, acidification crystallization treatment, extraction treatment, or other extraction and purification methods commonly used in the art.

[0037] Compared with the prior art, the present invention has the following positive effects:

[0038] This invention utilizes Pediococcus lactis fermentation to produce lactic acid. This strain has good adaptability to pretreated lignocellulose biomass systems and can ferment under high solids content conditions. At the same time, by supplementing growth factors, the lactic acid yield can be increased, which has broad industrialization prospects.

[0039] Information on the preservation of biological materials

[0040] The *Pediococcus acidilactici* Cath501 of this invention was deposited on November 4, 2021, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Luojia Mountain, Wuchang District, Wuhan, Hubei Province, China 430072, with accession number CCTCC NO: M20211364 and culture name *Pediococcus acidilactici* Cath501. Detailed Implementation

[0041] The present invention will be described in detail below through examples. In the following examples, unless otherwise specified, all raw materials used are commercially available.

[0042] Example 1

[0043] Take 2 kg of wheat straw, crush it, add 10 kg of 2% NaOH solution, and treat it at 120℃ for 20 min to obtain a material with a solid content of 50% (w / w).

[0044] The above materials were added to the reactor, and cellulase solution was added to the reactor at a ratio of 10 U / g relative to wheat straw. The temperature was controlled at 50℃ and the pH at 6.5. Saccharification was carried out for 3 hours to obtain the saccharification solution.

[0045] Fermentation medium containing 10 g / L peptone, 10 g / L yeast extract, 2 g / L diammonium citrate, 5 g / L anhydrous sodium acetate, 2 g / L dipotassium hydrogen phosphate, 0.3 g / L magnesium sulfate heptahydrate, and 0.25 g / L manganese sulfate monohydrate is added to a reactor containing saccharification solution. A seed culture of *Pediococcus lactis* Cath501 is inoculated into the fermentation medium at an inoculum size of 10%. Fermentation is carried out at 50°C and pH 5.5. Further saccharification is achieved during fermentation using cellulase, thus realizing simultaneous saccharification and fermentation.

[0046] During fermentation, the following ingredients were added: 0.05 g / L vitamin B1, 0.18 g / L vitamin B12, 0.2 g / L vitamin D2, 0.1 g / L methionine, 0.05 g / L leucine, 0.2 g / L phenylalanine, and 1.5 g / L ammonium sulfate. Fermentation was stopped when the glucose content in the fermentation broth was almost zero.

[0047] Example 2

[0048] Take 2 kg of wheat straw, crush it, add 10 kg of 2% NaOH solution, and treat it at 120℃ for 20 min to obtain a material with a solid content of 50% (w / w).

[0049] The above materials were added to the reactor, and cellulase solution was added to the reactor at a ratio of 10 U / g relative to wheat straw. The temperature was controlled at 50℃ and the pH at 6. Saccharification was carried out for 3 hours to obtain the saccharification solution.

[0050] Fermentation medium containing 10 g / L peptone, 10 g / L yeast extract, 2 g / L diammonium citrate, 5 g / L anhydrous sodium acetate, 2 g / L dipotassium hydrogen phosphate, 0.3 g / L magnesium sulfate heptahydrate, and 0.25 g / L manganese sulfate monohydrate was added to the reactor containing the saccharification treatment solution. A seed culture of *Pediococcus lactis* Cath501 was inoculated into the fermentation medium at an inoculum size of 10%. Fermentation was carried out at a temperature of 45°C and a pH of 5.5. Further saccharification was achieved during fermentation using cellulase, thus realizing simultaneous saccharification and fermentation.

[0051] During fermentation, the following ingredients were added: 0.03 g / L vitamin B1, 0.15 g / L vitamin B12, 0.1 g / L vitamin D2, 0.12 g / L methionine, 0.06 g / L leucine, 0.15 g / L phenylalanine, and 1 g / L ammonium sulfate. Fermentation was stopped when the glucose content in the fermentation broth was almost zero.

[0052] Example 3

[0053] Take 2 kg of wheat straw, crush it, add 10 kg of 2% NaOH solution, and treat it at 120℃ for 20 min to obtain a material with a solid content of 50% (w / w).

[0054] The above materials were added to the reactor, and cellulase solution was added to the reactor at a ratio of 10 U / g relative to wheat straw. The temperature was controlled at 50℃ and the pH at 6. Saccharification was carried out for 2 hours to obtain the saccharification solution.

[0055] Fermentation medium containing 10 g / L peptone, 10 g / L yeast extract, 2 g / L diammonium citrate, 5 g / L anhydrous sodium acetate, 2 g / L dipotassium hydrogen phosphate, 0.3 g / L magnesium sulfate heptahydrate, and 0.25 g / L manganese sulfate monohydrate was added to the fermentation medium. A seed culture of *Pediococcus lactis* Cath501 was inoculated into the fermentation medium at an inoculum size of 10%. Fermentation was carried out at a temperature of 52.5°C and a pH of 6.5. Further saccharification was achieved during fermentation using cellulase, thus realizing simultaneous saccharification and fermentation.

