ASPERGILLUS NIGER SEED CONTINUOUS CULTURE AND METHOD FOR PRODUCING CITRIC ACID THEREFROM

Abstract

Disclosed is an Aspergillus niger seed continuous culture method, comprising the steps of: (1) at a startup stage, Aspergillus niger spores are inoculated into a seed culture medium to obtain a seed liquid; (2) at a seed continuous culture stage, continuous dispersion treatment is performed on the seed liquid obtained in step (1), continuous culture is performed on the seed liquid obtained by dispersion, and meanwhile, a fresh seed feed medium is replenished; and (3) at a stop stage, the replenishment of the fresh seed feed medium and the dispersion treatment are stopped, continuous culture is performed to obtain a seed liquid, and then the seed liquid is transferred into the fermentation medium for fermentation culture. The method according to the present invention makes breakthrough to solve problems that multi-cellular filamentous bacteria grow slowly and mycelium pellets are easy to lose in continuous culture, thus fully achieving seed continuous culture.

Claims

1. An Aspergillus niger seed continuous culture method, comprising the steps of: (1) at a startup stage, inoculating Aspergillus niger spores into a seed culture medium, and culturing for 1636 h to obtain a seed liquid; (2) at a seed continuous culture stage, performing continuous dispersion treatment on the seed liquid obtained in step (1), performing continuous culturing on the seed liquid obtained by dispersion, performing fermentation culture after the seed liquid flows out continuously into a fermentation medium in the culturing process, and meanwhile replenishing a fresh seed feed medium at the same rate with the outflow seed liquid; and (3) at a stop stage, stopping the replenishment of the fresh seed feed medium and the dispersion treatment, performing continuous culture to obtain a seed liquid, and then transferring the seed liquid into the fermentation medium for fermentation culture.

2. The method according to claim 1, wherein the final concentration of the Aspergillus niger spores in step (1) after incubation is 1910.sup.5 spores/mL; the total sugar of the seed culture medium in step (1) is 100180 g/L, C/N is 2040; the seed culture conditions in step (1) are as follows: the temperature is 3539 C., the air volume is 0.20.4 vvm, the tank pressure is 0.050.1 Mpa, and the agitation rate is 100200 rpm.

3. The method according to claim 1, wherein the total sugar of the fresh seed feed medium replenished in step (2) is 150200 g/L, C/N is 1530; the continuous culture conditions in step (2) are as follows: the temperature is 3537 C., the air volume is 0.30.6 vvm, the tank pressure is 0.050.07 Mpa, and the agitation rate is 150200 rpm.

4. The method according to claim 1, wherein the continuous culture conditions in step (3) are as follows: the temperature is 3539 C., the air volume is 0.30.6 vvm, the tank pressure is 0.050.1 Mpa, and the agitation rate is 150200 rpm.

5. The method according to claim 1, wherein the continuous dispersion treatment method of the seed liquid in step (2) is to disperse mycelium pellets in the seed liquid into 1080 m of flocculent hyphae by adopting a disperser.

6. The method according to claim 1, wherein the replenishing rate of the fresh seed feed medium and the outflow rate of the seed liquid in step (2) are F=V/t, wherein V is the volume of the seed liquid in a seed tank and is determined by different tanks; t is the residence time of the seed liquid, and is adjusted according to cell concentration feedback of the flocculent hyphae, is usually taken a value of 6-24 h.

7. The method according to claim 1, wherein the fermentation inoculation proportion in step (2) and step (3) is 525% (v/v).

8. The method according to claim 1, wherein the total sugar of the fermentation medium in step (2) and step (3) is 160200 g/L, C/N is 5090; the fermentation culture conditions in step (2) and step (3) are as follows: the temperature is 3539 C., the air volume is 0.10.4 vvm, the tank pressure is 0.050.1 Mpa, the agitation rate is 100200 rpm, and the fermentation ends when the concentration of a reducing sugar is less than 5 g/L.

9. The method according to claim 1, wherein the seed culture medium, the fresh seed feed medium and the fermentation medium are formulated with a starchy raw material liquefying liquid and a nitrogen source respectively; wherein the starchy raw materials at least comprise one of corn flour, cassava flour, sorghum flour and wheat starch; the nitrogen source at least comprises one of ammonium sulfate, urea, soybean meal powder and corn steep liquor.

10. A method for producing citric acid, comprising the step of: fermenting the Aspergillus niger obtained by the seed continuous culture method according to the claim 1 to produce citric acid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 is a schematic diagram of the culture method according to CN201410329652.X.

