Method for producing citric acid by fermentation

Abstract

The invention relates to the technical field of microbial fermentation, and discloses a method for producing citric acid through fermentation, according to the method, a seed solution with high mycelium concentration can be obtained in a seed culture stage, separated mycelia are directly inoculated to a fermentation culture medium, the proliferation time of the mycelia in a fermentation tank can be shortened, and the yield of citric acid is increased. In addition, fermentation clear liquid and extraction waste liquid are refluxed, so that the pressure of wastewater treatment can be relieved, and green production is realized.

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 fermentation. Background Art

[0002] Citric acid is an important organic acid, which is mainly produced by deep fermentation. It is widely used in food, medicine, chemical industry and other fields. At present, domestic citric acid is generally prepared by Aspergillus niger strain to culture spores. The spores are inoculated into seed culture medium and then transferred to fermentation medium after 18-30 hours of culture. The citric acid is fermented in a fermenter and then the fermentation liquid is separated to obtain mycelium and fermentation clear liquid. The mycelium is used to make feed or other by-products, and the fermentation clear liquid is extracted and refined to obtain citric acid products.

[0003] The traditional methods commonly used in the industry have the following problems: in the cultivation of Aspergillus niger spores in the seed tank, due to the limitations of culture medium nutrient preparation and process control, the concentration of dry mycelium in the seed liquid is low when the seed liquid is transferred to the fermentation tank; the ratio of seed liquid to fermentation tank culture volume is 8-12%. After the seed culture medium is connected to the fermentation tank, the mycelium content is only 0.8-1.44g / L. The amount of mycelium for general fermentation to produce citric acid is 15-20g / L, and the fermentation cycle is 60-70 hours. The early 0-10 hours of fermentation is the period of further expansion of the mycelium, and the actual acid production time of the mycelium is only 50-60 hours, which reduces the utilization rate of the fermentation tank; some unused proteins, inorganic phosphorus, metal ions, etc. in the seed culture medium are brought into the fermentation tank, which changes the nutritional components in the fermentation medium, leads to secondary growth of mycelium, affects the sugar-acid conversion rate, and causes the waste liquid after citric acid extraction to contain more ammonia nitrogen, inorganic phosphorus, metal ions, etc., increasing the pressure of environmental protection treatment. CN104087624B discloses a method for producing citric acid by continuous fermentation of Aspergillus niger, wherein the pellets in part of the fermentation liquid are dispersed to obtain hyphae of a certain size, and then the hyphae are cultured and fermented to produce citric acid. However, the step of dispersing the hyphae pellets into hyphae is difficult to control to obtain uniform hyphae fragments, and at the same time, the hyphae needs to be re-cultured to grow into the pellets, and there is still the problem that the culture time needs to be increased in the early stage of the fermentation tank to proliferate the mycelium. Summary of the invention

[0004] The purpose of the present invention is to overcome the problems existing in the prior art and provide a method for producing lemons by fermentation. The method can obtain a seed liquid with a high mycelium concentration, directly inoculate the separated mycelium into a fermentation medium, and reduce the proliferation time of the mycelium in a fermentation tank. In addition, the fermentation clear liquid and the extraction waste liquid are refluxed, which can reduce the pressure of wastewater treatment and realize green production.

[0005] In order to achieve the above object, the present invention provides a method for producing citric acid by fermentation, the method comprising: S1, inoculating Aspergillus niger spores into a seed culture medium for culturing to obtain a seed solution; S2, performing solid-liquid separation on the seed liquid to obtain the first mycelium and culture supernatant; S3, inoculating the first mycelium into the first fermentation medium to perform a first fermentation, and performing solid-liquid separation on the fermentation liquid after the first fermentation to obtain a second mycelium and a fermentation clear liquid; S4, inoculating the obtained second mycelium into the second fermentation medium for second fermentation, and performing solid-liquid separation on the fermentation liquid after the second fermentation to obtain the third mycelium and the fermentation clear liquid; S5. Optionally, take a third mycelium and repeat the step of S4 at least once; S6. Collect the obtained fermentation clear liquid, and obtain extraction waste liquid and citric acid through extraction.

[0006] Through the above technical solution, the present invention has at least the following beneficial effects: (1) The seed solution culture method provided by the present invention shortens the growth time of mycelium, improves mycelium activity, and can obtain seed solution with a high mycelium concentration.

[0007] (2) The present invention directly inoculates the separated mycelium into the fermentation medium, which reduces the mycelium reproduction time in the early stage of fermentation and shortens the fermentation cycle.

