CN110699344A - A comprehensive utilization process of citric acid fermentation tailings - Google Patents

Abstract

The invention belongs to the field of comprehensive utilization of waste resources, and particularly relates to a comprehensive process of citric acid fermentation tailings. Firstly, the citric acid fermentation tailings are subjected to the cascade reaction of steam explosion and ionic liquid, and the crystal structure of the chitin and corn cellulose is fully destroyed while the cell components are fully released, which is a cellulase enzyme. At the same time, the by-products such as soluble sugar and protein produced in the pretreatment process are fermented and produced by chitin deacetylase, realizing a synergistic green production. Easy industrial application.

Description

A comprehensive utilization process of citric acid fermentation tailings

Technical field:

The invention belongs to the field of comprehensive utilization of waste resources, and particularly relates to a comprehensive process of citric acid fermentation tailings.

Background technique:

Citric acid is the organic acid with the largest microbial fermentation yield at present, and is widely used in beverages, food, washing and daily chemicals and other industries. The microorganisms used are mainly Aspergillus niger. After citric acid fermentation, a large amount of Aspergillus niger silk residue was produced. According to industry statistics, about 0.15 tons of dry mycelium is produced for every ton of citric acid produced. Currently, global citric acid production is estimated at more than 2 million tons per year, which means about 300,000 tons of black fungus per year. Typically, these tailings are devalued and used as feed for ruminants.

It has been reported that about 15% of the chitin in the dry weight of Aspergillus niger somatic cells. Therefore, Aspergillus niger mycelium is a good raw material for the production of chitin and its derivatives, including chitosan, chitosan oligosaccharide, N-acetyl-D-glucosamine (GlcNAc) and glucosamine (GlcN), in biological It has a wide range of applications in the pharmaceutical, food, health care and cosmetic industries. However, the current mainstream process still adopts chemical extraction, and there are problems such as the use of strong acid, strong alkali and environmental pollution. The enzymatic hydrolysis of chitin and its derivatives has received increasing attention as an eco-friendly method. However, the enzymatic production of chitin and its derivatives remains challenging, one of the main reasons being that the crystallinity of chitin makes it insoluble in water. Therefore, it is of great significance to develop reasonable pretreatment methods to improve the crystal structure of chitin and its derivatives before bioconversion into chitosan.

At the same time, most of the industrial citric acid fermentation currently uses coarse material fermentation. In addition to mycelium, the fermentation tailings using corn as raw materials also contain a large amount of corn fiber (including 382 grams of cellulose, 445 grams per Kg of dry matter) g hemicellulose, 66 g lignin, 19 g protein and 28 g ash). Therefore, in order to fully recycle the mycelium waste, the mycelium and corn fiber should be studied at the same time. At present, there is no report on the comprehensive utilization of the residual fiber, protein and mycelium in the citric acid fermentation tailings by a reasonable pretreatment method, which is also an urgent problem to be discussed and solved in the current environmental protection situation.

The invention develops a process of steam explosion and ionic liquid cascade pretreatment of citric acid fermentation tailings and comprehensive utilization of different components thereof, which provides a possible way for comprehensive utilization of citric acid fermentation industrial tailings.

Invention content:

Aiming at the deficiencies of the prior art, the present invention aims to provide an efficient comprehensive treatment technology for citric acid fermentation tailings and its application. When the ingredients are fully released, the crystal structure of the bacterial chitin and corn cellulose is fully destroyed, which provides a good foundation for the action of cellulase and chitin deacetylase. The by-products such as soluble sugar and protein are fermented and produced by chitin deacetylase, which realizes a coordinated green production and is easy for industrial application.

