A method for producing citric acid by continuous fermentation of Aspergillus niger cells by membrane separation with residual sugar recovery

Abstract

The present invention discloses a method for producing citric acid by continuous fermentation of membrane-separated Aspergillus niger cells with residual sugar recovery. The method described in the present invention is: the Aspergillus niger seed liquid is connected to the fermentation medium, and the fermentation culture is used to produce citric acid; when the citric acid concentration reaches a certain concentration, the circulation pump is turned on, and the membrane separation liquid containing citric acid is continuously separated from the fermentation tank by using the membrane component, and the feed matrix is ​​continuously added to the fermentation tank at the same speed to keep the volume of the fermentation liquid balanced; citric acid is extracted from the membrane separation liquid; after adding pullulanase to the remaining extraction waste liquid for enzymolysis, glucose is added to prepare a glucose solution as a feed matrix, and then re-flowed to the fermentation tank. The present invention separates the fermentation process products and intercepts the cells through membrane separation technology, realizes the continuous fermentation of Aspergillus niger cells; the feed matrix is ​​prepared by using the extraction waste liquid and re-flowed to the fermentation tank, realizing the recovery of residual sugar.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to a method for producing citric acid by circulating and continuous fermentation of Aspergillus niger cells separated by membrane with residual sugar recovery. Background Art

[0002] Citric acid, also known as citric acid, has a chemical name of 3-carboxyl-3-hydroxyglutaric acid, a chemical formula of C6H8O7, and a relative molecular weight of 192.13Da. It is an important metabolite in the tricarboxylic acid cycle and is widely present in nature. It is a natural and harmless organic compound. The unique physical and chemical properties of citric acid, as well as its natural properties that are harmless to the human body and the environment, make it widely used in various fields. Due to its pleasant sour taste, it is often used to supplement fruit flavors in the food and beverage industry. Citric acid also helps prevent oxidation, avoid changes in taste and color, and maintain stable quality. At the same time, citric acid's excellent buffering capacity and outstanding metal ion chelating properties make it very suitable for use in cosmetics, health products and pharmaceuticals. In addition, citric acid also has medium-strong acidity, and has both hydroxyl and three carboxyl functional groups, as well as unique spatial chemical structure characteristics. It is widely used in the industrial production of organic compounds and is considered an important six-carbon platform compound. As the world's largest edible organic acid and a key chemical in the emerging bioeconomy, the efficient production of citric acid is of great practical significance.

[0003] At present, citric acid is mainly obtained by batch fermentation of Aspergillus niger, which has the disadvantages of low efficiency, much auxiliary work, long auxiliary time, low equipment utilization, and low degree of intelligence. The defects of the fermentation mode have become the key factors restricting the improvement of citric acid production efficiency, expansion of production capacity, and intelligent upgrading. In comparison, the continuous fermentation mode has greater advantages, but the special morphological structure of Aspergillus niger makes it difficult to maintain the concentration of bacteria in conventional continuous fermentation, and the use of Aspergillus niger to produce citric acid by continuous fermentation has become an industry problem. At the same time, a large amount of extraction waste liquid will be generated in the extraction section of citric acid industrial production. The extraction waste liquid contains rich nutrients and residual sugars, and is usually discharged after anaerobic and aerobic biological treatment meets the standards, which requires a large-scale land occupation and equipment investment, increases operating costs and causes waste of resources and energy. Therefore, it is an urgent problem to develop a process for producing citric acid by continuous fermentation of Aspergillus niger based on residual sugar recovery. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a method for producing citric acid by continuous fermentation of Aspergillus niger cells by membrane separation with residual sugar recovery. The present invention uses membrane separation technology to separate the fermentation products and trap the cells, thereby realizing continuous fermentation of Aspergillus niger cells; and uses the waste liquid extracted to prepare a feed matrix and re-flow it into the fermentation tank, thereby realizing residual sugar recovery.