[0008] (3) In the present invention, the culture supernatant obtained after the solid-liquid separation of the seed liquid and the extraction waste liquid after the citric acid extraction can be recycled again to achieve green production. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 is a schematic diagram of the fermentation process of Example 1; Figure 2 It is a schematic diagram of the fermentation process of Comparative Examples 1 and 2. DETAILED DESCRIPTION

[0010] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0011] The present invention provides a method for producing citric acid by fermentation, the method comprising: S1, inoculating Aspergillus niger spores into a seed culture medium for culturing to obtain a seed solution; S2, performing solid-liquid separation on the seed liquid to obtain the first mycelium and culture supernatant; S3, inoculating the first mycelium into the first fermentation medium to perform a first fermentation, and performing solid-liquid separation on the fermentation liquid after the first fermentation to obtain a second mycelium and a fermentation clear liquid; S4, inoculating the obtained second mycelium into the second fermentation medium for second fermentation, and performing solid-liquid separation on the fermentation liquid after the second fermentation to obtain the third mycelium and the fermentation clear liquid; S5. Optionally, take a third mycelium and repeat step S4 at least once; S6. Collect the obtained fermentation clear liquid, and obtain extraction waste liquid and citric acid through extraction.

[0012] In the present invention, the separated mycelium is directly inoculated into the fermentation medium, which can reduce the growth time of the mycelium in the fermentation medium and shorten the culture cycle.

[0013] According to the present invention, there is no specific limitation on the inoculation method of Aspergillus niger spores, as long as the Aspergillus niger spores can be inoculated into the seed culture medium.

[0014] In some specific embodiments of the present invention, the inoculation method of Aspergillus niger spores is to take out the sterilized seed culture medium from the seed tank with a sterile steel cylinder, inoculate the Aspergillus niger spores into the steel cylinder to prepare a spore suspension, and further transfer the spore suspension to the seed culture medium for culturing to obtain a seed liquid.

[0015] According to the present invention, the inoculation amount of Aspergillus niger spores is 1×10 5 -9×10 5 indivual.

[0016] According to the present invention, the Aspergillus niger spores are subjected to heat shock treatment before being inoculated into the seed culture medium.

[0017] According to the present invention, the Aspergillus niger spores are subjected to heat shock treatment after being inoculated into the seed culture medium.

[0018] Furthermore, the heat shock treatment temperature is 40-45°C, and the heat shock treatment time is 20-40 minutes.

[0019] According to the present invention, in step S1, the seed culture medium is a mixture of a first solvent, a first carbon source, a first nitrogen source and a first phosphorus source, wherein the dosage of the first carbon source, the first nitrogen source and the first phosphorus source is such that the content of reducing sugar in the seed culture medium is 50-70 g / L, the molar ratio of inorganic nitrogen to organic nitrogen is 1:1-4, the concentration of total nitrogen is 2-2.5 g / L, the concentration of total phosphorus is 700-800 mg / L, and the pH is 6-6.5.

[0020] Furthermore, the first solvent is water and / or culture supernatant.

[0021] In some specific embodiments of the present invention, the first solvent is culture supernatant.

[0022] Furthermore, the first carbon source is glucose, which can be provided by a saccharified liquid obtained by saccharifying a starchy raw material liquid.

[0023] Furthermore, the first nitrogen source is at least one of nitrogen-containing inorganic salts, corn steep liquor and soybean meal hydrolyzate.

[0024] Furthermore, the nitrogen-containing inorganic salt is ammonium sulfate and / or ammonium nitrate.

[0025] Furthermore, the first phosphorus source is potassium dihydrogen phosphate.

[0026] According to the present invention, in step S1, the culturing method is: when the pH of the culture system is reduced to 4.5-5, by adding an alkaline compound, a second nitrogen source, a second carbon source and a second phosphorus source, the concentration of reducing sugar in the culture medium is maintained at 45-70 g / L, the concentration of total nitrogen is maintained at 1.6-2.5 g / L, the concentration of total phosphorus is maintained at 450-600 mg / L, and the pH is not lower than 4.5.

[0027] In the present invention, through the above control process, the growth time of mycelium is shortened, the vitality of mycelium is improved, and the seed liquid with high mycelium concentration can be obtained.

