A method for producing lactic acid by combining enzymes and bacteria to enhance the fermentation of agricultural waste

Abstract

The present 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. 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 compound of pig manure and molasses has a positive promoting effect on lactic acid fermentation production, and the use of cellulase and acid protease for enzymatic pretreatment can effectively increase the release of soluble organic matter in the fermentation raw materials, providing sufficient materials for subsequent lactic acid production. The control of the organic load during the fermentation process significantly increases the concentration of organic matter in the fermentation system and controls it within a range suitable for lactic acid fermentation production, thereby effectively improving the fermentation efficiency and lactic acid yield. The method has the advantages of simple operation, easy implementation, stability and high efficiency, and has good application value in the production of lactic acid by anaerobic fermentation of agricultural waste.

Description

Technical Field

[0001] The present 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 biochemical. It can serve as a precursor for biodegradable plastics and has significant applications in the materials field, as well as in food, pharmaceuticals, cosmetics, and other fields. Currently, the fermentation process used to produce lactic acid from agricultural waste is primarily batch fermentation. While simple to operate and easy to control, it is only suitable for small-scale production. Semi-continuous fermentation, by continuously adding material to the reactor while removing some of the acidified liquid, ensures continuous fermentation. It has high production efficiency and is more suitable for large-scale production.

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

[0004] The present 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 to perform semi-continuous fermentation;

[0007] Wherein, the fermentation raw materials include pig manure and molasses in a mass ratio of 1: (1-6);

[0008] The enzymes used in the enzymolysis include cellulase and acid protease;

[0009] 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 5-8g VS / L / d.

[0010] The present invention has found that compared with using other crop waste feedstocks (such as pig manure and apple waste), using a mixture of pig manure and molasses in a mass ratio of 1:(1-6) as the fermentation feedstock, followed by enzymatic pretreatment with cellulase and acid protease, and continuous daily feeding and unloading starting on day 8-12 of fermentation while controlling the organic load to 5-8g VS / L / day, significantly increases lactic acid production. The pig manure and molasses work together to provide nutrients necessary for lactic acid bacteria growth and more efficiently convert the fermentation feedstock into the target product, lactic acid. The 1:(1-6) mass ratio of pig manure to molasses performed particularly well in semi-continuous fermentation.

[0011] In the fermentation raw materials mentioned above, the mass ratio of pig manure to molasses is 1:(1.5-6).

[0012] Preferably, the volatile solids content of the fermentation feedstock is 4%-20% VS, more preferably 6%-7% VS.

[0013] Preferably, the initial concentration of the fermentation is 2%-8% VS, more preferably 5%-6% VS.

[0014] In some embodiments of the present invention, water is added after inoculation of lactic acid bacteria to adjust the initial concentration of fermentation to 2%-8% VS.

[0015] Before mixing pig manure with molasses, impurities such as stones are first removed from the pig manure.

[0016] 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 5-8g VS / L / d, and the hydraulic retention time is controlled to be 8-12 days.

[0017] In the above continuous feeding and discharging, the feed is the enzymatic fermentation raw material, and the discharge is the fermentation liquid. The volume of the feed and discharge is the same every day.

[0018] In the above method, the fermentation tank is continuously fed with fermentation raw materials that have been 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 5-8g VS / L / d.

[0019] 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.

[0020] With respect to 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.

[0021] Preferably, the enzymatic hydrolysis is carried out at 45-55° C. for 3-12 hours. Preferably, the enzymatic hydrolysis time is 5-7 hours.

[0022] 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.

[0023] 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.

[0024] Preferably, the inoculation amount of the lactic acid bacteria is 5%-10% of the effective volume of the fermentation tank.

[0025] 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.

[0026] Preferably, the initial pH of the fermentation is 6.3-6.7, the fermentation temperature is controlled at 30-55° C. and the fermentation time is preferably 18-25 days.

[0027] Preferably, the OD of the bacterial solution of the inoculated lactic acid bacteria is 600 It is 1.4-1.8.

[0028] In some embodiments of the present invention, the method for producing lactic acid by fermentation of agricultural waste comprises the following steps:

[0029] (1) Pig manure and molasses are mixed in a mass ratio of 1:(1.5-6), and then cellulase and acid protease are added for enzymatic hydrolysis at a temperature of 45-55°C for 3-12 hours to obtain an enzymatic fermentation raw material;

[0030] (2) The enzymatic fermentation raw materials of step (1) are placed in a continuous stirred tank reactor (CSTR), and 5% to 10% of the effective volume of the fermentation tank is inoculated with activated lactic acid bacteria liquid (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;

[0031] (3) Adjust the initial fermentation concentration to 2%-8% VS, the initial pH to 6.3-6.7, and the fermentation temperature to 30-55°C. After the fermentation stabilizes for 8-10 days, operate the system in a continuous feed and discharge mode every day, control the hydraulic retention time to 8-12 days, and control the organic load to 5-8g VS / L / d to achieve fermentation with a medium solid concentration.

[0032] 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, increase 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 of 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 fermentation efficiency and lactic acid production, while ensuring the stability of the fermentation process, and ultimately 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

[0033] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0034] 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 for fermentation inoculation is as follows: before 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 liquid is regularly tested. 600 Value, when OD 600When the value reaches 1.4~1.8, lactic acid activation is completed, and the obtained lactic acid bacteria liquid is used as the inoculum liquid for subsequent fermentation to produce lactic acid.