[0005] The technical solution of the present invention is as follows:

[0006] A method for producing citric acid by membrane separation of Aspergillus niger fungi and continuous fermentation with residual sugar recovery, comprising the following steps:

[0007] (1) inoculating Aspergillus niger seed liquid into a fermentation medium to ferment and produce citric acid;

[0008] (2) When the citric acid concentration reaches 120-160 g / L, the circulation pump is turned on to continuously separate the membrane separation liquid containing citric acid from the fermentation tank using the membrane assembly at a rate of 0.4-0.75 L / h, and at the same time, the feed matrix is ​​continuously added to the fermentation tank at the same rate to keep the volume of the fermentation liquid balanced;

[0009] (3) extracting citric acid from the membrane separation liquid of step (2); adding pullulanase to the remaining extraction waste liquid for enzymatic hydrolysis, and then adding glucose to prepare a 150-165 g / L glucose solution as the feed matrix in step (2), which is re-added to the fermentation tank.

[0010] Furthermore, in step (1), the volume ratio of the Aspergillus niger seed liquid to the fermentation medium is 2:25 to 4:25.

[0011] Furthermore, in step (1), a seed culture medium with a total sugar content of 100 to 180 g / L and a C / N ratio of 15 to 30 is prepared, and Aspergillus niger spores are inoculated into the seed culture medium, and the spore concentration in the seed solution is 1×10 5 ~9×10 5 CFU / mL. The Aspergillus niger seed liquid was obtained by culturing at a temperature of 35-39°C, a stirring speed of 100-300 r / min, an air volume of 0.5-1.0 vvm, and a tank pressure of 0.07-0.1 MPa.

[0012] Furthermore, in step (1), the fermentation medium has an initial total sugar concentration of 150-170 g / L and a C / N of 55-90.

[0013] Furthermore, in step (1), the fermentation culture conditions are: fermentation temperature is 35-39°C, stirring speed is 100-300r / min, air volume is 0.5-1.0vvm, and tank pressure is 0.07-0.1Mpa.

[0014] Furthermore, in step (2), the circulation pump is a high-pressure centrifugal pump; the membrane assembly is a ceramic membrane with a pore size of 50 to 200 nm, a number of channels of 19, a channel diameter of 4 mm, and a membrane outer diameter of 30 mm.

[0015] Furthermore, in step (3), the method for extracting citric acid is calcium hydrogen method or chromatography.

[0016] Furthermore, in step (3), the extraction waste liquid contains 30-50 g / L residual total sugar, 20-40 g / L glucose, 0.3-0.8 g / L total nitrogen, and 0.5-5.0 g / L total acid.

[0017] Furthermore, in step (3), the enzymatic hydrolysis conditions of the pullulanase are: pH 4.0-7.5, and temperature 55-65°C.

[0018] Furthermore, in step (3), the pullulanase addition amount is 200 to 1000 U / L, and the enzymolysis time is 0.5 to 2 h.

[0019] The beneficial technical effects of the present invention are:

[0020] (1) The method of the present invention is based on residual sugar recovery and membrane separation technology to construct a green and efficient continuous fermentation mode; it realizes the recycling of fermentation residual sugar, avoids substrate loss, improves raw material utilization, and the fermentation conversion rate reaches 102.1%, which is 3.6% higher than that of batch fermentation.

[0021] (2) In the method of the present invention, the small molecule nutrient source is returned to the fermentation system to promote the synthesis of citric acid. The fermentation intensity of the new process reaches 3.66 g / L / h, which is 31.6% higher than that of batch fermentation.

[0022] (3) The present invention realizes water recycling, and the water consumption per unit of citric acid fermentation is reduced by about 80%.

[0023] (4) The present invention uses membrane separation technology to separate the fermentation products and retain the bacteria, thereby maintaining the concentration of the bacteria, recycling the retained bacteria, and extending the cycle of the bacteria to synthesize citric acid.

[0024] (5) The present invention first adds pullulanase to the fermentation residual sugar for treatment and then recycles it. The pullulanase can effectively hydrolyze the α-1,6 glycosidic bond, which is beneficial to the conversion of limit dextrin into glucose, thereby improving the utilization rate of residual sugar and avoiding the accumulation of residual sugar. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the citric acid concentration variation curve during the continuous fermentation process of the present invention.