[0028] In some specific embodiments of the present invention, in step S1, the culturing method is: when the pH of the culture system is reduced to 4.5-5, by adding an alkaline compound, a second nitrogen source, a second carbon source and a second phosphorus source, the concentration of reducing sugar in the culture medium is maintained at 50-60 g / L, the concentration of total nitrogen is maintained at 1.6-2 g / L, the concentration of total phosphorus is maintained at 500-550 mg / L, and the pH is not lower than 4.5.

[0029] In the present invention, the culture conditions of the seed culture medium are not particularly limited, as long as the purpose of the present invention can be achieved.

[0030] In some specific embodiments of the present invention, the culture conditions include: a stirring speed of 100-150 rpm, a pressure of 0.1-0.15 MPa, a temperature of 36-37°C, a ventilation volume of 700-900 Nm³ / h, and the ventilation volume makes the dissolved oxygen value in the seed culture medium not less than 20 vol%.

[0031] Furthermore, the alkaline compound is ammonia water and / or sodium hydroxide.

[0032] Furthermore, the second nitrogen source is ammonium sulfate.

[0033] Furthermore, the second carbon source is glucose.

[0034] Furthermore, the second phosphorus source is potassium dihydrogen phosphate.

[0035] According to the present invention, the culturing method makes the concentration of mycelium in the seed solution be 50-70 g / L.

[0036] According to the present invention, the method for solid-liquid separation of the seed liquid is not particularly limited, as long as it can be used for solid-liquid separation of culture supernatant and mycelium.

[0037] In some specific embodiments of the present invention, the solid-liquid separation is achieved by high-speed centrifugation.

[0038] According to the present invention, the time interval between the seed liquid entering the solid-liquid separation device and the mycelium being inoculated into the fermentation medium is within 10 minutes, and preferably the seed liquid enters directly without stopping.

[0039] In the present invention, the separated mycelium does not need to be further screened and cultured, but is directly inoculated into the fermentation medium. According to the present invention, the culture supernatant obtained after the solid-liquid separation of the seed liquid can be refluxed and used as the solvent for the next batch of seed culture medium.

[0040] According to the present invention, in steps S3-S5, the inoculation amount of the mycelium is independently 12-18 g per liter of fermentation medium.

[0041] According to the present invention, the fermentation medium used in steps S3-S5 is independently a mixture of a second solvent and a third carbon source, wherein the amount of the third carbon source is such that the content of reducing sugar in the fermentation medium is 150-220 g / L and the pH is 2.5-3.5.

[0042] Furthermore, the second solvent is water and / or extraction waste liquid.

[0043] Furthermore, the third carbon source is glucose, which can be provided by a saccharified liquid obtained by saccharifying a starchy raw material liquid.

[0044] According to the present invention, the fermentation conditions in steps S3-S5 independently include: The fermentation temperature is 36-38°C, the pressure is 0.08-0.10MPa, and the stirring speed is 80-100rpm; The ventilation rate is 4500-5500Nm³ / h, and the ventilation rate makes the oxygen concentration in the exhaust gas 16-17vol%.

[0045] According to the present invention, the fermentation conditions in steps S3-S5 independently include: During the fermentation process, the ammonia nitrogen concentration in the fermenter was detected every 1-3 hours. When the ammonia nitrogen concentration was lower than 20-25 mg / L, the nitrogen source flow pump was turned on to pump the third nitrogen source into the fermenter.

[0046] Furthermore, the third nitrogen source is ammonium sulfate.

[0047] According to the present invention, the method for solid-liquid separation of the fermentation liquid is not particularly limited, as long as it can be used for solid-liquid separation of the fermentation liquid and the mycelium.

[0048] In some specific embodiments of the present invention, the solid-liquid separation is achieved by high-speed centrifugation.

[0049] According to the present invention, in step S5, taking the third mycelium and repeating step S4 at least once (preferably 1-5 times) means that the steps S3 and S4 can be repeated, the obtained Nth mycelium is inoculated into the Nth fermentation medium for the Nth fermentation, and the fermentation liquid after the Nth fermentation is subjected to solid-liquid separation to obtain the N+1th mycelium and the fermentation clear liquid (N is preferably 3-7).

