Method for producing lactic acid by fermenting agricultural wastes

Abstract

The invention relates to the technical field of resource utilization of agricultural wastes, in particular to a method for producing lactic acid by fermenting agricultural wastes. According to the invention, pig manure and molasses are used as fermentation raw materials, are subjected to enzymolysis pretreatment and then are subjected to semi-continuous fermentation, and the organic load is controlled in the fermentation process. Wherein the pig manure and the molasses are compounded to play a positive role in promoting lactic acid fermentation production, and the cellulase and the acid protease are used for enzymolysis pretreatment, so that the release of soluble organic matters in the fermentation raw materials can be effectively improved, and sufficient materials are provided for subsequent lactic acid production; the organic load control in the fermentation process significantly improves the organic matter concentration in the fermentation system and controls the organic matter concentration in a range suitable for lactic acid fermentation production, thereby effectively improving the fermentation efficiency and lactic acid yield. The method has the advantages of simplicity in operation, easiness in implementation, stability and high efficiency, and has better application value in production of lactic acid by anaerobic fermentation of agricultural wastes.

Description

Technical Field

[0001] The invention relates to the technical field of resource utilization of agricultural waste, and in particular to a method for producing lactic acid by fermenting agricultural waste. Background Art

[0002] Lactic acid is an important multi-purpose bio-based chemical. It can be used as a precursor for biodegradable plastics. It has important application value in the field of materials and has wide application value in the fields of food, pharmaceuticals, cosmetics, etc. At present, the fermentation process used to produce lactic acid from agricultural waste is mainly batch fermentation. Although it is simple to operate and easy to control, it is only suitable for small-scale production. Semi-continuous fermentation can ensure the continuous fermentation by continuously adding materials to the reactor and removing part of the acidified liquid. It has high production efficiency and is more suitable for large-scale production.

[0003] However, there is currently limited research on the semi-continuous fermentation of lactic acid using agricultural waste, and the lactic acid yield and production efficiency still need to be improved. Summary of the invention

[0004] The invention provides a method for producing lactic acid by fermenting agricultural waste.

[0005] Specifically, the present invention provides the following technical solutions.

[0006] The present invention provides a method for producing lactic acid by fermenting agricultural waste, the method comprising: performing enzymatic pretreatment on a fermentation raw material to obtain an enzymatic fermentation raw material; inoculating lactic acid bacteria into the enzymatic fermentation raw material for semi-continuous fermentation; Wherein, the fermentation raw materials include pig manure and molasses in a mass ratio of 1:(1-6); The enzymes used in the enzymolysis include cellulase and acid protease; The semi-continuous fermentation includes: starting from the 8th to 12th day of fermentation, continuously feeding and discharging materials every day and controlling the organic load to 5-8 g VS / L / d.

[0007] The present invention found that compared with other crop waste raw materials (such as pig manure and apple waste), a mixture of pig manure and molasses in a mass ratio of 1: (1-6) was used as a fermentation raw material, and fermentation was performed after enzymatic pretreatment with cellulase and acid protease, and continuous feeding and discharging of materials every day from 8-12 days of fermentation and controlling the organic load to 5-8 g VS / L / d, which can significantly increase the yield of lactic acid. Among the above fermentation raw materials, pig manure and molasses can work together to provide nutrients required for the growth of lactic acid bacteria, and can more efficiently convert the fermentation raw materials into the target product lactic acid. The pig manure and molasses in a mass ratio of 1: (1-6) as a fermentation raw material performed particularly well in semi-continuous fermentation.

[0008] In the fermentation raw materials mentioned above, the mass ratio of pig manure to molasses is 1:(1.5-6).

[0009] Preferably, the volatile solid content of the fermentation raw material is 4%-20% VS, more preferably 6%-7% VS.

[0010] Preferably, the initial concentration of the fermentation is 2%-8% VS, more preferably 5%-6% VS.

[0011] In some embodiments of the present invention, water is added after inoculating lactic acid bacteria to adjust the initial concentration of fermentation to 2%-8% VS.

[0012] Before mixing pig manure with molasses, impurities such as stones are first removed from the pig manure.