[0026] Figure 2 This is the residual sugar concentration change curve during the continuous fermentation process. DETAILED DESCRIPTION

[0027] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0028] The present invention separates the fermentation process products and intercepts the thalline through membrane separation technology, and realizes the continuous fermentation of the Aspergillus niger thalline. However, research has found that in the continuous fermentation process, in order to maintain a higher citric acid synthesis rate, the substrate concentration needs to be greater than 20g / L, and the unused substrate and part of the nutrients are separated along with the citric acid membrane, causing raw material loss. In order to avoid the loss of substrate and nutrients and improve the raw material utilization rate, the present invention will extract the waste liquid back to the fermentation tank, realize the recovery of fermentation residual sugar and the recycling of the Aspergillus niger thalline. After the starchy raw materials (corn, wheat, cassava, sorghum) are liquefied and the Aspergillus niger secretes glucose amylase, the starch polymer is mostly hydrolyzed into glucose and then absorbed and utilized, but a large amount of limit dextrins remaining in the fermentation residual sugar cannot be hydrolyzed, and the accumulation of residual sugar is easily caused after the extraction waste liquid is reused.

[0029] In one embodiment of the present invention, in order to improve the utilization rate of residual sugar and avoid the accumulation of residual sugar, the fermented residual sugar is first treated with pullulanase and then recycled. Pullulanase can effectively hydrolyze α-1,6 glycosidic bonds, which is beneficial to the conversion of limit dextrin to glucose, thereby improving the utilization rate of residual sugar.

[0030] The present invention is further described below by way of examples and comparative examples.

[0031] The raw materials and reagents involved in the following examples are all commercially available products; Aspergillus niger is from the China Industrial Microbiological Culture Collection Center (CICC), with the collection number CICC 40021. The total sugar and reducing sugar were determined by Fehling titration; the nitrogen source was determined by Kjeldahl nitrogen determination; the citric acid was determined by 0.1429 mol / L NaOH titration; and the spore count was performed by a hemocytometer. Unless otherwise specified, the commonly used equipment and process methods in the field were used.

[0032] Example 1

[0033] A method for producing citric acid by membrane separation of Aspergillus niger fungi and continuous fermentation with residual sugar recovery, comprising the following steps:

[0034] (1) Prepare seed culture medium, control the total sugar content to 100 g / L, and the C / N ratio to 15; inoculate Aspergillus niger spores into the seed culture medium so that the final spore concentration is 9 × 10 5 CFU / mL; the Aspergillus niger seed liquid was obtained by culturing at a temperature of 35°C and a stirring speed of 100r / min.

[0035] (2) preparing a fermentation medium, controlling the total sugar content to be 150 g / L and the C / N content to be 55; inoculating 4 L of Aspergillus niger seed liquid into 25 L of fermentation medium; and producing citric acid under a fermentation culture at a temperature of 35° C., a stirring speed of 100 r / min, an air volume of 1.0 vvm, and a tank pressure of 0.1 MPa.

[0036] (3) When the citric acid concentration reaches 120 g / L, the circulation pump (the circulation pump is a high-pressure centrifugal pump) is turned on, and the membrane separation liquid containing citric acid is continuously separated from the fermentation tank at a rate of 0.75 L / h using a 50 nm membrane assembly (the membrane assembly is a ceramic membrane with 19 channels, a channel diameter of 4 mm, and a membrane outer diameter of 30 mm). At the same time, a feed matrix containing 150 g / L glucose is continuously added to the fermentation tank at the same rate to keep the volume of the fermentation liquid balanced.

[0037] (4) Citric acid was extracted from the membrane separation liquid using the calcium hydrogen method, and the obtained extraction waste liquid contained 50 g / L of residual total sugar, 40 g / L of glucose, 0.3 g / L of total nitrogen, and 0.5 g / L of total acid. The pH of the remaining extraction waste liquid was adjusted to 7.5, and pullulanase was added at a ratio of 1000 U / L, and enzymolysis was performed at 65°C for 0.5 h. Then, glucose was added to the extraction waste liquid after enzymolysis to prepare a feed matrix containing 150 g / L of glucose, and the feed matrix was re-added to the fermentation tank.