The comprehensive utilization method of the citric acid fermentation tailings is for the citric acid fermentation tailings using corn as raw material and Aspergillus niger as fermentation strains, and the steps are as follows:

(1) carry out gas explosion treatment with citric acid fermentation tailings, obtain soluble part and solid residual part, wherein, solid residual part carries out ionic liquid pretreatment;

(2) drying the material treated with the ionic liquid, enzymatic hydrolysis with cellulase, the soluble part after enzymatic hydrolysis is for use, and the solid residue is enzymatically hydrolyzed with chitin deacetylase to produce partially deacetylated chitosan Sugar precursor, which provides the basis for the subsequent further deacetylation to prepare chitosan and chitosan oligosaccharide with high degree of deacetylation;

(3) the soluble part after gas explosion and cellulase enzymolysis is used for the fermentation production of chitin deacetylase, and the chitin deacetylase obtained by production is used for the enzymolysis in step (2) again, with This realizes the cyclic production and utilization of chitin deacetylase;

Further, the conditions of the gas explosion pretreatment are: the moisture content of the citric acid fermentation tailings is 25-40%, the steam explosion pressure is 2.0-2.5MPa, and the time is 60-90s;

Further, the conditions of the ionic liquid pretreatment are: the solid residue after the steam explosion pretreatment is dried to a moisture content of 10-12%, the ionic liquid treatment temperature is 60-80 ° C, the treatment time is 6-24 h, and the ionic liquid The addition amount is 10-20mL per 5g of drying material;

Further, the ionic liquid is tetrabutylammonium hydroxide, and the ionic liquid concentration is 20-25% aqueous solution;

Further, the enzymatic hydrolysis conditions of the cellulase are as follows: the material is dried to a moisture content of 10-12%, the material concentration is adjusted to 80-140 g/L, the enzymatic hydrolysis temperature is 40-60 °C, the enzymatic hydrolysis time is 20-32 h, and the enzymatic hydrolysis pH4.0-5.8, the amount of cellulase added is 20-35U/g (drying the substrate);

Further, the enzymatic hydrolysis conditions of the chitin deacetylase: the solid residue after cellulose enzymatic hydrolysis is dried to a moisture content of 10-12%, the material concentration is adjusted to 20-50 g/L, and the enzymatic hydrolysis temperature is 25-37 °C, The enzymatic hydrolysis time is 6-12h, the enzymatic hydrolysis pH is 5.5-7.5, and the addition amount of chitin deacetylase is 3000-3700 U/g (drying the substrate).

Further, the liquid residue obtained by the solid-liquid separation after the steam explosion treatment and the liquid residue obtained by the solid-liquid separation after cellulose enzymolysis are combined, and dried to a water content of 10-12% for use in chitin removal. Fermentation substrates produced by acetylase (hereinafter referred to as "by-products");

Further, the fermentation production conditions of chitin deacetylase are: the inoculum amount is 2-10%, the stirring speed is 160-200rpm, the temperature is 30-40°C, and the fermentation is 12-30h;

Preferably, the fermentation strain is Rhodococcus, yeast, Escherichia coli, Mortierella or anthrax;

More preferably, the fermentation strain is Rhodococcus equi CGMCC NO.14861;

Further, the fermentation medium is composed of: yeast extract powder 5-10g/L, by-product 0.5-2.0g/L, magnesium sulfate 1.0g/L, potassium dihydrogen phosphate 0.3g/L, dipotassium hydrogen phosphate 1.0g /L, sodium chloride 0.5-2.0g/L, pH6.0-7.0.

In the above treatment process, the release rate of soluble components of citric acid fermentation tailings after steam explosion is increased by 15-30%; the content of soluble sugar after cellulase enzymatic hydrolysis reaches 0.4-0.53g/g; after chitin deacetylase enzymatic hydrolysis The content of acetic acid produced by chitin deacetylase deacetylation reached 480-554 mg/L; after 24 hours of fermentation, the content of chitin deacetylase in the fermentation broth reached 4854.7 U/mL.

Beneficial effects:

The cascade pretreatment and comprehensive utilization technology of the citric acid fermentation tailings provided by the invention can realize the efficient release of various components in the citric acid fermentation tailings, and provide more suitable substrates for subsequent enzymatic hydrolysis. Through the optimization of pretreatment conditions and subsequent enzymatic hydrolysis conditions, the release rate of soluble components of citric acid tailings after steam explosion increased by 30.2%, the soluble sugar content after cellulose enzymatic hydrolysis reached 0.53 g/g, and the cellulose enzymatic hydrolysis efficiency increased by 26.97%. Chitin deacetylase showed high activity from the inactivity of unpretreated substrates, and the acetic acid content released by deacetylation reached 554.48 mg/L. At the same time, the by-products generated during pretreatment and enzymatic hydrolysis were applied For the fermentation production of chitin deacetylase, the fermentative enzyme production increased by 1.5 times, reaching 4854.7 U/mL. Compared with the prior art, the cascade pretreatment process provided by the present invention can realize the efficient pretreatment of the citric acid fermentation tailings, realize the co-production of chitin deacetylase and chitin derivatives, and realize the citric acid fermentation. The comprehensive utilization of tailings resources provides a good reference and has a wide range of industrial application prospects.

