Citric acid fermentation process

Abstract

The invention discloses a citric acid fermentation process, and belongs to the technical field of citric acid fermentation, and the citric acid fermentation process comprises the following steps: pretreating corn straw to obtain a pretreatment solution, then adjusting the pH to 5, adding a mixture, reacting at 25-30 DEG C for 2-4 days to obtain a saccharified solution, and carrying out filter pressing on the saccharified solution to obtain the citric acid. Separating out saccharification clear liquid and enzymolysis residues; adding water into the saccharified clear liquid to dilute the saccharified clear liquid until the total sugar content is 10wt% to obtain a seed culture medium, putting the seed culture medium into a seed tank, sterilizing, cooling to room temperature, inoculating an aspergillus niger strain, and stopping culturing when the acidity is greater than or equal to 1g / 100mL to obtain a seed liquid; preparing a fermentation culture medium, sterilizing, adding the fermentation culture medium into a fermentation tank, and then adding the seed liquid obtained in the step A2 for fermentation to obtain the citric acid fermentation liquid. The process method is high in citric acid conversion rate and suitable for large-scale production of citric acid.

Description

Technical Field

[0001] The invention belongs to the technical field of citric acid fermentation, and in particular relates to a citric acid fermentation process. Background Art

[0002] Citric acid is one of the main metabolites of organisms. It is widely used in food, medicine, chemical industry and other fields. At present, Aspergillus niger is mainly used commercially to ferment citric acid with starchy grains as substrates. The conversion rate is generally higher than 70%, and better Aspergillus niger can achieve a conversion rate of more than 95%. However, traditional citric acid fermentation requires a large amount of grain as a substrate. Therefore, in order to save food and reduce costs, it is considered to use agricultural residues or industrial waste as raw materials to ferment and produce citric acid. Lignocellulose is rich in available cellulose and hemicellulose components and is the first choice for citric acid production substrates.

[0003] Industrial bioconversion of lignocellulose requires the application of high temperature and acidic or alkaline pretreatment conditions to degrade lignin, reduce crystallinity, increase pore capacity, and dissolve cellulose and hemicellulose into hydrolyzable polysaccharides. This process is expensive and inefficient. Secondly, pretreatment will generate inhibitors, including organic acid compounds such as formic acid and acetic acid, furan compounds such as furfural and hydroxymethylfurfural, phenolic compounds such as vanillin and p-hydroxybenzaldehyde, etc. These inhibitors will inhibit the subsequent hydrolysis process and have an inhibitory effect on the growth and production of subsequent fermentation bacteria. Therefore, it is necessary to degrade these inhibitors by detoxification. There are many ways to detoxify, such as water washing, over-alkalization treatment, vacuum distillation, ion adsorption, activated carbon decolorization, etc. However, these methods have many disadvantages. For example, a large amount of fresh water is required, wastewater is generated, lignocellulose particles and fermentable sugars are lost, and inhibitor detoxification is incomplete. For example, water washing is one of the common methods of detoxification, which will lose a large amount of pretreated lignocellulose particles, thereby reducing fermentation yield. Moreover, this will also lead to the generation of a large amount of wastewater during downstream treatment, wasting a large amount of water resources. Therefore, a more effective detoxification method is needed to overcome the problems caused by the above detoxification methods and increase the efficiency of detoxification.

[0004] Patent publication number CN104805136B discloses a method for producing citric acid using lignocellulose raw materials. The patent mixes dried corn stalks with a 5% dilute sulfuric acid solution at a mass ratio of 2:1, and then pre-treats the mixture at 190°C and 1.2MPa for 3 minutes. After pre-treatment, the pH of the material is adjusted to 5.0, and the strain Amorphotheca resinae used for biological detoxification is added. ZN1, with a preservation number of CGMCC7452, was solid-state detoxified at 25°C for 10 days; the detoxified material was added to a fermentation tank, the solid content was adjusted to 25%, the amount of cellulase was 15 FPU / g dry solid, and saccharification was performed at 50°C and pH 4.8 for 48 hours; the supernatant obtained by centrifugation was the straw hydrolysate; 2.5 g / L ammonium chloride, 2.5 g / L potassium dihydrogen phosphate, 0.25 g / L magnesium sulfate heptahydrate, 0.25 mg / L copper sulfate pentahydrate, 1.1 mg / L zinc sulfate heptahydrate, 6.5 mg / L ferrous sulfate heptahydrate, and 3.6 mg / L manganese chloride were added to the hydrolysate, and the cultured Aspergillus niger was inoculated at a 10% inoculation amount. nigerM288, fermented at 33°C and 200rpm for 168 hours; the initial sugar concentration in the hydrolyzate was 89.89 g / L, the final citric acid concentration was 58.04 g / L, and the citric acid yield was 64.56 g / g glucose. However, the conversion rate of citric acid in this patent is still poor. Summary of the invention