[0038] Example 2

[0039] A method for producing citric acid by membrane separation of Aspergillus niger fungi and continuous fermentation with residual sugar recovery, comprising the following steps:

[0040] (1) Prepare seed culture medium, control the total sugar content to 140 g / L, and the C / N ratio to 25; inoculate Aspergillus niger spores into the seed culture medium so that the final spore concentration is 5 × 10 5 CFU / mL; the Aspergillus niger seed liquid was obtained by culturing at a temperature of 37°C and a stirring speed of 200r / min.

[0041] (2) preparing a fermentation medium, controlling the total sugar content to 160 g / L and the C / N content to 70; inoculating 3 L of Aspergillus niger seed liquid into 25 L of fermentation medium; and producing citric acid under fermentation conditions of a temperature of 37° C., a stirring speed of 200 r / min, an air volume of 0.8 vvm, and a tank pressure of 0.08 MPa.

[0042] (3) When the citric acid concentration reaches 140 g / L, the circulation pump (the circulation pump is a high-pressure centrifugal pump) is turned on, and the membrane separation liquid containing citric acid is continuously separated from the fermentation tank at a rate of 0.5 L / h using a 100 nm membrane assembly (the membrane assembly is a ceramic membrane with 19 channels, a channel diameter of 4 mm, and a membrane outer diameter of 30 mm). At the same time, a feed matrix containing 160 g / L glucose is continuously added to the fermentation tank at the same rate to keep the volume of the fermentation liquid balanced.

[0043] (4) Citric acid was extracted from the membrane separation liquid using the calcium hydrogen method, and the obtained extraction waste liquid contained 35 g / L of residual total sugar, 30 g / L of glucose, 0.5 g / L of total nitrogen, and 2.5 g / L of total acid. The pH of the remaining extraction waste liquid was adjusted to 6.0, and pullulanase was added at a ratio of 500 U / L, and enzymolysis was performed at 60°C for 1 hour. Then, glucose was added to the extraction waste liquid after enzymolysis to prepare a feed matrix containing 160 g / L of glucose, and the feed matrix was re-added to the fermentation tank.

[0044] Example 3

[0045] A method for producing citric acid by membrane separation of Aspergillus niger fungi and continuous fermentation with residual sugar recovery, comprising the following steps:

[0046] (1) Prepare seed culture medium, control the total sugar content to 180 g / L, and the C / N ratio to 30; inoculate Aspergillus niger spores into the seed culture medium so that the final spore concentration is 1×10 5 CFU / mL; the Aspergillus niger seed liquid was obtained by culturing at a temperature of 39°C and a stirring speed of 300r / min.

[0047] (2) preparing a fermentation medium, controlling the total sugar content to 170 g / L and the C / N content to 90; inoculating 2 L of Aspergillus niger seed liquid into 25 L of fermentation medium; and producing citric acid under fermentation conditions of a temperature of 39° C., a stirring speed of 300 r / min, an air volume of 0.5 vvm, and a tank pressure of 0.07 MPa.

[0048] (3) When the citric acid concentration reaches 160 g / L, the circulation pump (the circulation pump is a high-pressure centrifugal pump) is turned on, and the membrane separation liquid containing citric acid is continuously separated from the fermentation tank at a rate of 0.4 L / h using a 200 nm membrane assembly (the membrane assembly is a ceramic membrane with 19 channels, a channel diameter of 4 mm, and a membrane outer diameter of 30 mm). At the same time, a feed matrix containing 165 g / L glucose is continuously added to the fermentation tank at the same rate to keep the volume of the fermentation liquid balanced.

[0049] (4) Citric acid was extracted from the membrane separation liquid by chromatography, and the obtained extraction waste liquid contained 30 g / L of residual total sugar, 20 g / L of glucose, 0.8 g / L of total nitrogen, and 5.0 g / L of total acid. The pH of the remaining extraction waste liquid was adjusted to 4.0, and pullulanase was added at a ratio of 200 U / L, and enzymolysis was performed at 55°C for 2 h. Then, glucose was added to the extraction waste liquid after enzymolysis to prepare a feed matrix containing 165 g / L of glucose, and the feed matrix was re-added to the fermentation tank.