[0050] According to a preferred embodiment of the present invention, when the reducing sugar in the first fermentation medium is consumed to 5-10g / L, the fermentation liquid is separated into solid and liquid. If the acid production intensity of the mycelium is not less than 2.5g / h / L at this time, the separated mycelium directly enters the second fermentation medium for the second fermentation, and the separated fermentation clear liquid is used as the second fermentation medium after supplementing the carbon source and adjusting the pH, and the cycle is repeated to carry out fermentation processes including but not limited to the third fermentation, the fourth fermentation, and the fifth fermentation. When the reducing sugar in the fermentation medium is consumed to 5-10g / L, the acid production intensity of the mycelium is less than 2.5g / h / L, and the fermentation process of this batch is terminated. The separated fermentation clear liquid enters the extraction process to obtain citric acid and extraction waste liquid.

[0051] According to the present invention, the method for extracting citric acid is not particularly limited, as long as it can be used to extract citric acid.

[0052] In some specific embodiments of the present invention, the extraction method is calcium hydrogen method.

[0053] According to the present invention, the Aspergillus niger is deposited in the General Microbiological Center of China Microorganism Culture Collection Committee (abbreviated as CGMCC), with the deposit number CGMCC No.40040, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, which has been disclosed in CN117925414A.

[0054] The inventors of the present invention found that the mycelium content in the seed liquid was significantly increased when the method of the present invention was combined with the preferred strain, and during the fermentation process, the conversion rate of citric acid was also significantly improved.

[0055] The present invention will be described in detail below by way of examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0056] Unless otherwise specified, the reagents and materials used in the following examples were purchased from regular chemical reagent suppliers and were of analytical grade.

[0057] The spore suspension was prepared as follows: Use a sterile steel bottle to take out 50L of sterilized seed culture medium from the seed tank, and inoculate Aspergillus niger spores into the steel bottle to prepare a spore suspension. The spore suspension needs to be heat-shocked at 42°C for 30 minutes before inoculation. The number of spores per milliliter of spore suspension is 1.0×10 13 -1.5×10 13 indivual.

[0058] In the following embodiments, The reducing sugar was detected by Fehling's reagent method.

[0059] The detection method of total nitrogen refers to GB / T 609-2006.

[0060] The test method for total phosphorus refers to GB / T 11893-1989 Water quality - Determination of total phosphorus - Ammonium molybdate spectrophotometry.

[0061] The mycelium concentration was calculated as follows: Take 200 mL of the cultured seed liquid and use a high-speed centrifuge at 6000 rpm for solid-liquid separation to obtain mycelium W1g. Take 5 g of mycelium and dry it at 105°C to a constant weight of W2g. Calculate the mycelium concentration per unit volume = W2÷5×W1÷200×1000, unit: g / L.

[0062] The detection method of ammonia nitrogen in the fermentation process is as follows: after the fermentation liquid is filtered with filter paper, 2 mL of the filtered clear liquid is taken, and the ammonia nitrogen concentration W1 mg / L in the fermentation liquid is detected by a fully automatic Kjeldahl nitrogen analyzer. The ammonia nitrogen control concentration during the fermentation process is W2 mg / L. When the ammonia nitrogen concentration is lower than W2 mg / L, the ammonium sulfate solution flow pump is turned on. The ammonia nitrogen concentration in the ammonium sulfate solution is 100 g / L. The volume of ammonium sulfate solution pumped into the fermentation tank is calculated based on the amount of ammonia nitrogen missing, that is, the volume of ammonium sulfate pumped (L) is (W2-W1) mg / L×450m³×1000L / m³÷100 mg / L.

[0063] The acidity detection method during the fermentation process is as follows: filter the fermentation broth with filter paper, take 1 mL of the filtered clear liquid, add phenolphthalein indicator and then use 0.1429 mol / L sodium hydroxide standard solution for neutralization titration. The volume of sodium hydroxide standard solution consumed × 10 is the acid concentration of the fermentation broth at that time point, in g / L.

[0064] The acidity difference and time difference between two adjacent sample points can be used to calculate the acid production per unit fermentation liquid per unit time, that is, fermentation intensity (g / L / h) = acid concentration difference ÷ time difference.

[0065] Conversion rate (%) = (fermentation liquid acidity at the end of fermentation ÷ reducing sugar concentration at 0 hours of fermentation) × 100% The specific operation of extracting citric acid is carried out according to the method for extracting citric acid by the calcium hydrogen method disclosed in CN102617333A.

[0066] Example 1 according to Figure 1 The fermentation process diagram was used for the experiment.