[0034] FIG. 2 is a schematic diagram of the culture method according to the present invention.

DETAILED DESCRIPTION

[0035] The present invention will be specifically described with reference to the accompanying drawings and examples.

[0036] All the raw materials and the reagents involved in the following examples and comparative examples are commercially available; Aspergillus niger is from the China Industrial Microbiological Culture Collection Center with the Accession No. CICC 40021. The total sugar and the reducing sugar are both determined with Fehling's titration method, the nitrogen source is determined by Kjeldahl determination, the citric acid assay adopts 0.1429 mol/L NaOH titration, and the spore count adopts blood counting chamber. Unless specially stated, devices and process methods commonly used in the art are employed.

Example 1

[0037] Corn flour and tap water were uniformly mixed according to the mass ratio of 1:3, the pH of the slurry was adjusted to 6.0 by Ca(OH).sub.2, and thermostable a-amylase was added in an addition amount of 20 U/g corn flour; ejection liquefaction was carried out, and qualified corn liquefied liquid was obtained after the iodine test was light brown; 80% of the corn liquefied liquid was filtered through a plate frame to remove filter residues to obtain corn syrup.

[0038] Startup stage: corn liquefied liquid and ammonium sulfate were formulated into a seed culture medium (total sugar was 100 g/L, C/N was 20), Aspergillus niger spores were inoculated into the seed culture medium after sterilized and cooled, and the final concentration of the spores was 9*10.sup.5 spores/mL, and cultured for 16 h at a temperature of 35 C., an air volume of 0.2 vvm, a tank pressure of 0.05 Mpa, and an agitation rate of 100 rpm to obtain a seed liquid.

[0039] Continuous phase: corn liquefied liquid and ammonium sulfate were formulated into a seed feed culture medium (total sugar was 150 g/L, C/N was 15). The obtained seed liquid was performed continuous dispersion treatment to disperse mycelium pellets into 10 m of flocculent hyphae by adopting a disperser; and meanwhile, the speed of continuously replenishing the fresh seed feed medium was controlled with a residence time of 6 h, and the seed liquid flowed out continuously at the same speed into a fermentation medium for fermentation culture. At the seed continuous culture stage, the culture temperature was 35 C., the air volume was 0.3 vvm, the tank pressure was 0.05 Mpa, and the agitation rate was 150 rpm.

[0040] Stop stage, when detecting that the seed vitality tends to decline, the replenishment of the fresh seed feed medium and the dispersion treatment were stopped, and then the remaining seed liquid was transferred into the fermentation medium for fermentation culture. The seed culture conditions at this stage: the temperature was 35 C., the air volume was 0.3 vvm, the tank pressure was 0.05 Mpa, and the agitation rate was 150 rpm.

[0041] The fermentation medium were formulated with corn liquefied liquid, corn syrup and ammonium sulfate (total sugar was 160 g/L, C/N was 50); the fermentation inoculation proportion was 25% (v/v); the fermentation culture conditions were as follows: the temperature was 35 C., the air volume was 0.1 vvm, the tank pressure was 0.05 Mpa, the agitation rate was 100 rpm, and the fermentation ended when the concentration of a reducing sugar was less than 5 g/L, acidity and residual sugar corresponding to each batch of fermentation were determined, and indexes such as conversion rate and fermentation strength were calculated.

Example 2

[0042] Corn flour and tap water were uniformly mixed according to the mass ratio of 1:3, the pH of the slurry was adjusted to 6.0 by Ca(OH).sub.2, and thermostable a-amylase was added in an addition amount of 20 U/g corn flour; ejection liquefaction was carried out, and qualified corn liquefied liquid was obtained after the iodine test was light brown; 80% of the corn liquefied liquid was filtered through a plate frame to remove filter residues to obtain corn syrup. The cassava flour and tap water were uniformly mixed according to the mass ratio of 1:3, the pH of the slurry was adjusted to 6.0 by Ca(OH).sub.2, and thermostable a-amylase was added in an addition amount of 20 U/g cassava flour; ejection liquefaction was carried out, and qualified cassava syrup was obtained after the iodine test was light brown.

[0043] Startup stage: corn liquefied liquid and urea were formulated into a seed culture medium (total sugar was 140 g/L, C/N was 30), Aspergillus niger spores were inoculated into the seed culture medium after sterilized and cooled, and the final concentration of the spores was 4.5*10.sup.5 spores/mL, and cultured for 26 h at a temperature of 37 C., an air volume of 0.3 vvm, a tank pressure of 0.075 Mpa, and an agitation rate of 150 rpm to obtain a seed liquid.