[0050] Comparative Example 1

[0051] A method for producing citric acid by fermentation comprises the following steps:

[0052] (1) Prepare seed culture medium, control the total sugar content to 140 g / L, and the C / N ratio to 25; inoculate Aspergillus niger spores into the seed culture medium so that the final spore concentration is 5 × 10 5 CFU / mL; the Aspergillus niger seed liquid was obtained by culturing at a temperature of 37°C and a stirring speed of 200r / min.

[0053] (2) preparing a fermentation medium, controlling the total sugar content to 160 g / L and the C / N content to 70; inoculating 3 L of Aspergillus niger seed liquid into 25 L of fermentation medium; producing citric acid under a fermentation culture at a temperature of 37°C, a stirring speed of 200 r / min, an air volume of 0.8 vvm, and a tank pressure of 0.08 MPa; and stopping the fermentation when the residual sugar concentration is lower than 0.5 g / L.

[0054] Comparative Example 2

[0055] A method for producing citric acid by fermentation comprises the following steps:

[0056] (1) Prepare seed culture medium, control the total sugar content to 140 g / L, and the C / N ratio to 25; inoculate Aspergillus niger spores into the seed culture medium so that the final spore concentration is 5 × 10 5 CFU / mL; the Aspergillus niger seed liquid was obtained by culturing at a temperature of 37°C and a stirring speed of 200r / min.

[0057] (2) preparing a fermentation medium, controlling the total sugar content to 160 g / L and the C / N content to 70; inoculating 3 L of Aspergillus niger seed liquid into 25 L of fermentation medium; and producing citric acid under fermentation conditions of a temperature of 37° C., a stirring speed of 200 r / min, an air volume of 0.8 vvm, and a tank pressure of 0.08 MPa.

[0058] (3) Deionized water was added with glucose to prepare a 160 g / L glucose solution as a feed matrix. When the citric acid concentration reached 140 g / L, the circulation pump (the circulation pump was a high-pressure centrifugal pump) was turned on, and the membrane separation liquid containing citric acid was continuously separated from the fermenter at a rate of 0.45 L / h using a 100 nm membrane assembly (the membrane assembly was a ceramic membrane with 19 channels, a channel diameter of 4 mm, and a membrane outer diameter of 30 mm). At the same time, 160 g / L of glucose solution was continuously added to the fermenter at the same rate to keep the volume of the fermentation liquid balanced.

[0059] Comparative Example 3

[0060] A method for producing citric acid by fermentation comprises the following steps:

[0061] (1) Prepare seed culture medium, control the total sugar content to 100 g / L, and the C / N ratio to 15; inoculate Aspergillus niger spores into the seed culture medium so that the final spore concentration is 9 × 10 5CFU / mL; the Aspergillus niger seed liquid was obtained by culturing at a temperature of 35°C and a stirring speed of 100r / min.

[0062] (2) preparing a fermentation medium, controlling the total sugar content to be 150 g / L and the C / N content to be 55; inoculating 4 L of Aspergillus niger seed liquid into 25 L of fermentation medium; and producing citric acid under a fermentation culture at a temperature of 35° C., a stirring speed of 100 r / min, an air volume of 1.0 vvm, and a tank pressure of 0.1 MPa.

[0063] (3) When the citric acid concentration reaches 120 g / L, the circulation pump (the circulation pump is a high-pressure centrifugal pump) is turned on, and the membrane separation liquid containing citric acid is continuously separated from the fermenter at a rate of 0.9 L / h using a 50 nm membrane assembly (the membrane assembly is a ceramic membrane with 19 channels, a channel diameter of 4 mm, and a membrane outer diameter of 30 mm). At the same time, a feed matrix containing 150 g / L glucose is continuously added to the fermenter at the same rate to keep the volume of the fermentation liquid balanced.