[0067] (1) Corn starch slurry liquefaction, slurry dry matter concentration of 30wt%, starch slurry volume of 200m 3 Calcium carbonate was added to fine-tune the pH to 5.2, 18 kg of Novozymes a-glucoamylase (II) was added, and the mixture was sprayed to 108°C through a jet liquefier and then flashed to 93°C for 120 minutes. The iodine test showed no blue reaction when sampling, and a liquefied liquid was obtained, the reducing sugar concentration of which was 327 g / L.

[0068] 15.3m 3 Add the above liquefied liquid to the seed tank, add 5.8m³ corn slurry, 27.2kg dipotassium hydrogen phosphate, 123kg ammonium sulfate, and then add the clear liquid separated from the seed tank to make the volume 95m³ 3 , raise the temperature to 121°C, keep warm for 25 minutes, then cool to 36°C for use.

[0069] The reducing sugar content was 50.1 g / L, the total nitrogen content was 2 g / L, and the volume of the tank was 100 m³.

[0070] (3) The spore suspension was transferred to the prepared seed tank for culture. The seed tank culture volume was 100m³ and inoculated with Aspergillus niger spores with the preservation number of CGMCC No.40040. The spore concentration was 1.2×10 5 / mL, the initial conditions of the seed tank were a stirring speed of 100rpm, a tank pressure of 0.12Mpa, a temperature of 36℃, an initial ventilation volume of 800Nm³ / h, and the dissolved oxygen value was maintained at 20vol% during the culture process.

[0071] (4) After culturing in the seed tank for 10 hours, when the pH drops to 4.5, turn on the sodium hydroxide dissolved oxygen flow pump and adjust the culture medium by adding 40wt% ammonium sulfate, 20wt% ammonia water, 30wt% potassium dihydrogen phosphate, 20wt% sodium hydroxide solution, 50wt% glucose solution, etc. The total nitrogen, reducing sugar and total phosphorus were tested every 2 hours to maintain the total nitrogen, reducing sugar and total phosphorus concentrations in the culture medium at 1.8g / L, 45g / L and 450mg / L, respectively.

[0072] (5) After 28 hours of cultivation in the seed tank, the mycelium concentration is 51 g / L. Immediately stop all feed pumps and pump the seed liquid into the prepared disc centrifuge for solid-liquid separation. The first mycelium obtained by separation is directly inoculated into the first fermentation medium for fermentation and acid production.

[0073] (6) After the first fermentation medium was inoculated, the reducing sugar content was 151 g / L, the pH was 2.5, and the inoculation amount of the mycelium was 10.5 g / L. The ammonia nitrogen concentration was tested every 2 hours, and the flow acceleration and flow addition amount of ammonium sulfate were adjusted according to the ammonia nitrogen concentration to maintain the ammonia nitrogen concentration in the culture medium at 20 mg / L. The acidity and reducing sugar content during the fermentation process were tested, and the fermentation intensity at two time points was calculated. At 44 hours, the acidity was 142 g / L and the reducing sugar content was 27 g / L. At 48 hours, the acidity was 154 g / L and the reducing sugar content was 15 g / L. The fermentation intensity in the period from 44 hours to 48 hours was 3 g / L / h. At 51 hours, the acidity was 160 g / L and the reducing sugar content was 9 g / L. The fermentation intensity at this time was 3.1 g / L / h, stop adding ammonium sulfate, pump the fermentation liquid into the disc centrifuge for solid-liquid separation, and the mycelium obtained by centrifugation directly enters the second fermentation medium, and the second, third, fourth, fifth and sixth fermentation processes are carried out in sequence. In the sixth fermentation process, the acidity is 143g / L and the reducing sugar is 21g / L at 56 hours, the acidity is 152g / L and the reducing sugar is 13g / L at 60 hours, and the fermentation intensity is 2.25g / L / h. Continue to ferment until 64 hours, the acidity is 161g / L and the reducing sugar is 3g / L, then stop fermentation, the fermentation liquid enters the disc centrifuge for separation, the mycelium is dried, and the clear liquid is subjected to the calcium hydrogen method to extract citric acid. This continuous fermentation ends.

[0074] Example 2 (1) Corn starch was liquefied to a slurry concentration of 30 wt% and a volume of 200 m³. Calcium carbonate was added to fine-tune the pH to 5.2. 18 kg of Novozymes α-glucoamylase (II) was added. The slurry was sprayed to 108°C by a jet liquefier and then flashed to 93°C for 120 minutes. The iodine test of the sample showed no blue reaction, and a liquefied liquid was obtained. The reducing sugar concentration of the liquefied liquid was 327 g / L.