[0044] Continuous phase: corn liquefied liquid and urea were formulated into a seed feed culture medium (total sugar was 175 g/L, C/N was 23). The obtained seed liquid was performed continuous dispersion treatment to disperse mycelium pellets into 45 m of flocculent hyphae by adopting a disperser; and meanwhile, the speed of continuously replenishing the fresh seed feed medium was controlled with a residence time of 15 h, and the seed liquid flowed out continuously at the same speed into a fermentation medium for fermentation culture. At the seed continuous culture stage, the culture temperature was 36 C., the air volume was 0.45 vvm, the tank pressure was 0.06 Mpa, and the agitation rate was 175 rpm.

[0045] Stop stage, when detecting that the seed vitality tends to decline, the replenishment of the fresh seed feed medium and the dispersion treatment were stopped, and then the remaining seed liquid was transferred into the fermentation medium for fermentation culture. The seed culture conditions at this stage: the temperature was 37 C., the air volume was 0.45 vvm, the tank pressure was 0.075 Mpa, and the agitation rate was 175 rpm.

[0046] The fermentation medium were formulated with corn liquefied liquid, corn syrup, cassava syrup and urea (total sugar was 180 g/L, C/N was 70); the fermentation inoculation proportion was 15% (v/v); the fermentation culture conditions were as follows: the temperature was 37 C., the air volume was 0.25 vvm, the tank pressure was 0.075 Mpa, the agitation rate was 150 rpm, and the fermentation ended when the concentration of a reducing sugar was less than 5 g/L, acidity and residual sugar corresponding to each batch of fermentation were determined, and indexes such as conversion rate and fermentation strength were calculated.

Example 3

[0047] Corn flour and tap water were uniformly mixed according to the mass ratio of 1:3, the pH of the slurry was adjusted to 6.0 by Ca(OH).sub.2, and thermostable a-amylase was added in an addition amount of 20 U/g corn flour; ejection liquefaction was carried out, and qualified corn liquefied liquid was obtained after the iodine test was light brown; 80% of the corn liquefied liquid was filtered through a plate frame to remove filter residues to obtain corn syrup. The wheat starch and tap water were uniformly mixed according to the mass ratio of 1:3, the pH of the slurry was adjusted to 6.0 by Ca(OH).sub.2, and thermostable a-amylase was added in an addition amount of 20 U/g wheat starch; ejection liquefaction was carried out, and qualified wheat syrup was obtained after the iodine test was light brown.

[0048] Startup stage: corn liquefied liquid and soybean meal powder were formulated into a seed culture medium (total sugar was 180 g/L, C/N was 40), Aspergillus niger spores were inoculated into the seed culture medium after sterilized and cooled, and the final concentration of the spores was 1*10.sup.5 spores/mL, and cultured for 36 h at a temperature of 39 C., an air volume of 0.4 vvm, a tank pressure of 0.1 Mpa, and an agitation rate of 200 rpm to obtain a seed liquid.

[0049] Continuous phase: corn liquefied liquid and soybean meal powder were formulated into a seed feed culture medium (total sugar was 200 g/L, C/N was 30). The obtained seed liquid was performed continuous dispersion treatment to disperse mycelium pellets into 80 m of flocculent hyphae by adopting a disperser; and meanwhile, the speed of continuously replenishing the fresh seed feed medium was controlled with a residence time of 24 h, and the seed liquid flowed out continuously at the same speed into a fermentation medium for fermentation culture. At the seed continuous culture stage, the culture temperature was 37 C., the air volume was 0.6 vvm, the tank pressure was 0.07 Mpa, and the agitation rate was 200 rpm.

[0050] Stop stage, when detecting that the seed vitality tends to decline, the replenishment of the fresh seed feed medium and the dispersion treatment were stopped, and then the remaining seed liquid was transferred into the fermentation medium for fermentation culture. The seed culture conditions at this stage: the temperature was 39 C., the air volume was 0.6 vvm, the tank pressure was 0.1 Mpa, and the agitation rate was 200 rpm.