[0064] (4) Citric acid was extracted from the membrane separation liquid by the calcium hydrogen method to obtain an extraction waste liquid. The pH of the remaining extraction waste liquid was adjusted to 7.5, and pullulanase was added at a ratio of 1000 U / L, and enzymatic hydrolysis was performed at 65°C for 0.5 h. Then, glucose was added to the extraction waste liquid after enzymatic hydrolysis to prepare a feed matrix containing 150 g / L glucose, and the feed matrix was re-added to the fermentation tank.

[0065] Comparative Example 4

[0066] A method for producing citric acid by fermentation comprises the following steps:

[0067] (1) Prepare seed culture medium, control the total sugar content to 100 g / L, and the C / N ratio to 15; inoculate Aspergillus niger spores into the seed culture medium so that the final spore concentration is 9 × 10 5 CFU / mL; the Aspergillus niger seed liquid was obtained by culturing at a temperature of 35°C and a stirring speed of 100r / min.

[0068] (2) preparing a fermentation medium, controlling the total sugar content to be 150 g / L and the C / N content to be 55; inoculating 4 L of Aspergillus niger seed liquid into 25 L of fermentation medium; and producing citric acid under a fermentation culture at a temperature of 35° C., a stirring speed of 100 r / min, an air volume of 1.0 vvm, and a tank pressure of 0.1 MPa.

[0069] (3) When the citric acid concentration reaches 120 g / L, the circulation pump (the circulation pump is a high-pressure centrifugal pump) is turned on, and the membrane separation liquid containing citric acid is continuously separated from the fermentation tank at a rate of 0.2 L / h using a 50 nm membrane assembly (the membrane assembly is a ceramic membrane with 19 channels, a channel diameter of 4 mm, and a membrane outer diameter of 30 mm). At the same time, a feed matrix containing 150 g / L glucose is continuously added to the fermentation tank at the same rate to keep the volume of the fermentation liquid balanced.

[0070] (4) Citric acid was extracted from the membrane separation liquid by the calcium hydrogen method to obtain an extraction waste liquid. The pH of the remaining extraction waste liquid was adjusted to 7.5, and pullulanase was added at a ratio of 1000 U / L, and enzymatic hydrolysis was performed at 65°C for 0.5 h. Then, glucose was added to the extraction waste liquid after enzymatic hydrolysis to prepare a feed matrix containing 150 g / L glucose, and the feed matrix was re-added to the fermentation tank.

[0071] Comparative Example 5

[0072] A method for producing citric acid by fermentation comprises the following steps:

[0073] (1) Prepare seed culture medium, control the total sugar content to 140 g / L, and the C / N ratio to 25; inoculate Aspergillus niger spores into the seed culture medium so that the final spore concentration is 5 × 10 5 CFU / mL; the Aspergillus niger seed liquid was obtained by culturing at a temperature of 37°C and a stirring speed of 200r / min.

[0074] (2) preparing a fermentation medium, controlling the total sugar content to 160 g / L and the C / N content to 70; inoculating 3 L of Aspergillus niger seed liquid into 25 L of fermentation medium; and producing citric acid under fermentation conditions of a temperature of 37° C., a stirring speed of 200 r / min, an air volume of 0.8 vvm, and a tank pressure of 0.08 MPa.

[0075] (3) When the citric acid concentration reaches 140 g / L, the circulation pump (the circulation pump is a high-pressure centrifugal pump) is turned on, and the membrane separation liquid containing citric acid is continuously separated from the fermentation tank at a rate of 0.5 L / h using a 100 nm membrane assembly (the membrane assembly is a ceramic membrane with 19 channels, a channel diameter of 4 mm, and a membrane outer diameter of 30 mm). At the same time, a feed matrix containing 160 g / L glucose is continuously added to the fermentation tank at the same rate to keep the volume of the fermentation liquid balanced.

[0076] (4) Citric acid was extracted from the membrane separation liquid using the calcium hydrogen method, and the obtained extraction waste liquid contained 35 g / L of residual total sugar, 30 g / L of glucose, 0.5 g / L of total nitrogen, and 2.5 g / L of total acid. The pH of the remaining extraction waste liquid was adjusted to 6.0, and glucoamylase was added at a ratio of 500 U / L, and enzymolysis was performed at 60°C for 1 hour. Then, glucose was added to the extraction waste liquid after enzymolysis to prepare a feed matrix containing 160 g / L of glucose, which was re-added to the fermentation tank.