[0075] (2) 21.4m 3 Add the above liquefied liquid to the seed tank, add 5.8m³ corn syrup, 27.20kg dipotassium hydrogen phosphate, 311kg ammonium sulfate, and then add the clear liquid separated from the seed tank to make the volume 95m³ 3 , raise the temperature to 121°C, keep warm for 25 minutes, then cool to 36°C for use.

[0076] The sampling measured reducing sugar content of 69.5g / L, total nitrogen content of 2.4g / L, and the volume of the tank was 100m³.

[0077] (3) The spore suspension was transferred to the prepared seed tank for culture. The culture volume of the seed tank was 100 m³ and inoculated with Aspergillus niger spores with the preservation number of CGMCC No. 40040. The spore concentration was 1.1×10 5 The initial conditions of the seed tank were a stirring speed of 100 rpm, a tank pressure of 0.12 MPa, a temperature of 36 °C, an initial ventilation volume of 800 Nm³ / h, and the process was controlled according to the dissolved oxygen value to maintain the dissolved oxygen at 25 vol%.

[0078] (4) After culturing in the seed tank for 10 hours, when the pH drops to 4.5, turn on the sodium hydroxide dissolved oxygen flow pump and adjust the culture medium by adding 40wt% ammonium sulfate, 20wt% ammonia water, 30wt% potassium dihydrogen phosphate, 20wt% sodium hydroxide solution, 50wt% glucose solution, etc. The total nitrogen, reducing sugar and total phosphorus were tested every 2 hours to maintain the concentrations of total nitrogen, reducing sugar and total phosphorus in the culture medium at 2g / L, 50g / L and 500mg / L respectively.

[0079] (5) After culturing in the seed tank for 20 hours, the mycelium concentration reaches 70 g / L. Immediately stop all feed pumps and pump the seed liquid into the prepared disc centrifuge for solid-liquid separation. The first mycelium obtained by separation is directly inoculated into the first fermentation medium for fermentation and acid production.

[0080] (6) After the first fermentation medium was inoculated, the reducing sugar content was 221 g / L, the pH was 2.7, and the inoculation amount of the mycelium was 17.6 g / L. The ammonia nitrogen concentration was tested every 2 hours, and the flow acceleration and flow addition amount of ammonium sulfate were adjusted according to the ammonia nitrogen concentration to maintain the ammonia nitrogen concentration in the culture medium at 20 mg / L. The acidity and reducing sugar during the fermentation process were tested, and the fermentation intensity at two time points was calculated. The acidity at 64 hours was 211.3 g / L and the reducing sugar was 27 g / L. The acidity at 68 hours was 225.5 g / L and the reducing sugar was 15 g / L. The fermentation intensity in the period from 64 hours to 68 hours was 3.0 g / L / h. The acidity at 72 hours was 238.2 g / L and the reducing sugar was 8 g / L. The fermentation intensity at this time was 3.3 g / L / h. The addition of ammonium sulfate was stopped, and the fermentation liquid was pumped into the disc centrifuge. Solid-liquid separation was performed, and the mycelium obtained by centrifugation directly entered the second fermentation medium, and the second, third, fourth and fifth fermentation processes were carried out in sequence. In the fifth fermentation process, the acidity was 217.5g / L and the reducing sugar was 21g / L at 84 hours, the acidity was 227g / L and the reducing sugar was 13g / L at 88 hours, and the fermentation intensity in the 84-88 hour period was 2.375g / L / h, which was lower than 2.50g / L / h. After the end of this tank, the mycelium separated was no longer connected to the next tank. The fifth fermentation continued until 91 hours, when the acidity was 234g / L and the reducing sugar was 2g / L, the fermentation was stopped, the fermentation liquid entered the disc centrifuge for separation, the mycelium was dried, and the clear liquid was subjected to the calcium hydrogen method to extract citric acid. This continuous fermentation was terminated.

[0081] Example 3 Fermentation was carried out in the same manner as in Example 1, except that the Aspergillus niger used was replaced by a strain with a deposit number of CGMCCNO.19029 (disclosed in CN113667607A).

[0082] Comparative Example 1 according to Figure 2 The fermentation process diagram was used for the experiment.

[0083] (1) Corn starch slurry liquefaction, slurry dry matter concentration of 30wt%, starch slurry volume of 200m³, add 15kg of Novozymes a-glucoamylase (II), spray to 108℃ through the jet liquefier, then flash to 93℃, maintain for 120 minutes, take a sample and pass the iodine test, and obtain liquefied liquid 212m 3 The reducing sugar concentration of the liquefied liquid is 328g / L.