Description of drawings:

Figure 1. The whole flow chart of cascade pretreatment process and comprehensive utilization;

Figure 2. Characterization of the effect of different steam explosion conditions for pretreatment of citric acid fermentation tailings

Among them, A: color change; B: scanning electron microscope picture; C: porosity change; D: soluble component change;

Fig. 3 SEM images of different cascade pretreatment citric acid fermentation tailings;

Among them, A: magnify 2000 times; B: magnify 10000 times;

Figure 4. Fermentation production curve of chitin deacetylase.

Detailed ways:

In order to make the purpose, technical solutions and advantages of the present patent more clear, the present patent will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present patent, but not to limit the present invention.

The process flow of the present invention is shown in FIG. 1 , and the present invention will be further explained below with reference to specific embodiments.

Embodiment 1 Steam explosion pretreatment

The moisture content of the citric acid fermentation tailings with corn as the raw material and Aspergillus niger as the fermentation strain was adjusted to 30%, the steam explosion pressure was 1.0, 1.5, 2.0, 2.5MPa, and the time was 60s. The materials before and after the steam explosion were subjected to scanning electron microscopy. , pore size distribution analyzer to characterize, and to detect the release ratio of soluble components.

The results are shown in Figure 2. From Figure 2-A, it can be seen that with the increase of the steam explosion pressure, the color of the material gradually deepens; from Figure 2-B, it can be seen that with the increase of the steam explosion pressure, the proportion of small particles of the material is obvious. It can be seen from Figure 2-C that the pore area difference of the material at the average pore diameter of 2-10nm increases significantly; from Figure 2-D, it can be seen that the increase of the steam explosion pressure and the release of the soluble components of the citric acid bacteria are also positive These changes all indicate that the steam explosion pretreatment has a positive effect on the expansion and fragmentation of the citric acid fermentation residue.

Example 2 Ionic liquid cascade pretreatment

The solid residual moisture of the citric acid fermentation tailings after steam explosion pretreatment in Example 1 was dried to a content of 10%, and the ionic liquid was treated with tetrabutylammonium hydroxide. The ionic liquid concentration was a 20% aqueous solution, and the ionic liquid treatment temperature was 80 ℃, treatment time 6h, the amount of ionic liquid added is 10mL per 5g material, the material before and after the cascade pretreatment was further characterized by scanning electron microscopy, the results are shown in Figure 3, after the cascade pretreatment of ionic liquid, the surface of the material The compact structure becomes more loose.

Example 3 Cellulase treatment of material after cascade pretreatment

The material after the 2.5MPa steam explosion and ionic liquid cascade treatment in Example 2 was dried to a moisture content of 10%, the material concentration was adjusted to 120g/L, and cellulase was used to carry out enzymolysis, the enzymolysis temperature was 60°C, and the enzymolysis was carried out. The time was 30h, the pH of enzymolysis was 5.0, and the amount of cellulase added was 20U/g. The change of soluble sugar content was detected by liquid chromatography, and the total sugar reached 64.49g/L (0.53g/g (drying substrate)) after cellulose enzymolysis after cascade pretreatment.

At the same time, the blank sample without pretreatment, the sample pretreated only by 2.5MPa steam explosion in Example 1, and the sample pretreated by ionic liquid under the same conditions in Example 2 were used as controls respectively. The change of soluble sugar content was measured, and the results are shown in Table 1. Both the steam explosion pretreatment and ionic liquid pretreatment alone can promote the enzymatic hydrolysis of cellulase. Compared with the effect, the enzymatic effect of cellulase increased by 26.97%.