[0077] Test Example 1

[0078] The effects of Examples 1-3 and Comparative Examples 1-2 were compared, and the results are shown in Table 1.

[0079] Table 1 Technical effects of Examples 1-3 and Comparative Examples 1-2

[0080]

[0081]

[0082] As shown in Table 1, comparative example 1 adopts batch fermentation, and 3 batches of fermentation can be carried out within 210 hours. The auxiliary time of the fermentation tank is 52 hours in total, 3 batches of seeds need to be cultured, and 120 mL of spore suspension is consumed; the final fermentation conversion rate is 98.5%, the fermentation intensity is 2.78 g / L / h, and the fermentation water consumption is 4.82 m 3 / t citric acid.

[0083] Comparative Example 2 uses membrane separation of Aspergillus niger for continuous fermentation, but does not recycle residual sugar and wastewater. The technical effect shows that the fermentation tank auxiliary time is 14h, only one batch of seeds needs to be cultured, and 40mL of spore suspension is consumed; the final fermentation conversion rate is 99.5%, the fermentation intensity is 3.10g / L / h, and the fermentation water consumption is 4.67m 3 / t citric acid.

[0084] In Example 1-3, membrane separation of Aspergillus niger bacteria with residual sugar recovery was used to produce citric acid by continuous fermentation. Compared with Comparative Example 1, Examples 1-3 achieved remarkable results. Continuous fermentation significantly shortened the auxiliary time of the fermentation tank; achieved bacterial recycling, and greatly reduced spore consumption and seed culture batches; achieved residual sugar recovery, and increased the fermentation conversion rate by 1.5-3.6%; small molecule nutrient sources returned to the fermentation system to promote citric acid synthesis, and the fermentation intensity increased by 14.9-31.6%; achieved water recycling, and the unit water consumption of citric acid fermentation decreased by 76.8-79.7%.

[0085] Compared with Comparative Example 2, the fermentation conversion rate, fermentation intensity and water consumption of Examples 1-3 are significantly improved, indicating that the method of the present invention has the advantages of high efficiency and greenness, and the use of the method of the present invention plays an important role in promoting the technological improvement of the citric acid industry.

[0086] Test Example 2

[0087] The technical effects of Example 1 and Comparative Examples 3-4 are compared. Figure 1 and Figure 2 shown.

[0088] The main difference between Example 1 and Comparative Examples 3 and 4 is the feeding rate. The results show that the feeding rate has a significant effect on the steady-state control of continuous fermentation. Comparative Example 3 shows that when the feeding rate is fast, the sugar replenishment rate is greater than the sugar consumption rate, and the residual sugar concentration gradually increases; at the same time, the citric acid separation rate is greater than the citric acid synthesis rate, causing the citric acid concentration to gradually decrease until a new equilibrium point is reached. Comparative Example 4 shows that when the feeding rate is slow, the sugar replenishment rate is less than the sugar consumption rate, and the residual sugar concentration gradually decreases; at the same time, the citric acid separation rate is less than the citric acid synthesis rate, causing the citric acid concentration to gradually increase until a new equilibrium point is reached. Compared with Comparative Examples 3 and 4, the feeding rate of Example 1 is controlled at an appropriate level, and the continuous fermentation is in a relatively stable state.

[0089] Test Example 3

[0090] The effects of Example 2 and Comparative Example 5 are compared, and the results are shown in Table 2.