[0035] Example 1

[0036] This embodiment provides a method for producing lactic acid by fermenting agricultural waste, which comprises the following steps:

[0037] (1) Pig manure and molasses were mixed in a mass ratio of 2:3 (volatile solids content was 6% VS), and then cellulase and acid protease were added for enzymatic hydrolysis. The addition amount of cellulase was 12.5U / g VS fermentation raw material, and the addition amount of acid protease was 37.5U / g VS fermentation raw material. The enzymatic hydrolysis was carried out at 55℃ for 6h to obtain the enzymatic fermentation raw material.

[0038] (2) The enzymatic fermentation raw material of step (1) is placed in a continuous stirred tank reactor (CSTR), and the lactic acid bacteria liquid is inoculated at an inoculum amount of 6% of the effective volume of the fermenter. Water is added to adjust the concentration of the material in the fermenter to 5% VS;

[0039] (3) The initial pH of the fermentation was adjusted to about 6.5, and the fermentation temperature was controlled at 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 5 g VS / L / d.

[0040] After 20 days of fermentation, the average lactic acid concentration was 28.3 g / L.

[0041] Example 2

[0042] This embodiment provides a method for producing lactic acid by fermenting agricultural waste, which comprises the following steps:

[0043] (1) Pig manure and molasses were mixed in a mass ratio of 1:6 (volatile solids content was 7% VS), and then cellulase and acid protease were added for enzymatic hydrolysis. The addition amount of cellulase was 50 U / g VS fermentation raw material, and the addition amount of acid protease was 150 U / g VS fermentation raw material. The enzymatic hydrolysis was carried out at 55°C for 6 h to obtain the enzymatic fermentation raw material.

[0044] (2) The enzymatic fermentation raw material of step (1) is placed in a continuous stirred tank reactor (CSTR), and the lactic acid bacteria liquid is inoculated at an inoculum amount of 5% of the effective volume of the fermenter. Water is added to adjust the concentration of the material in the fermenter to 6% VS;

[0045] (3) The initial pH of the fermentation was adjusted to about 6.5, and the fermentation temperature was controlled at 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 6 g VS / L / d.

[0046] After 20 days of fermentation, the average lactic acid concentration was 25.3 g / L.

[0047] Comparative Example 1

[0048] This comparative example provides a method for producing lactic acid by fermenting agricultural waste, which comprises the following steps:

[0049] (1) Pig manure and molasses were mixed in a mass ratio of 6:1 (volatile solids content was 9% VS), and then cellulase and acid protease were added for enzymatic hydrolysis. The addition amount of cellulase was 100 U / g VS fermentation raw material, and the addition amount of acid protease was 300 U / g VS fermentation raw material. The enzymatic hydrolysis was carried out at 55°C for 6 h to obtain the enzymatic fermentation raw material.

[0050] (2) The enzymatic fermentation raw material of step (1) is placed in a continuous stirred tank reactor (CSTR), and the lactic acid bacteria liquid is inoculated at an inoculum amount of 10% of the effective volume of the fermenter. Water is added to adjust the concentration of the material in the fermenter to 8% VS;

[0051] (3) The initial pH of the fermentation was adjusted to about 6.5, and the fermentation temperature was controlled at 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.

[0052] After 20 days of fermentation, the average lactic acid concentration was 0.2 g / L.

[0053] Comparative Example 2

[0054] This comparative example provides a method for producing lactic acid by fermenting agricultural waste. The only difference between the method in Example 1 is 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.

[0055] Comparative Example 3

[0056] This comparative example provides a method for producing lactic acid using fermentation of agricultural waste. This method differs from the method in Example 2 only in that the pig manure and molasses were mixed directly before fermentation without enzymatic pretreatment. After 20 days of fermentation, the average lactic acid concentration obtained was 22.7 g / L.

[0057] Comparative Example 4

[0058] This comparative example provides a method for producing lactic acid using agricultural waste fermentation. This method differs from the method in Example 2 only in that the pig manure is removed from the fermentation feedstock, resulting in only molasses. After 20 days of fermentation, the average lactic acid concentration achieved was 13.8 g / L.

[0059] 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 various 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 a fermentation raw material to obtain an enzymatic fermentation raw material; inoculating lactic acid bacteria into the enzymatic fermentation raw material to perform semi-continuous anaerobic fermentation; Wherein, the fermentation raw materials are pig manure and molasses in a mass ratio of 1: (1-6); The enzymes used in the enzymatic hydrolysis include cellulase and acid protease; in the enzymatic hydrolysis, the amount of cellulase added is 10-50 U / g VS fermentation raw material, and the amount of acid protease added is 30-150 U / g VS fermentation raw material; the enzymatic hydrolysis is performed at 45-55° C. for 3-12 hours; The semi-continuous anaerobic 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, and controlling the hydraulic retention time to 8-12 days; in the continuous feeding and discharging, the feed is the enzymatic fermentation raw material, and the discharge is the fermentation liquid.

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 The lactic acid bacteria include at least one selected from the group consisting of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Bifidobacterium, Lactobacillus rhamnosus and Streptococcus thermophilus.

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

7. The method according to any one of claims 1 to 3 and 6, characterized in that The initial pH of the fermentation is 6.0-7.0, and the fermentation temperature is controlled at 30-55°C.