[0005] The object of the present invention is to provide a citric acid fermentation process to solve the problem of poor conversion rate of citric acid.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A citric acid fermentation process comprises the following steps:

[0008] Step A1: pretreating corn stalks to obtain a pretreatment liquid, then adjusting the pH to 5, adding the mixture, reacting at 25-30° C. for 2-4 days to obtain a saccharification liquid, filtering the saccharification liquid to separate the saccharification clear liquid and the enzymatic hydrolysis residue; the mixture consists of lignin peroxidase, manganese peroxidase, swollenin, and cellulase.

[0009] The volume ratio of the pretreatment liquid to the lignin peroxidase, manganese peroxidase, swollenin and cellulase is 15-20: 0.3: 0.15-0.2: 0.15-0.2: 0.3; and the solid content of the enzymolysis residue is 49-51% by weight.

[0010] The main components of corn straw are cellulose, hemicellulose and lignin. Cellulose is a polymer compound composed of glucose linked by β-1,4-glycosidic bonds, and hemicellulose is an amorphous substance composed of different pentoses. Lignin is an amorphous polymer composed of phenylpropane as the basic structural unit. It has a dense structure and acts as a skeleton structure surrounding the outside of cellulose and hemicellulose. The hydrolysis and conversion of cellulose and hemicellulose is the key to the efficient utilization of corn straw. Pretreatment can degrade the lignin in corn straw to a certain extent, while better retaining the cellulose and hemicellulose components, thereby improving the comprehensive utilization efficiency of corn straw.

[0011] Lignin peroxidase and manganese peroxidase can catalyze the breakage of phenolic and non-phenolic structures in the lignin of pretreated corn stalks; swelling agent can act on cellulose and hemicellulose. These enzymes act together on the pretreated corn stalks to make its structure looser; the three enzymes work synergistically to make it easier for cellulase to contact the target substrate, thereby improving the saccharification efficiency and the fermentation yield of citric acid.

[0012] Manganese peroxidase can also degrade substances such as furfural and 5-hydroxymethylfurfural in corn straw, thereby reducing the inhibitory effect of the above substances on subsequent fermentation bacteria, promoting the growth of Aspergillus niger, and increasing the citric acid conversion rate. After degrading corn straw, manganese peroxidase will leave some divalent manganese ions in the saccharification liquid. Divalent manganese ions have a significant activating effect on the key enzyme for citrate production - phosphofructokinase, which can promote the subsequent production of citric acid.

[0013] Step A2: diluting the saccharification clear liquid with water to a total sugar content of 10% by weight to obtain a seed culture medium, placing the seed culture medium into a seed tank, sterilizing it, cooling it to room temperature, and then inoculating Aspergillus niger species, and stopping the culture when the acidity is ≥1 g / 100 mL to obtain a seed liquid;

[0014] Step A3: prepare a fermentation medium, sterilize it, add the fermentation medium into a fermentation tank, and then add the seed liquid obtained in step A2 for fermentation to obtain a citric acid fermentation liquid.

[0015] Furthermore, in step A1, the specific steps of pretreatment are: mixing corn stalks with mixed solution A, and pretreating at 130°C for 40-80 minutes; wherein the usage ratio of corn stalks to mixed solution A is 1kg:15-20L, and mixed solution A consists of 2% calcium carbonate solution and 1% hydrogen peroxide solution.

[0016] Furthermore, in step A2, the volume percentage of the Aspergillus niger inoculation amount is 8-12%.

[0017] Furthermore, in step A2, the conditions for seed tank culture are: seed culture is carried out at 35°C and an initial pH value of 5, the tank pressure is controlled at 0.04-0.06MPa, the ventilation volume is controlled at 0.3-0.5 volume: (volume·min), and the stirring speed is controlled at 250-350rpm; after 10-15h of seed culture, the tank pressure is controlled at 0.06-0.08MPa, the ventilation volume is controlled at 0.6-0.8 volume: (volume·min), and the stirring speed is controlled at 300-450rpm.