Table 1: Cellulase hydrolysis effect table after cascade pretreatment

The chitin deacetylase treatment of material after embodiment 4 pretreatment

The solid residue obtained after the cascade pretreatment and cellulase enzymatic hydrolysis in Example 3 was centrifugally washed, then dried to a moisture content of 10%, adjusted to a material concentration of 40 g/L, and chitin deacetylase was used for enzymatic hydrolysis , the enzymatic hydrolysis temperature is 30 °C, the enzymatic hydrolysis time is 6 h, the enzymatic hydrolysis pH is 7.0, and the addition amount of chitin deacetylase is 3200 U/mL. The content of acetic acid released after deacetylase hydrolysis reached 554.48 mg/L.

At the same time, the blank sample without pretreatment, the sample pretreated only by 2.5MPa steam explosion in Example 1, and the sample pretreated by ionic liquid under the same conditions in Example 2 were used as controls respectively, and the cellulose of Example 3 was used. The enzymatic hydrolysis method and the chitin deacetylase enzymatic hydrolysis method of the present embodiment were used to measure the change of the acetic acid content. The results are shown in Table 2. The chitin of the liquid pretreated bacteria was inactive, and the steam explosion pretreatment played a significant role in promoting the effect of chitin deacetylase. After the cascade pretreatment of steam explosion and ionic liquid, the chitin deacetylase The effect of enzyme was further improved by 29.07%.

Table 2: Enzymatic hydrolysis effect of chitin deacetylase after pretreatment

Example 5 Utilization of by-products produced in cascade pretreatment and cellulose enzymatic hydrolysis

Collect the soluble components produced in the steam explosion process of the above-mentioned embodiment and the soluble sugar produced in the enzymatic hydrolysis of cellulose (respectively centrifuged to remove the insolubles and mixed) and dried to a moisture content of 10% (hereinafter referred to as "by-products"), used for several Fermentative production of butyl deacetylase.

The fermentation conditions were as follows: using Rhodococcus equi CGMCC NO.14861, the inoculum amount was 10%, the stirring speed was 200 rpm, and the temperature was 37° C. to carry out fermentation.

Fermentation medium: yeast extract powder 8g/L, by-product addition 2.0g/L, magnesium sulfate 1.0g/L, potassium dihydrogen phosphate 0.3g/L, dipotassium hydrogen phosphate 1.0g/L, sodium chloride 2.0g /L, pH 6.8.

The maximum enzyme activity was reached at 30h fermentation, which was determined to be 4854.7U/mL.

The fermentation curve is shown in Figure 4. The fermentation medium without by-products was used as a blank. Compared with the blank control, both of them reached the maximum enzyme production within 30h of fermentation, and then entered the stable phase of enzyme production. Compared with the increase of 61.82%.

Embodiment 6 A kind of comprehensive utilization of citric acid fermentation tailings

(1) the citric acid fermentation tailings with corn as raw material and Aspergillus niger as fermentation strain are adjusted to water content of 25%, the steam explosion pressure is 2.0MPa, and the time is 70s;

(2) soluble part and solid residual part are obtained after gas explosion, wherein, the solid residual part is subjected to ionic liquid pretreatment: the solid residual after the steam explosion pretreatment is dried to a moisture content of 10%, and the ionic liquid treatment temperature is 60 ° C , the treatment time is 10h, and the amount of ionic liquid added is 15mL per 5g of drying materials;

The ionic liquid is tetrabutylammonium hydroxide, and the ionic liquid concentration is 22% aqueous solution;

(3) Dry the material treated with the ionic liquid, and use cellulase for enzymatic hydrolysis. The conditions for enzymatic hydrolysis are: drying the material to a moisture content of 12%, adjusting the material concentration to 80 g/L, enzymatic hydrolysis temperature of 40 °C, and enzymatic hydrolysis time. 20h, enzymolysis pH 4.0, cellulase addition 25U/g (drying substrate);

(4) The soluble part after enzymatic hydrolysis is ready for use, and the solid residue is enzymatically hydrolyzed with chitin deacetylase. The enzymatic hydrolysis conditions are: the solid residue after enzymatic hydrolysis of cellulose is dried to a moisture content of 10%, and the concentration of the material is adjusted. 20g/L, enzymolysis temperature 25°C, enzymolysis time 8h, enzymolysis pH 5.5, addition amount of chitin deacetylase 3000U/g (drying substrate);