[0084] (2) 13.7m 3 The liquefied liquid is connected to the seed tank, and 2.7m³ corn slurry, 40kg ammonium sulfate, 7.9kg potassium dihydrogen phosphate are added, and then water is added to make the volume 43m 3, raise the temperature to 121℃, keep it warm for 25 minutes, then cool it down to 36℃ for use. The reducing sugar content and total nitrogen content of the sample were 100g / L and 2g / L, respectively, and the volume of the tank was 45m³.

[0085] (3) Inoculate spores of Aspergillus niger with a collection number of CGMCC No. 40040 at a spore concentration of 3 × 10 5 / mL, at a ventilation rate of 1600Nm 3 / h, stirring rate 95rpm, tank pressure 0.10MPa, culture for 30 hours until the seeds mature, at which time the pH is 1.95, the acidity is 15g / L, the mycelium concentration is 9g / L, and it is ready to be transferred to the fermentation tank.

[0086] (4) The remaining 198.3m 3 The liquefied liquid is connected to the fermentation tank, 9.57m³ corn slurry is added, and then water is added to make the volume 405m 3 , the temperature was raised to 80℃, kept warm for 30 minutes and then cooled to 37℃, and the mature seed liquid was inoculated at a ratio of 10vol% of the fermentation medium. After inoculation, the reducing sugar was 149g / L, the total nitrogen was 0.86g / L, and the ventilation volume was 4500Nm 3 / h, stirring speed 95rpm, tank pressure 0.08Mpa, culture until the reducing sugar is less than 2g / L to stop fermentation, fermentation cycle 55 hours, acidity is 146g / L. Fermentation is stopped, the fermentation liquid enters the disc centrifuge for separation, the mycelium is dried, and the clear liquid is subjected to the calcium hydrogen method to extract citric acid.

[0087] Comparative Example 2 according to Figure 2 The fermentation process diagram was used for the experiment.

[0088] (1) Corn starch slurry was adjusted and liquefied. The dry matter concentration of the slurry was 32wt%, and the volume of the starch slurry was 300m³. 16kg of Novozymes a-glucoamylase (II) was added. The slurry was sprayed to 108℃ by a jet liquefier and then flashed to 93℃ for 150 minutes. The sample was tested and passed the iodine test. 310m³ of liquefied liquid was obtained. 3 The reducing sugar concentration of the liquefied liquid is 342 g / L.

[0089] (2) 16.5m 3 The liquefied liquid is connected to the seed tank, and 1.7m³ corn slurry, 127kg ammonium sulfate, 7.9kg potassium dihydrogen phosphate are added, and then water is added to make the volume 43m 3 , raise the temperature to 121℃, keep it warm for 25 minutes, then cool it down to 36℃ for use. The sample was measured to have 120g / L reducing sugar, 2.4g / L total nitrogen, and 45m³ tank volume.

[0090] (3) Inoculate spores of Aspergillus niger with a collection number of CGMCC No. 40040 at a spore concentration of 5 × 10 5 / mL culture medium was inoculated and the ventilation volume was 1600Nm 3 / h, stirring rate 95rpm, tank pressure 0.10MPa, culture for 28 hours until the seeds mature, at which time the pH is 2.00, the acidity is 1.60g / 100mL, the mycelium concentration is 11g / L, and it is ready to be transferred to the fermentation tank.

[0091] (4) The remaining 293.5m 3 The liquefied liquid is connected to the fermentation tank, 13.7m³ corn slurry is added, and then water is added to make the volume 405m 3 , raise the temperature to 80℃, keep it warm for 30 minutes, then cool it down to 37℃, inoculate the mature seed liquid at a ratio of 10vol% of the fermentation medium, and the reducing sugar after inoculation is 221g / L and the total nitrogen is 1.3g / L, ventilation volume 4500Nm 3 / h, stirring speed of 95rpm, tank pressure of 0.08Mpa, fermentation was stopped when the reducing sugar concentration was lower than 3g / L, the fermentation cycle was 84 hours, the acidity was 215g / L, the fermentation liquid entered the disc centrifuge for separation, the mycelium was dried, and the clear liquid was subjected to calcium hydrogen method to extract citric acid.