[0018] Furthermore, in step A3, the fermentation medium includes saccharification supernatant, saccharification liquid, and urea, and the mass ratio of the saccharification supernatant, saccharification liquid, and urea is 6:1.5:0.6.

[0019] Furthermore, in step A3, the culture conditions of the fermentation tank fermentation are: initial pH 4-5, temperature 30-40°C, ventilation volume 0.3-0.8 volume: (volume.min), rotation speed 350-450rpm, and time 3-4 days.

[0020] Beneficial effects of the present invention:

[0021] 1. The present invention uses lignin as raw material for citric acid fermentation, which effectively reduces the production cost and can meet the needs of large-scale production.

[0022] 2. The present invention can obtain a saccharification liquid with a good saccharification effect by controlling the amount of the mixture added, which is beneficial to the subsequent fermentation of citric acid. DETAILED DESCRIPTION

[0023] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only 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.

[0024] In the present invention, the Aspergillus niger species is Aspergillus niger Co827; the concentration of total sugar is determined according to the method of GB / T5009.8-2003.

[0025] Example 1

[0026] A citric acid fermentation process comprises the following steps:

[0027] Step A1: 1 kg of corn stalks is mixed with 15 L of a mixture consisting of 2% calcium carbonate solution and 1% hydrogen peroxide solution, and pretreated at 130° C. for 40 min to obtain a pretreated solution, and then the pH is adjusted to 5, and a mixture consisting of 0.3 L of lignin peroxidase, 0.15 L of manganese peroxidase, 0.15 L of swollenin, and 0.3 L of cellulase is added, and the mixture is reacted at 25° C. for 2 days to obtain a saccharified liquid, and the saccharified liquid is filtered to separate the saccharified liquid and the enzymolysis residue, wherein the solid content of the enzymolysis residue is 49% by weight;

[0028] Step A2: dilute the saccharification clear liquid with water to a total sugar content of 10% by weight to obtain a seed culture medium, put the seed culture medium into a seed tank, sterilize, cool to room temperature, and then inoculate Aspergillus niger, with the volume percentage of Aspergillus niger inoculation being 8%. Carry out seed culture at 35°C and an initial pH value of 5, control the tank pressure at 0.04MPa, the ventilation volume at 0.3 volume: (volume·min), and the stirring speed at 250rpm; after 10h of seed culture, control the tank pressure at 0.06MPa, the ventilation volume at 0.6 volume: (volume·min), and the stirring speed at 300rpm, stop the culture when the acidity is ≥1g / 100mL, and obtain a seed solution;

[0029] Step A3: preparing a fermentation medium, the fermentation medium including 120 kg of saccharification liquid, 30 kg of saccharification liquid, and 12 kg of urea, sterilizing the fermentation medium, adding the fermentation medium into a fermentation tank, and then adding the seed liquid obtained in step A2, and fermenting for 3 days under the conditions of an initial pH of 4-5, a temperature of 30° C., an aeration volume of 0.3 volume: (volume.min), and a rotation speed of 350 rpm to obtain a citric acid fermentation liquid.

[0030] Example 2

[0031] A citric acid fermentation process comprises the following steps:

[0032] Step A1: 1 kg of corn stalks is mixed with 20 L of a mixture consisting of 2% calcium carbonate solution and 1% hydrogen peroxide solution, and pretreated at 130° C. for 80 min to obtain a pretreated solution, and then the pH is adjusted to 5, and a mixture consisting of 0.3 L of lignin peroxidase, 0.18 L of manganese peroxidase, 0.18 L of swollenin, and 0.3 L of cellulase is added, and the mixture is reacted at 30° C. for 3 days to obtain a saccharified liquid, and the saccharified liquid is filtered to separate the saccharified liquid and the enzymolysis residue, wherein the solid content of the enzymolysis residue is 51% by weight;

[0033] Step A2: dilute the saccharification clear liquid with water to a total sugar content of 10% by weight to obtain a seed culture medium, put the seed culture medium into a seed tank, sterilize, cool to room temperature, and then inoculate Aspergillus niger, with the volume percentage of Aspergillus niger inoculation being 12%. Carry out seed culture at 35°C and an initial pH value of 5, control the tank pressure at 0.06MPa, the ventilation volume at 0.5 volume: (volume·min), and the stirring speed at 350rpm; after 15h of seed culture, control the tank pressure at 0.08MPa, the ventilation volume at 0.8 volume: (volume·min), and the stirring speed at 450rpm, stop the culture when the acidity is ≥1g / 100mL, and obtain a seed solution;

[0034] Step A3: preparing a fermentation medium, the fermentation medium including 120 kg of saccharification clear liquid, 30 kg of saccharification liquid, and 12 kg of urea, sterilizing, adding the fermentation medium into a fermentation tank, and then adding the seed liquid obtained in step A2, fermenting for 4 days under the conditions of an initial pH of 4-5, a temperature of 40° C., a ventilation volume of 0.8 volume: (volume.min), and a rotation speed of 450 rpm to obtain a citric acid fermentation liquid.