[0051] The fermentation medium were formulated with corn liquefied liquid, corn syrup, wheat syrup and soybean meal powder (total sugar was 200 g/L, C/N was 90); the fermentation inoculation proportion was 5% (v/v); the fermentation culture conditions were as follows: the temperature was 39 C., the air volume was 0.4 vvm, the tank pressure was 0.1 Mpa, the agitation rate was 200 rpm, and the fermentation ended when the concentration of a reducing sugar was less than 5 g/L, acidity and residual sugar corresponding to each batch of fermentation were determined, and indexes such as conversion rate and fermentation strength were calculated.

Comparative Example 1 (Example 1 in CN201410329652.X)

[0052] Glucose and soybean meal powder were formulated into seed culture medium (the total sugar was 80 g/L, the total nitrogen was 1.5 g/L), and the fermentation medium (the total sugar was 160 g/L, the total nitrogen was 1.5 g/L), respectively; after the seeds were transplanted, the spore concentration was 550,000 spores/mL inoculation; cultured for 20 h to obtain a mature seed solution, seed liquid hyphae were treated by a disperser, mycelium pellets after treatment had an average diameter of 100 m, and inoculated into the next stage of seed culture medium and the fermentation culture; where the next stage was a fermentation culture, the fermentation ended when the concentration of the reducing sugar in the fermentation culture was reduced to be less than 5 g/L. Such cycles were repeated until the seed viability declined significantly. Acidity and residual sugar corresponding to each batch of fermentation were determined, and indexes such as conversion rate and fermentation strength were calculated.

Comparative Example 2

[0053] The fermentation medium had a total sugar of 180 g/L and a total nitrogen of 1.5 g/L); other conditions were the same as in Example 1.

Comparative Example 3

[0054] The preparation of corn flour liquefying liquid and corn syrup was the same as in Example 1. Corn liquefied liquid and ammonium sulfate were formulated into a seed culture medium (the total sugar was 120 g/L, the total nitrogen was 2 g/L) and a fermentation medium (the total sugar was 160 g/L, the total nitrogen was 1.0 g/L). Aspergillus niger spores were inoculated into the seed culture medium, the final concentration of the spores was 4*10.sup.5 spores/mL, and cultured for 20 h to obtain a primary mature seed liquid. 70% of the mature seed liquid was transferred into the first batch of fermentation medium for culture and the fermentation ended when the concentration of the reducing sugar was less than 5 g/L. The remaining 30% of the seed liquid was treated by a disperser, the obtained mycelium pellets or fungus blocks with an average diameter of 62 Lm were transferred to the seed culture medium and cultured for 16 h to regain a secondary mature seed liquid. Such cycles were repeated until the seed viability declined significantly. Acidity and residual sugar corresponding to each batch of fermentation were determined, and indexes such as conversion rate and fermentation strength were calculated.

Comparative Example 4

[0055] The preparation of corn flour liquefying liquid and corn syrup was the same as in Example 1. Corn liquefied liquid and ammonium sulfate were formulated into a seed culture medium (the total sugar was 120 g/L, the total nitrogen was 2 g/L) and a fermentation medium (the total sugar was 160 g/L, the total nitrogen was 1.0 g/L). Aspergillus niger spores were inoculated into the seed culture medium, the final concentration of the spores was 4*10.sup.5 spores/mL, and cultured for 20 h to obtain a primary mature seed liquid. 70% of the mature seed liquid was transferred into the first batch of fermentation medium for culture and the fermentation ended when the concentration of the reducing sugar was less than 5 g/L. The remaining 30% of the seed liquid was treated by a disperser to obtain mycelium pellets or fungus blocks with an average diameter of 50 m, the fresh seed feed medium were replenished into the remaining seed oil, and cultured for 16 h to regain a secondary mature seed liquid. Such cycles were repeated until the seed viability declined significantly. Acidity and residual sugar corresponding to each batch of fermentation were determined, and indexes such as conversion rate and fermentation strength were calculated.

Comparative Example 5

[0056] The speed of continuously replenishing the fresh seed feed medium at the seed continuous culture stage was controlled with a residence time of 4 h; other conditions were the same as in Example 1.

Comparative Example 6

[0057] The C/N of the seed feed culture medium at the seed continuous culture stage was 40; other conditions were the same as in Example 1.

Test Example 1

[0058] The technical effects of Example 1 and Comparative examples 1, 3 and 4 are compared, as shown in Table 1.