[0056] During fermentation, the following ingredients were added: 0.06 g / L vitamin B1, 0.17 g / L vitamin B12, 0.03 g / L vitamin D2, 0.2 g / L methionine, 0.05 g / L leucine, 0.1 g / L phenylalanine, and 1.8 g / L ammonium sulfate. Fermentation was stopped when the glucose content in the fermentation broth was almost zero.

[0057] Example 4

[0058] Take 2 kg of wheat straw, crush it, add 10 kg of 2% NaOH solution, and treat it at 120℃ for 20 min to obtain a material with a solid content of 50% (w / w).

[0059] The above materials were added to the reactor, and cellulase solution was added to the reactor at a ratio of 10 U / g relative to wheat straw. The temperature was controlled at 50℃, the pH at 5.5, and the saccharification was carried out for 3.5 hours to obtain the saccharification solution.

[0060] Fermentation medium containing 10 g / L peptone, 10 g / L yeast extract, 2 g / L diammonium citrate, 5 g / L anhydrous sodium acetate, 2 g / L dipotassium hydrogen phosphate, 0.3 g / L magnesium sulfate heptahydrate, and 0.25 g / L manganese sulfate monohydrate is added to a reactor containing saccharification solution. A seed culture of *Pediococcus lactis* Cath501 is inoculated into the fermentation medium at an inoculum size of 10%. Fermentation is carried out at 50°C and pH 5.5. Further saccharification is achieved during fermentation using cellulase, thus realizing simultaneous saccharification and fermentation.

[0061] During fermentation, the following ingredients were added: 0.08 g / L vitamin B1, 0.2 g / L vitamin B12, 0.03 g / L vitamin D2, 0.1 g / L methionine, 0.05 g / L leucine, 0.2 g / L phenylalanine, and 2 g / L ammonium sulfate. Fermentation was stopped when the glucose content in the fermentation broth was almost zero.

[0062] Example 5

[0063] Take 2 kg of wheat straw, crush it, add 10 kg of 2% NaOH solution, and treat it at 120℃ for 20 min to obtain a material with a solid content of 50% (w / w).

[0064] The above materials were added to the reactor, and cellulase solution was added to the reactor at a ratio of 10 U / g relative to wheat straw. The temperature was controlled at 50℃ and the pH at 6. Saccharification was carried out for 4 hours to obtain the saccharification solution.

[0065] Fermentation medium containing 10 g / L peptone, 10 g / L yeast extract, 2 g / L diammonium citrate, 5 g / L anhydrous sodium acetate, 2 g / L dipotassium hydrogen phosphate, 0.3 g / L magnesium sulfate heptahydrate, and 0.25 g / L manganese sulfate monohydrate is added to a reactor containing saccharification solution. A seed culture of *Pediococcus lactis* Cath501 is inoculated into the fermentation medium at an inoculum size of 10%. Fermentation is carried out at 50°C and pH 5.5. Further saccharification is achieved during fermentation using cellulase, thus realizing simultaneous saccharification and fermentation.

[0066] During fermentation, the following ingredients were added: 0.04 g / L of vitamin B1, 0.17 g / L of vitamin B12, 0.05 g / L of vitamin D2, 0.1 g / L of methionine, 0.1 g / L of leucine, 0.1 g / L of phenylalanine, and 2 g / L of ammonium sulfate. Fermentation was stopped when the glucose content in the fermentation broth was almost zero.

[0067] Example 6

[0068] Take 2 kg of wheat straw, crush it, add 10 kg of 2% NaOH solution, and treat it at 100℃ for 10 min to obtain a material with a solid content of 35% (w / w).

[0069] The above materials were added to the reactor, and cellulase solution was added to the reactor at a ratio of 10 U / g relative to wheat straw. The temperature was controlled at 50℃ and the pH at 6. Saccharification was carried out for 3 hours to obtain the saccharification solution.

[0070] Fermentation medium containing 10 g / L peptone, 10 g / L yeast extract, 2 g / L diammonium citrate, 5 g / L anhydrous sodium acetate, 2 g / L dipotassium hydrogen phosphate, 0.3 g / L magnesium sulfate heptahydrate, and 0.25 g / L manganese sulfate monohydrate is added to a reactor containing saccharification solution. A seed culture of *Pediococcus lactis* Cath501 is inoculated into the fermentation medium at an inoculum size of 10%. Fermentation is carried out at 50°C and pH 5.5. Further saccharification is achieved during fermentation using cellulase, thus realizing simultaneous saccharification and fermentation.