[0092] Table 1

[0093] Table 2

[0094] As can be seen from Table 1, the seed culture method provided by the present invention is used in combination with Aspergillus niger with a deposit number of CGMCC No. 40040, which can greatly improve the mycelium concentration of the seed liquid, thereby eliminating the time required for the mycelium to continue to grow after being connected to the fermenter, and significantly improving the fermentation intensity of the fermenter. At the same time, as can be seen from Table 2, after the mycelium is inoculated, the mycelium directly produces acid in the fermenter, reducing the carbon source consumption caused by the growth of the mycelium, so that the sugar-acid conversion rate of the fermenter is significantly improved.

[0095] In addition, since the present invention only contains carbon source and ammonia nitrogen in the fermentation tank culture medium, the culture supernatant after solid-liquid separation of the seed tank culture medium and the extracted waste liquid after citric acid extraction and separation are reused, and no waste liquid is discharged in the entire fermentation process, thereby achieving clean and green production.

[0096] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for producing citric acid by fermentation, characterized in that: The method comprises: S1, inoculating Aspergillus niger spores into a seed culture medium for culturing to obtain a seed solution; S2, performing solid-liquid separation on the seed liquid to obtain the first mycelium and culture supernatant; S3, inoculating the first mycelium into the first fermentation medium to perform a first fermentation, and performing solid-liquid separation on the fermentation liquid after the first fermentation to obtain a second mycelium and a fermentation clear liquid; S4, inoculating the obtained second mycelium into the second fermentation medium for second fermentation, and performing solid-liquid separation on the fermentation liquid after the second fermentation to obtain the third mycelium and the fermentation clear liquid; S5. Optionally, take a third mycelium and repeat the step of S4 at least once; S6. Collect the obtained fermentation clear liquid, and obtain extraction waste liquid and citric acid through extraction.

2. The method according to claim 1, wherein: In step S1, the Aspergillus niger spores are subjected to heat shock treatment before being inoculated into the seed culture medium; And / or, the Aspergillus niger spores are subjected to heat shock treatment after being inoculated into the seed culture medium.

3. The method according to claim 1 or 2, wherein: In step S1, the seed culture medium is a mixture of a first solvent, a first carbon source, a first nitrogen source and a first phosphorus source, wherein the first carbon source, the first nitrogen source and the first phosphorus source are used in such amounts that the content of reducing sugar in the seed culture medium is 50-70 g / L, the molar ratio of inorganic nitrogen to organic nitrogen is 1:1-4, the concentration of total nitrogen is 2-2.5 g / L, the concentration of total phosphorus is 700-800 mg / L, and the pH is 6-6.5; And / or, the first solvent is water and / or culture supernatant.

4. The method according to claim 1 or 2, wherein: In step S1, the culturing method is: when the pH of the culture system is reduced to 4.5-5, by adding an alkaline compound, a second nitrogen source, a second carbon source and a second phosphorus source, the concentration of reducing sugar in the culture medium is maintained at 45-70 g / L, the concentration of total nitrogen is maintained at 1.6-2.5 g / L, the concentration of total phosphorus is maintained at 450-600 mg / L, and the pH is not lower than 4.

5.

5. The method according to claim 1 or 2, wherein: The culture method makes the concentration of mycelium in the seed liquid be 50-70 g / L.

6. The method according to claim 1 or 2, wherein: In steps S3-S5, the inoculation amount of mycelium is independently 12-18 g per liter of fermentation medium.

7. The method according to claim 1 or 2, wherein: The fermentation medium used in steps S3-S5 is independently a mixture of the second solvent and the third carbon source, wherein the amount of the third carbon source is such that the content of reducing sugar in the fermentation medium is 150-220 g / L and the pH is 2.5-3.5; And / or, the second solvent is water and / or extraction waste liquid.

8. The method according to claim 1 or 2, wherein: The fermentation conditions in steps S3-S5 independently include: The fermentation temperature is 36-38°C, the pressure is 0.08-0.1MPa, and the stirring speed is 80-100rpm; The ventilation rate is 4500-5500Nm³ / h, and the ventilation rate makes the oxygen concentration in the exhaust gas 16-17vol%.

9. The method according to claim 1 or 2, wherein: The fermentation conditions in steps S3-S5 independently include: During the fermentation process, the ammonia nitrogen concentration in the fermentation broth was detected every 1-3 hours. When the ammonia nitrogen concentration was lower than 20-25 mg / L, the third nitrogen source was added to the fermentation broth.

10. The method according to claim 1 or 2, wherein: The deposit number of the Aspergillus niger is CGMCC No.40040.

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