[0035] Example 3

[0036] A citric acid fermentation process comprises the following steps:

[0037] Step A1: 1 kg of corn stalks is mixed with 18 L of a mixture consisting of 2% calcium carbonate solution and 1% hydrogen peroxide solution, and pretreated at 130° C. for 60 min to obtain a pretreated solution, and then the pH is adjusted to 5, and a mixture consisting of 0.3 L of lignin peroxidase, 0.2 L of manganese peroxidase, 0.2 L of swollenin, and 0.3 L of cellulase is added, and the mixture is reacted at 28° C. for 4 days to obtain a saccharified liquid, and the saccharified liquid is filtered to separate the saccharified liquid and the enzymolysis residue, wherein the solid content of the enzymolysis residue is 50% by weight;

[0038] Step A2: dilute the saccharification clear liquid with water to a total sugar content of 10% by weight to obtain a seed culture medium, put the seed culture medium into a seed tank, sterilize, cool to room temperature, and then inoculate Aspergillus niger, with the volume percentage of Aspergillus niger inoculation being 10%. Carry out seed culture at 35°C and an initial pH value of 5, control the tank pressure at 0.05MPa, the ventilation volume at 0.4 volume: (volume·min), and the stirring speed at 300rpm; after 13h of seed culture, control the tank pressure at 0.07MPa, the ventilation volume at 0.7 volume: (volume·min), and the stirring speed at 380rpm, stop the culture when the acidity is ≥1g / 100mL, and obtain a seed solution;

[0039] Step A3: preparing a fermentation medium, the fermentation medium including 120 kg of saccharification clear liquid, 30 kg of saccharification liquid, and 12 kg of urea, sterilizing the fermentation medium, adding the fermentation medium into a fermentation tank, and then adding the seed liquid obtained in step A2, and fermenting for 3.5 days under the conditions of an initial pH of 4-5, a temperature of 35° C., a ventilation volume of 0.5 volume: (volume.min), and a rotation speed of 400 rpm to obtain a citric acid fermentation liquid.

[0040] Comparative Example 1

[0041] Compared with Example 1, the difference between this comparative example is that the "0.3L lignin peroxidase, 0.15L manganese peroxidase, 0.15L swollenin" in step A1 of comparative example 1 is replaced by "0.3L lignin peroxidase, 0.15L manganese peroxidase", and the remaining raw materials and steps are the same as Example 1.

[0042] Comparative Example 2

[0043] Compared with Example 1, the difference between this comparative example is that the "0.3L lignin peroxidase, 0.15L manganese peroxidase, 0.15L swollenin" in step A1 of comparative example 1 is replaced by "0.3L lignin peroxidase, 0.15L swollenin", and the remaining raw materials and steps are the same as Example 1.

[0044] Comparative Example 3

[0045] Compared with Example 1, the difference between this comparative example is that the "0.3L lignin peroxidase, 0.15L manganese peroxidase, 0.15L swollenin" in step A1 of comparative example 1 is replaced by "0.15L manganese peroxidase, 0.15L swollenin", and the remaining raw materials and steps are the same as Example 1.

[0046] Comparative Example 4

[0047] The difference between this comparative example and Example 1 is that the "0.3L lignin peroxidase, 0.15L manganese peroxidase, 0.15L swollenin" in step A1 of comparative example 1 is replaced by "0.25L lignin peroxidase, 0.1L manganese peroxidase, 0.1L swollenin", and the remaining raw materials and steps are the same as Example 1.

[0048] Comparative Example 5

[0049] Compared with Example 3, this comparative example is different in that "0.3L lignin peroxidase, 0.2L manganese peroxidase, and 0.2L swollenin" in step A1 of comparative example 1 is replaced with "0.35L lignin peroxidase, 0.25L manganese peroxidase, and 0.25L swollenin", and the remaining raw materials and steps are the same as those in Example 3.