[0071] During fermentation, the following ingredients were added: 0.04 g / L of vitamin B1, 0.17 g / L of vitamin B12, 0.05 g / L of vitamin D2, 0.1 g / L of methionine, 0.1 g / L of leucine, 0.1 g / L of phenylalanine, and 2 g / L of ammonium sulfate. Fermentation was stopped when the glucose content in the fermentation broth was almost zero.

[0072] Comparative Example 1

[0073] The same process as in Example 1 was used, except that vitamin B1 and vitamin B12 were not added, and all other conditions were the same as in Example 1.

[0074] Comparative Example 2

[0075] The same process as in Example 1 was used, except that vitamin D2 was not added, and all other conditions were the same as in Example 1.

[0076] Comparative Example 3

[0077] The same process as in Example 1 was used, except that methionine, leucine and phenylalanine were not added, and other conditions were the same as in Example 1.

[0078] Comparative Example 4

[0079] The same process as in Example 1 was used, except that ammonium sulfate was not added, and other conditions were the same as in Example 1.

[0080] Comparative Example 5

[0081] The same process as in Example 1 was used, except that the fermentation temperature was 42°C, and the other conditions were the same as in Example 1.

[0082] Table 1

[0083]

[0084]

[0085] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A process for producing lactic acid, characterized in that, Includes the following steps: (1) The treated lignocellulose raw material is saccharified to obtain a saccharification solution; (2) Lactic acid is produced by fermentation in a fermentation medium containing saccharification solution, and growth factors are added during the fermentation process. The growth factors are B vitamins, D vitamins, amino acids and ammonium sulfate. The amino acids are methionine, leucine and phenylalanine. The B vitamins are vitamin B1 and vitamin B12. The D vitamin is vitamin D2. The addition amount of vitamin B1 is 0.03–0.08 g / L, the addition amount of vitamin B12 is 0.15–0.2 g / L, the addition amount of vitamin D derivatives is 0.03–0.2 g / L, the addition amount of ammonium sulfate is 1–2 g / L, the addition amount of methionine is 0.1–0.3 g / L, the addition amount of leucine is 0.05–0.1 g / L, and the addition amount of phenylalanine is 0.1–0.2 g / L. During the fermentation process, the temperature is controlled at 45–55°C and the pH at 4.5–6.

5. The fermentation medium also contains the following components: 5-15 g / L peptone, 5-15 g / L yeast extract, 1-3 g / L diammonium citrate, 1-10 g / L anhydrous sodium acetate, 1-3 g / L dipotassium hydrogen phosphate, 0.1-0.3 g / L magnesium sulfate heptahydrate, and 0.1-0.5 g / L manganese sulfate monohydrate. The fermentation medium is inoculated with seed culture at a rate of 5-25% (by volume). The bacterial cells in the seed culture are selected from Pediococcus lactis, specifically Cath501, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20211364.

2. The process according to claim 1, characterized in that, The amount of vitamin D added is 0.1–0.2 g / L; and / or The amount of ammonium sulfate added is 1.5–2 g / L.

3. The process according to claim 1, characterized in that, The solid content of the processed lignocellulose raw material is 30%–70%; and / or The lignocellulose raw materials include at least one of straw, rice straw, rice husk, bagasse, wood, and sawdust.

4. The process according to claim 1, characterized in that, The solid content of the processed lignocellulose raw material is 40% to 50%.

5. The process according to claim 1, characterized in that, Saccharification is performed using cellulase, with the amount of cellulase solution relative to the lignocellulose raw material being 10–50 U / g; and / or During saccharification, the temperature is controlled at 40–60℃, and / or the pH value is 4.5–6.

5.

6. The process according to claim 5, characterized in that, The amount of cellulase solution relative to the lignocellulose raw material is 10–20 U / g; and / or During saccharification, the temperature is controlled at 45–55°C, and / or the pH value is 5–6.

7. The process according to claim 1, characterized in that, During fermentation, the temperature is controlled at 45–52℃; and / or the pH is controlled at 5–6.

8. The process according to claim 1, characterized in that, During fermentation, the temperature is controlled at 48–51℃.

9. The process according to claim 1, characterized in that, The inoculation amount of the seed solution is 10-20%.

10. The process according to any one of claims 1 to 9, characterized in that, The yield of lactic acid obtained was above 1.5 g / L / h.

11. The process according to claim 10, characterized in that, The yield of lactic acid obtained was above 1.7 g / L / h.

12. The process according to claim 10, characterized in that, The yield of lactic acid obtained was above 1.9 g / L / h.

13. The process according to claim 10, characterized in that, The yield of lactic acid obtained was above 2.0 g / L / h.

14. The process according to claim 10, characterized in that, The yield of lactic acid obtained was above 2.3 g / L / h.

15. A method for preparing lactic acid, characterized in that, include: (1) Lactic acid fermentation broth is prepared by the process described in any one of claims 1 to 14; (2) Lactic acid is obtained by extracting and refining the obtained lactic acid fermentation broth.

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