[0013] In the above method, starting from the 8th to 12th day of fermentation, the material is continuously fed and discharged every day and the organic load is controlled to be 5-8 g VS / L / d, and the hydraulic retention time is controlled to be 8-12 days.

[0014] In the above continuous feeding and discharging, the feeding is the enzymatic fermentation raw material, and the discharging is the fermentation liquid. The volume of the feeding and discharging is the same every day.

[0015] In the above method, the fermentation tank is continuously fed with fermentation raw materials pretreated by enzymatic hydrolysis every day, and the fermentation liquid of the same feed volume is discharged, the hydraulic retention time is controlled to 8-12 days, and the organic load is set to 5-8 g VS / L / d.

[0016] In the above method, in the enzymatic hydrolysis, the added amount of cellulase is 10-50 U / g VS fermentation raw material, and the added amount of acid protease is 30-150 U / g VS fermentation raw material.

[0017] For the fermentation raw material of the present invention, controlling the addition amount of cellulase and acid protease within the above range is more conducive to increasing the final yield of lactic acid.

[0018] Preferably, the enzymolysis is carried out at 45-55° C. for 3-12 hours. Preferably, the enzymolysis time is 5-7 hours.

[0019] In the above method, the lactic acid bacteria include at least one selected from Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Bifidobacterium, Lactobacillus rhamnosus and Streptococcus thermophilus.

[0020] In some embodiments of the present invention, the lactic acid bacteria include Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Bifidobacterium, Lactobacillus rhamnosus and Streptococcus thermophilus. Wherein, the ratio of the viable count of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Bifidobacterium, Lactobacillus rhamnosus and Streptococcus thermophilus is 1:1:1:1:1:1:1:1.

[0021] Preferably, the inoculation amount of the lactic acid bacteria is 5%-10% of the effective volume of the fermentation tank.

[0022] In the above method, the initial pH of the fermentation is 6.0-7.0, and the fermentation temperature is controlled at 30-55°C.

[0023] Preferably, the initial pH of the fermentation is 6.3-6.7, and the fermentation temperature is controlled at 30-55° C. The fermentation time is preferably 18-25 days.

[0024] Preferably, the OD of the inoculated lactic acid bacteria solution is 600 It is 1.4-1.8.

[0025] In some embodiments of the present invention, the method for producing lactic acid by fermentation of agricultural waste comprises the following steps: (1) mixing pig manure and molasses in a mass ratio of 1:(1.5-6), adding cellulase and acid protease for enzymatic hydrolysis at a temperature of 45-55°C for 3-12 hours, and obtaining an enzymatic fermentation raw material; (2) The enzymatic fermentation raw material of step (1) is placed in a continuous stirred tank reactor (CSTR), and 5% to 10% of the effective volume of the fermentation tank is inoculated with an activated lactic acid bacteria solution (OD 600 is 1.4-1.8); the lactic acid bacteria include one or more of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Bifidobacterium, Lactobacillus rhamnosus and Streptococcus thermophilus; (3) The initial fermentation concentration was adjusted to 2%-8% VS, the initial pH was adjusted to 6.3-6.7, and the fermentation temperature was controlled to 30-55°C. After the fermentation was stable for 8-10 days, a continuous feed and discharge operation mode was implemented every day, the hydraulic retention time was controlled to 8-12 days, and the organic load was controlled to 5-8 g VS / L / d to achieve fermentation with medium solid concentration.