[0091] Table 2 Technical effects of Example 2 and Comparative Example 5

[0092] project Example 2 Comparative Example 5 Total fermentation operation time (h) 210 210 Fermentation method continuous continuous Fermentation batch 1 1 Fermentation tank auxiliary time (h) 14 14 Spore usage (mL) 40 40 Seed culture batches 1 1 Total sugar consumption (g) 14210 14210 Total acid produced (g) 14208 14054 Fermentation conversion rate (%) 100.0 98.9 Fermentation intensity (g / h / L) 3.38 3.32 <![CDATA[Water consumption (m 3 / t citric acid)]]> 1.06 1.10

[0093] As shown in Table 2, the main difference between Example 2 and Comparative Example 5 is that the types of enzyme preparations used in the enzymatic treatment of the extraction waste liquid are different. As can be seen from Table 1, the use of enzyme preparations has a greater impact on the conversion rate. In Comparative Example 5, glucoamylase was used, which had a smaller enzymatic effect on the limit dextrin in the fermentation residual sugar, and the final fermentation conversion rate was 98.9%. In Example 2, pullulanase was used to further degrade the limit dextrin in the fermentation residual sugar, and the final fermentation conversion rate was 100%, the conversion rate was significantly improved, and the substrate utilization rate was higher.

[0094] Example 1 achieved the best fermentation conversion rate of 102.1% by optimizing the parameters, the fermentation intensity was 3.66 g / L / h, and the fermentation water consumption was 0.98 m 3 / t citric acid.

[0095] The present invention integrates membrane separation technology, fermentation residual sugar recovery and enzymatic hydrolysis technology to achieve stable operation of continuous fermentation. At the same time, fermentation residual sugar and water are recycled to avoid substrate loss, thus constructing an efficient and green citric acid fermentation process.

[0096] The above is only a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and changes directly derived or associated by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included in the protection scope of the present invention.

Claims

1. A method for producing citric acid by continuous fermentation of Aspergillus niger cells by membrane separation with residual sugar recovery, characterized in that: The method comprises the following steps: (1) Inoculating Aspergillus niger seed liquid into a fermentation medium to ferment and produce citric acid; (2) When the citric acid concentration reaches 120-160 g / L, the circulation pump is turned on to continuously separate the membrane separation liquid containing citric acid from the fermenter using the membrane assembly at a rate of 0.4-0.75 L / h. At the same time, the feed matrix is ​​continuously added to the fermenter at the same rate to keep the volume of the fermentation liquid balanced. (3) extracting citric acid from the membrane separation liquid of step (2); adding pullulanase to the remaining extraction waste liquid for enzymatic hydrolysis, and then adding glucose to prepare a 150-165 g / L glucose solution as the feed matrix in step (2), and re-adding it to the fermentation tank; In step (1), the fermentation medium is: the initial total sugar concentration is 150-170 g / L, and the C / N is 55-90; In step (2), the circulation pump is a high-pressure centrifugal pump; the membrane assembly is a ceramic membrane with a pore size of 50-200 nm, 19 channels, a channel diameter of 4 mm, and a membrane outer diameter of 30 mm; In step (3), the method for extracting citric acid is calcium hydrogen method or chromatography; In step (3), the extraction waste liquid contains 30-50 g / L of residual total sugar, 20-40 g / L of glucose, 0.3-0.8 g / L of total nitrogen, and 0.5-5.0 g / L of total acid.

2. The method according to claim 1, characterized in that In step (1), the volume ratio of the Aspergillus niger seed liquid to the fermentation medium is 2:25-4:

25.

3. The method according to claim 1, characterized in that In step (1), the Aspergillus niger seed solution is prepared by inoculating Aspergillus niger spores into a seed culture medium so that the final spore concentration is 1×10 5 ~9×10 5 CFU / mL culture is obtained; the total sugar of the seed culture medium is 100-180 g / L, and the C / N is 15-30.

4. The method according to claim 1, characterized in that In step (1), the fermentation culture conditions are: fermentation temperature is 35-39°C, stirring speed is 100-300 r / min, air volume is 0.5-1.0 vvm, and tank pressure is 0.07-0.1 Mpa.

5. The method according to claim 1, characterized in that In step (3), the enzymatic hydrolysis conditions of the pullulanase are: pH 4.0-7.5, and temperature 55-65°C.

6. The method according to claim 1, characterized in that In step (3), the pullulanase addition amount is 200-1000 U / L, and the enzymolysis time is 0.5-2 h.

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