[0050] The saccharification liquid obtained in step A1 of Example 1-Example 3 and Comparative Example 1-Comparative Example 5 was tested for glucose content by high performance liquid chromatography, and the chromatographic column was an American Bio-Rad Aminex-87H chromatographic column. Then, the citric acid content and conversion rate of the citric acid fermentation broth obtained in Example 1-Example 3 and Comparative Example 1-Comparative Example 5 were tested. The citric acid content test method was carried out according to GB 5009.157-2016, and the acid supply of a single tank = the concentration of the citric acid solution × the volume of the citric acid solution; conversion rate (%) = the acid supply of a single tank / the total weight of sugar used × 100%, where the total weight of sugar used includes the weight of sugar used in the seed tank and the weight of sugar used in the fermentation tank. The test results are shown in Table 1:

[0051] Table 1

[0052] project Glucose content (g) Citric acid content (g / 100mL) Conversion rate (%) Example 1 384.9 16.3 98.7 Example 2 388.9 16.5 99.3 Example 3 395.4 16.8 99.6 Comparative Example 1 338.1 14.9 96.3 Comparative Example 2 326.5 13.6 94.2 Comparative Example 3 334.6 14.4 95.1 Comparative Example 4 346.4 15.1 95.7 Comparative Example 5 354.9 15.7 97.1

[0053] It can be concluded from Table 1 that the citric acid fermentation processes of Examples 1 to 3 are superior to the fermentation processes of Comparative Examples 1 to 5.

[0054] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0055] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A citric acid fermentation process, characterized in that: The following steps are involved: Step A1: pretreating corn stalks to obtain a pretreatment liquid, then adjusting the pH to 5, adding the mixture, reacting at 25-30° C. for 2-4 days to obtain a saccharification liquid, filtering the saccharification liquid to separate the saccharification clear liquid and the enzymatic hydrolysis residue; the mixture comprises lignin peroxidase, manganese peroxidase, swollenin, and cellulase; Step A2: diluting the saccharification clear liquid with water to a total sugar content of 10% by weight to obtain a seed culture medium, placing the seed culture medium into a seed tank, sterilizing it, cooling it to room temperature, and then inoculating Aspergillus niger species, and stopping the culture when the acidity is ≥1 g / 100 mL to obtain a seed liquid; Step A3: prepare a fermentation medium, sterilize it, add the fermentation medium into a fermentation tank, and then add the seed liquid obtained in step A2 for fermentation to obtain a citric acid fermentation liquid.

2. A citric acid fermentation process according to claim 1, characterized in that: In step A1, the volume ratio of the pretreatment liquid to the lignin peroxidase, manganese peroxidase, swollenin and cellulase is 15-20:0.3:0.15-0.2:0.15-0.2:0.3; and the solid content of the enzymatic residue is 49-51% by weight.

3. A citric acid fermentation process according to claim 1, characterized in that: In step A1, the specific steps of pretreatment are: mixing corn stalks with mixed solution A, and pretreating at 130° C. for 40-80 minutes; wherein the usage ratio of corn stalks to mixed solution A is 1 kg: 15-20 L, and mixed solution A consists of 2% calcium carbonate solution and 1% hydrogen peroxide solution.

4. A citric acid fermentation process according to claim 1, characterized in that: In step A2, the volume percentage of the Aspergillus niger inoculation amount is 8-12%.

5. A citric acid fermentation process according to claim 1, characterized in that: In step A2, the conditions for seed tank culture are: seed culture is carried out at 35°C and an initial pH value of 5, the tank pressure is controlled at 0.04-0.06MPa, the ventilation volume is controlled at 0.3-0.5 volume: (volume·min), and the stirring speed is controlled at 250-350rpm; after 10-15h of seed culture, the tank pressure is controlled at 0.06-0.08MPa, the ventilation volume is controlled at 0.6-0.8 volume: (volume·min), and the stirring speed is controlled at 300-450rpm.

6. A citric acid fermentation process according to claim 1, characterized in that: In step A3, the fermentation medium includes saccharification supernatant, saccharification liquid, and urea; the mass ratio of saccharification supernatant, saccharification liquid, and urea is 6:1.5:0.

6.

7. A citric acid fermentation process according to claim 1, characterized in that: In step A3, the culture conditions of the fermentation tank fermentation are: initial pH 4-5, temperature 30-40° C., ventilation volume 0.3-0.8 volume: (volume.min), rotation speed 350-450 rpm, and time 3-4 days.