[0026] The beneficial effects of the present invention include at least: the present invention uses pig manure and molasses as fermentation raw materials, performs semi-continuous fermentation after enzymatic pretreatment, and controls the organic load during the fermentation process. Among them, the compounding of pig manure and molasses in a specific ratio has a positive promoting effect on lactic acid fermentation production, and the use of cellulase and acid protease for enzymatic pretreatment can destroy the stubborn structure of lignocellulose and crude protein in the raw materials, effectively promote the hydrolysis of organic matter in the fermentation system, improve the release of soluble organic matter in pig manure and molasses, and provide sufficient materials for subsequent microbial metabolism to produce lactic acid; because the fermentation raw materials used contain rich nutrients and lactic acid bacteria inoculation, high-content lactic acid can be produced without sterilizing the substrate and adding exogenous nutrients; the control of organic load during the fermentation process significantly increases the organic matter concentration in the fermentation system and controls it within a range suitable for lactic acid fermentation production, thereby improving the fermentation efficiency and lactic acid production, while ensuring the stability of the fermentation process, and finally realizing the resource utilization of agricultural organic waste. The method has the advantages of simple operation, easy implementation, environmental friendliness, low cost, stability and high efficiency, and has good application value in the production of lactic acid by anaerobic fermentation of agricultural waste. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] The lactic acid bacteria used in the following examples were purchased from the China Anaerobic Microbial Culture Collection Center, including Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Bifidobacterium, Lactobacillus rhamnosus and Streptococcus thermophilus. Each strain was cultured to a viable count of ≥1×10 8 CFU / mL, and mixed in a ratio of 1:1:1:1:1:1:1 before use. The culture method of lactic acid bacteria used for fermentation inoculation is as follows: before lactic acid bacteria inoculation, the lactic acid bacteria are activated by MRS culture medium, the temperature is controlled at 42°C in a constant temperature incubator, and the OD of the lactic acid bacteria solution is regularly tested. 600 Value, when OD 600 When the value reaches 1.4~1.8, lactic acid activation is completed, and the obtained lactic acid bacteria liquid is used as the inoculum for subsequent fermentation to produce lactic acid.

[0029] Example 1 This embodiment provides a method for producing lactic acid by fermenting agricultural waste, which comprises the following steps: (1) Pig manure and molasses were mixed in a mass ratio of 2:3 (volatile solid content was 6% VS), and then cellulase and acid protease were added for enzymatic hydrolysis. The amount of cellulase added was 12.5 U / g VS fermentation raw material, and the amount of acid protease added was 37.5 U / g VS fermentation raw material. The synergistic enzymatic hydrolysis was carried out at 55°C for 6 hours to obtain an enzymatic fermentation raw material. (2) placing the enzymatic fermentation raw material of step (1) in a continuous stirred tank reactor (CSTR), inoculating the lactic acid bacteria solution, the inoculation amount being 6% of the effective volume of the fermentation tank, and adding water to adjust the concentration of the material in the fermentation tank to 5% VS; (3) The initial pH of the fermentation was adjusted to about 6.5, and the fermentation temperature was controlled to 35°C. After the fermentation was stable for 10 days, a continuous feed and discharge operation mode was implemented (the feed was the enzymatic fermentation raw material in the above step (1), and the discharge was the fermentation liquid) with the hydraulic retention time controlled to 10 days and the organic load controlled to 5 g VS / L / d.

[0030] After 20 days of fermentation, the average lactic acid concentration was 28.3 g / L.

[0031] Example 2 This embodiment provides a method for producing lactic acid by fermenting agricultural waste, which comprises the following steps: (1) Pig manure and molasses were mixed in a mass ratio of 1:6 (volatile solid content was 7% VS), and then cellulase and acid protease were added for enzymatic hydrolysis. The amount of cellulase added was 50 U / g VS fermentation raw material, and the amount of acid protease added was 150 U / g VS fermentation raw material. The synergistic enzymatic hydrolysis was carried out at 55°C for 6 hours to obtain an enzymatic fermentation raw material. (2) placing the enzymatic fermentation raw material of step (1) in a continuous stirred tank reactor (CSTR), inoculating the lactic acid bacteria solution, the inoculation amount being 5% of the effective volume of the fermentation tank, and adding water to adjust the concentration of the material in the fermentation tank to 6% VS; (3) The initial pH of the fermentation was adjusted to about 6.5, and the fermentation temperature was controlled to 35°C. After the fermentation was stable for 10 days, a continuous feed and discharge operation mode was implemented (the feed was the enzymatic fermentation raw material in the above step (1), and the discharge was the fermentation liquid) with the hydraulic retention time controlled to 10 days and the organic load controlled to 6 g VS / L / d.

[0032] After 20 days of fermentation, the average lactic acid concentration was 25.3 g / L.