TABLE-US-00001 TABLE 1 Example Comparative Comparative Comparative 1 example 1 example 3 example 4 Seed continuous running 288 260 260 260 time (h) Number of seed tanks 1 3 3 1 Each batch of seed tank 0 12 9 1 auxiliary time (h) Bran koji usage amount 0.22 0.33 0.33 0.33 (barrel/fermentation tank) Number of corresponding 18 12 12 12 fermentation tanks Corresponding fermentation 160 160 160 160 initial total sugar mean (g/L) Corresponding fermentation 162.5 157.0 159.0 158.5 acid production mean (g/L) Corresponding fermentation 52.5 64.7 57.2 56.6 period mean (h) Corresponding fermentation 101.6 98.1 99.4 99.1 conversion rate mean (%) Corresponding fermentation 3.10 2.43 2.78 2.80 strength mean (g/(h .Math. L))

[0059] A comparison of the technical effects of Example 1 and Comparative examples 1, 3 and 4 shows that: (1) as compared with the patent CN201410329652.X, the method according to the present invention simplifies the device system, and reduces the usage amount of the seed tank to ; the method also reduces operation steps, significantly improves the production efficiency, and saves the seed culture auxiliary time corresponding to each batch of fermentation tank by 12 h. (2) The method according to the present invention has a longer continuous running time, the start-stop auxiliary time can be shortened, the usage amount of the Aspergillus niger spores can be reduced by , and the culture cost of the Aspergillus niger spores can be greatly reduced. (3) Compared with the patent CN201410329652.X, when the fermentation initial sugar concentration is 16%, the fermentation strength is increased by 27.5%, and the fermentation conversion rate is increased by 3.4%.

Test Example 2

[0060] The technical effects of Example 2 and Comparative example 2 are compared, as shown in Table 2.

TABLE-US-00002 TABLE 2 Comparative Example 2 example 2 Seed continuous running time (h) 360 260 Number of seed tanks 1 3 Single seed tank auxiliary time (h) 34 480 Bran koji usage amount (barrel/fermentation tank) 0.16 0.33 Number of corresponding fermentation tanks 25 12 Corresponding fermentation initial total sugar mean (g/L) 180 180 Corresponding fermentation acid production mean (g/L) 181.2 172.6 Corresponding fermentation period mean (h) 59.4 78.2 Corresponding fermentation conversion rate mean (%) 100.7 95.9 Corresponding fermentation strength mean (g/(h .Math. L)) 3.03 2.30

[0061] A comparison of the technical effects of Example 2 and Comparative example 2 shows that: as compared with the patent CN201410329652.X, the method according to the present invention has greater advantages under high concentration fermentation conditions, when the fermentation initial sugar concentration is increased to 18%, the fermentation acid production exceeds 18%, the fermentation period is less than 60 h, the fermentation conversion rate exceeds 100%, the fermentation strength is increased by 31.6%, and the fermentation conversion rate is increased by 4.8%. Therefore, the method according to the present invention can fully achieve high concentration, high conversion rate and high efficiency fermentation production of citric acid, and play an important promotion role on the technical improvement in the citric acid industry.

Test Example 3

[0062] performances of Example 1 of the present invention and Comparative examples 5 and 6 are compared, as shown in Table 3.

TABLE-US-00003 TABLE 3 Example Comparative Comparative 1 example 5 example 6 Seed continuous running time (h) 288 136 152 Number of seed tanks 1 1 1 Single seed tank auxiliary time (h) 24 24 24 Bran koji usage amount 0.22 0.50 0.44 (barrel/fermentation tank) Number of corresponding 18 8 9 fermentation tanks Corresponding fermentation initial 160 160 160 total sugar mean (g/L) Corresponding fermentation acid 162.5 158.7 157.3 production mean (g/L) Corresponding fermentation 52.5 55.9 58.1 period mean (h) Corresponding fermentation conversion 101.6 99.2 98.3 rate mean (%) Corresponding fermentation strength 3.10 2.84 2.71 mean (g/(h .Math. L))

[0063] A comparison of the performances of Example 1 of the present invention and Comparative examples 5 and 6 shows that, when parameters, such as the residence time and C/N of the feed culture medium at the seed continuous culture stage are changed, the seed continuous running time will be significantly affected, the running time will be shortened, seed vitality will be declined rapidly, and the indexes corresponding to fermentation conversion rate and fermentation strength also will be significantly reduced.

[0064] The embodiments described above are merely preferred embodiments of the present invention and do not limit the present invention. It can be understood that, any improvement and variation directly derived or conceived by persons skilled in the art without departing from the spirit and concept of the present invention, should be deemed to fall within the protection scope of the present invention.