(5) The soluble part after gas explosion and cellulase enzymatic hydrolysis is used for the fermentation production of chitin deacetylase: the liquid residue obtained by solid-liquid separation after the steam explosion treatment, and the solid-liquid residue obtained after the enzymatic hydrolysis of cellulose The liquid residues obtained by liquid separation are combined, dried to a water content of 10% and used as a fermentation substrate for the production of chitin deacetylase (hereinafter referred to as "by-product");

The fermentation production conditions of chitin deacetylase are: the inoculum amount is 5%, the stirring speed is 160rpm, the temperature is 30°C, and the fermentation is 24h;

The fermentation strain is Rhodococcus CGMCC NO.14861;

The fermentation medium is composed of: yeast extract powder 5g/L, by-product 0.5g/L, magnesium sulfate 1.0g/L, potassium dihydrogen phosphate 0.3g/L, dipotassium hydrogen phosphate 1.0g/L, sodium chloride 0.5g /L, pH 6.0.

In the above treatment process, the release rate of soluble components of citric acid fermentation tailings after steam explosion increased by 18.5%; the content of soluble sugar reached 0.48g/g after enzymatic hydrolysis by cellulase; The content of acetic acid generated by acetylase deacetylation reached 496 mg/L; after 24 hours of fermentation, the content of chitin deacetylase in the fermentation broth reached 3947.3 U/mL.

Embodiment 7 A kind of comprehensive utilization of citric acid fermentation tailings

(1) the citric acid fermentation tailings with corn as raw material and Aspergillus niger as fermentation strain are adjusted to water content of 40%, the steam explosion pressure is 2.5MPa, and the time is 90s;

(2) obtain soluble part and solid residual part after gas explosion, wherein, solid residual part is carried out ionic liquid pretreatment: the solid residue after steam explosion pretreatment is dried to moisture content 12%, and ionic liquid treatment temperature is 80 ℃, The treatment time is 24h, and the amount of ionic liquid added is 20mL per 5g of drying materials;

The ionic liquid is tetrabutylammonium hydroxide, and the ionic liquid concentration is 25% aqueous solution;

(3) Dry the material treated with the ionic liquid, and use cellulase for enzymatic hydrolysis. The conditions for enzymatic hydrolysis are: drying the material to a moisture content of 12%, adjusting the material concentration to 140 g/L, enzymatic hydrolysis temperature of 60 °C, and enzymatic hydrolysis time. 32h, enzymatic hydrolysis pH5.8, cellulase addition 35U/g (drying substrate);

(4) The soluble part after enzymatic hydrolysis is ready for use, and the solid residue is enzymatically hydrolyzed with chitin deacetylase. The enzymatic hydrolysis conditions are: the solid residue after enzymatic hydrolysis of cellulose is dried to a moisture content of 12%, and the concentration of the material is adjusted. 50g/L, enzymolysis temperature 37℃, enzymolysis time 12h, enzymolysis pH 7.5, addition amount of chitin deacetylase 3700U/g (drying substrate);

(5) The soluble part after gas explosion and cellulase enzymatic hydrolysis is used for the fermentation production of chitin deacetylase: the liquid residue obtained by solid-liquid separation after the steam explosion treatment, and the solid-liquid residue obtained after the enzymatic hydrolysis of cellulose The liquid residues obtained by liquid separation are combined, dried to a water content of 12% and used as a fermentation substrate for the production of chitin deacetylase (hereinafter referred to as "by-products");

The fermentation production conditions of chitin deacetylase are: the inoculum amount is 8%, the stirring speed is 200rpm, the temperature is 40°C, and the fermentation is 30h;

The fermentation strain is anthracis CF-6;

The fermentation medium consists of: yeast extract powder 10g/L, by-product addition 2.0g/L, magnesium sulfate 1.0g/L, potassium dihydrogen phosphate 0.3g/L, dipotassium hydrogen phosphate 1.0g/L, sodium chloride 2.0g/L, pH7.0.

In the above treatment process, the release rate of soluble components in the citric acid fermentation tailings after steam explosion increased by 27.2%; the soluble sugar content reached 0.51g/g after enzymatic hydrolysis by cellulase; The content of acetic acid generated by acetylase deacetylation reached 531 mg/L; after 30 hours of fermentation, the content of chitin deacetylase in the fermentation broth reached 4657.3 U/mL.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as a limitation on the scope of the patent. It should be noted that, for those skilled in the art, without departing from the concept of the present patent, the above-mentioned embodiments can also be modified, combined and improved, which all belong to the protection scope of the present patent. Therefore, the scope of protection of this patent should be subject to the claims.