[0033] Comparative Example 1 This comparative example provides a method for producing lactic acid by fermenting agricultural waste, which comprises the following steps: (1) Pig manure and molasses were mixed in a mass ratio of 6:1 (volatile solid content was 9% VS), and then cellulase and acid protease were added for enzymatic hydrolysis. The amount of cellulase added was 100 U / g VS fermentation raw material, and the amount of acid protease added was 300 U / g VS fermentation raw material. The synergistic enzymatic hydrolysis was carried out at 55°C for 6 hours to obtain an enzymatic fermentation raw material. (2) placing the enzymatic fermentation raw material of step (1) in a continuous stirred tank reactor (CSTR), inoculating the lactic acid bacteria solution, the inoculation amount being 10% of the effective volume of the fermentation tank, and adding water to adjust the concentration of the material in the fermentation tank to 8% VS; (3) The initial pH of the fermentation was adjusted to about 6.5, and the fermentation temperature was controlled to 35°C. After the fermentation was stable for 10 days, a continuous feed and discharge operation mode was implemented (the feed was the enzymatic fermentation raw material in the above step (1) and the discharge was the fermentation liquid). The hydraulic retention time was controlled to 10 days, and the organic load was controlled to 8 g VS / L / d.

[0034] After 20 days of fermentation, the average lactic acid concentration was 0.2 g / L.

[0035] Comparative Example 2 This comparative example provides a method for producing lactic acid by fermenting agricultural waste, which differs from the method in Example 1 only in that the fermentation raw materials are pig manure and apple waste in a mass ratio of 2:3; after 20 days of fermentation, the average lactic acid concentration obtained is 18.6 g / L.

[0036] Comparative Example 3 This comparative example provides a method for producing lactic acid by fermentation of agricultural waste, which differs from the method of Example 2 only in that the pig manure and molasses are not pre-treated by enzymatic hydrolysis and are directly mixed for fermentation. After 20 days of fermentation, the average lactic acid concentration obtained is 22.7 g / L.

[0037] Comparative Example 4 This comparative example provides a method for producing lactic acid by fermenting agricultural waste, which differs from the method in Example 2 only in that the pig manure in the fermentation raw material is removed, that is, the fermentation raw material is only molasses. After 20 days of fermentation, the average lactic acid concentration obtained is 13.8 g / L.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for producing lactic acid by fermenting agricultural waste, characterized in that: The method comprises: performing enzymatic pretreatment on the fermentation raw material to obtain an enzymatic fermentation raw material; inoculating lactic acid bacteria into the enzymatic fermentation raw material to perform semi-continuous fermentation; Wherein, the fermentation raw materials include pig manure and molasses in a mass ratio of 1:(1-6); The enzymes used in the enzymolysis include cellulase and acid protease; The semi-continuous fermentation includes: starting from the 8th to 12th day of fermentation, continuously feeding and discharging materials every day and controlling the organic load to 5-8 gVS / L / d.

2. The method according to claim 1, characterized in that: In the fermentation raw materials, the mass ratio of pig manure to molasses is 1:(1.5-6).

3. The method according to claim 1, characterized in that The volatile solid content of the fermentation raw material is 4%-20% VS.

4. The method according to any one of claims 1 to 3, characterized in that The initial concentration of the fermentation is 2%-8% VS.

5. The method according to any one of claims 1 to 3, characterized in that: Starting from the 8th to 12th day of fermentation, feed and discharge continuously every day and control the organic load to 5-8 g VS / L / d, and the hydraulic retention time to 8-12 days; And / or, in the continuous feeding and discharging, the feed is the enzymatic fermentation raw material, and the discharging is the fermentation liquid.

6. The method according to any one of claims 1 to 3, characterized in that: In the enzymolysis, the added amount of cellulase is 10-50 U / g VS fermentation raw material, and the added amount of acid protease is 30-150 U / g VS fermentation raw material.

7. The method according to claim 6, characterized in that The enzymolysis is carried out at 45-55° C. for 3-12 hours.

8. The method according to any one of claims 1 to 3 and 7, characterized in that: The lactic acid bacteria include at least one selected from Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Bifidobacterium, Lactobacillus rhamnosus and Streptococcus thermophilus.

9. The method according to claim 8, characterized in that The inoculation amount of the lactic acid bacteria is 5%-10% of the effective volume of the fermentation tank.

10. The method according to any one of claims 1 to 3, 7 and 9, characterized in that: The initial pH of the fermentation is 6.0-7.0, and the fermentation temperature is controlled at 30-55°C.