Claims (10)

1. A comprehensive utilization method of citric acid fermentation tailings is characterized in that the fermentation tailings are citric acid fermentation tailings taking corns as raw materials and aspergillus niger as fermentation strains, and the treatment method comprises the following steps:

(1) carrying out gas explosion treatment on the citric acid fermentation tailings to obtain a soluble part and a solid residual part, wherein the solid residual part is subjected to ionic liquid pretreatment;

(2) drying the material treated by the ionic liquid, performing enzymolysis by adopting cellulase, wherein the soluble part after enzymolysis is for later use, and performing enzymolysis on the solid residue by using chitin deacetylase to produce a partially deacetylated chitosan precursor;

(3) and (3) performing fermentation production on the chitin deacetylase after air explosion and enzymolysis by using cellulase, and performing enzymolysis in the step (2) again on the produced chitin deacetylase to realize cyclic production and utilization of the chitin deacetylase.

2. The comprehensive utilization method of citric acid fermentation tailings as claimed in claim 1, wherein the conditions of the gas explosion pretreatment are as follows: the water content of the citric acid fermentation tailings is 25-40%, the steam explosion pressure is 2.0-2.5MPa, and the time is 60-90 s.

3. The comprehensive utilization method of citric acid fermentation tailings as claimed in claim 1, wherein the ionic liquid pretreatment conditions are as follows: and drying the solid residues subjected to steam explosion pretreatment until the water content is 10-12%, treating the solid residues with an ionic liquid at the temperature of 60-80 ℃ for 6-24h, wherein the addition amount of the ionic liquid is 10-20mL per 5g of the dried material.

4. The method for comprehensively utilizing citric acid fermentation residues as claimed in claim 3, wherein the ionic liquid is tetrabutylammonium hydroxide, and the ionic liquid concentration is 20-25% aqueous solution.

5. The method for comprehensively utilizing the citric acid fermentation tailings as claimed in claim 1, wherein the cellulase enzymolysis conditions are as follows: drying the materials until the water content is 10-12%, adjusting the material concentration to 80-140g/L, carrying out enzymolysis at 40-60 ℃ for 20-32h, adjusting the pH value of enzymolysis to 4.0-5.8, and adding 20-35U/g of cellulase.

6. The method for comprehensively utilizing citric acid fermentation tailings as claimed in claim 1, wherein the enzymatic hydrolysis conditions of the chitin deacetylase are as follows: drying the solid residue after the cellulose enzymolysis until the water content is 10-12%, adjusting the material concentration to 20-50g/L, the enzymolysis temperature to 25-37 ℃, the enzymolysis time to 6-12h, the enzymolysis pHs to 5.5-7.5, and the addition amount of the chitin deacetylase to 3000-3700U/g.

7. The method of claim 1, wherein the liquid residue obtained by solid-liquid separation after steam explosion treatment and the liquid residue obtained by solid-liquid separation after cellulose hydrolysis are combined and dried until the water content is 10-12% and used as a fermentation substrate for chitin deacetylase production, which is hereinafter referred to as a "by-product";

the fermentation medium for producing chitin deacetylase consists of: 5-10g/L of yeast extract powder, 0.5-2.0g/L of by-product, 1.0g/L of magnesium sulfate, 0.3g/L of monopotassium phosphate, 1.0g/L of dipotassium phosphate, 0.5-2.0g/L of sodium chloride and pH 6.0-7.0.

8. The method for comprehensively utilizing citric acid fermentation residues as claimed in claim 7, wherein the fermentation conditions for producing chitin deacetylase by using the fermentation medium are as follows: the inoculation amount is 2-10%, the stirring speed is 160-.

9. The method of claim 8, wherein the fermentation broth is Rhodococcus, yeast, Escherichia coli, Mortierella or anthrax.

10. The method for comprehensive utilization of citric acid fermentation residues as claimed in claim 9, wherein the fermentation strain is Rhodococcus equi CGMCC NO. 14861.

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