A process for co-production of citric acid and microbial inoculant

Abstract

The application discloses a kind of citric acid-microbial inoculant coproduction process methods.The process method described in the application is as follows: (1) citric acid fermentation liquor is subjected to plate and frame filtration treatment, and mycelium and citric acid feed liquid are separated, the citric acid feed liquid is neutralized, and calcium citrate and raffinate are obtained after filtration, calcium citrate enters subsequent extraction process, and citric acid is prepared; and raffinate enters evaporator for concentration, and concentrated liquid and condensate are obtained; (2) the condensate is used as citric acid production raw material mixing water, and the concentrated liquid is used for culturing microorganisms after pretreatment, and microbial inoculant is obtained; (3) then the microbial inoculant is centrifuged, and concentrated microbial inoculant and tail liquid are obtained, the concentrated microbial inoculant is added with protective agent and carrier, and microbial inoculant is obtained by spray drying.The method described in the application uses citric acid production raffinate as microbial culture nutrient source, realizes high-value and resource utilization of nutrient substances in raffinate, significantly reduces water consumption, and reduces sewage discharge.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a process for the co-production of citric acid and microbial inoculants. Background Technology

[0002] Citric acid is the world's second-largest fermentation product and has become the world's largest producer of edible organic acids and a key green chemical in the emerging bioeconomy. my country is a major citric acid producer, with a total output exceeding 2 million tons per year, accounting for approximately 80% of the global total. However, citric acid fermentation is a typical "high-waste emission" industry, generating a large amount of residue liquid during extraction. This residue liquid is rich in nutrients, including amino acids, proteins, polysaccharides, monosaccharides, organic acids, and fats, with a COD concentration as high as 15,000-45,000 mg / L. To avoid environmental pollution, anaerobic and aerobic biological treatment methods are typically used to treat the residue liquid to meet standards before discharge. This method requires large-scale land use and equipment investment, and also results in a significant waste of water resources and nutrients. With increasingly stringent national and local environmental policies, traditional wastewater treatment technologies are no longer sufficient to meet the long-term development needs of enterprises. Given the characteristics of the residue liquid—high organic matter concentration, comprehensive nutrition, and good safety—developing high-value, resource-based utilization technologies for citric acid production residue liquid to achieve nutrient recovery and water resource recycling is of great significance.

[0003] Currently, technologies for the resource utilization of citric acid production residue have been developed successively. For example, patent 201810778332.0 describes the development of citric acid production residue as a supplementary carbon source for biological nitrogen and phosphorus removal processes in chemical or municipal wastewater treatment; patent 201911184067.4 describes the development of citric acid production residue as an organic nutrient solution for livestock and poultry farming; patent 201910156157.6 introduces a method for preparing citric acid organic liquid fertilizer from citric acid fermentation tail liquid; and patent 201910379373.7 discloses a method for concentrating citric acid fermentation tail liquid and adding Bacillus subtilis to formulate microbial fertilizer. However, the above-mentioned inventions require external systems to achieve resource utilization, and there are no reports on resource utilization through internal microbial fermentation and circulation.

[0004] Studies have found that citric acid production effluent is rich in nutrients and has good biodegradability, making it a suitable nutrient source for microbial culture. However, there are also many unfavorable factors in using citric acid production effluent for microbial culture, which pose challenges that need to be overcome. These include: (1) the presence of small amounts of inhibitors that affect microbial growth; (2) residual polysaccharides that are difficult to fully degrade and utilize; and (3) the low concentration of some nutrients, which cannot meet the requirements for high-concentration microbial culture. Therefore, how to overcome these technical challenges, utilize citric acid production effluent for microbial culture, and achieve resource utilization through microbial fermentation and internal circulation is an urgent problem to be solved. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a process for the co-production of citric acid and microbial inoculants. The process of this invention uses the citric acid production residue as a nutrient source for microbial cultivation, achieving high-value and resource-based utilization of the nutrients in the residue.

[0006] The technical solution of the present invention is as follows:

[0007] A process for the co-production of citric acid and microbial inoculant, the process comprising the following steps:

[0008] (1) The citric acid fermentation broth is subjected to plate and frame filtration to separate mycelium and citric acid broth. The citric acid broth is neutralized with calcium carbonate and filtered to obtain calcium citrate and raffinate. Calcium citrate is then used in the subsequent extraction process to produce citric acid. The raffinate is then used in the evaporator for concentration to obtain concentrate and condensate.

[0009] (2) The condensate was used as the mixing water for the raw material production of citric acid. The concentrate was pretreated and then used to cultivate microorganisms to obtain microbial inoculum.

[0010] (3) Then the microbial liquid is centrifuged to obtain concentrated liquid and tail liquid. The tail liquid is mixed with the residual liquid for concentration treatment. After adding a protectant and a carrier to the concentrated liquid, it is spray-dried to obtain microbial agent.

[0011] Further, in step (1), the method for preparing the citric acid fermentation broth is as follows: Aspergillus niger spores are inoculated into the seed culture medium and cultured for 20-32 hours to obtain a mature seed broth; then the mature seed broth is transferred to the fermentation culture medium at a volume inoculation ratio of 7-15% and cultured until the reducing sugar concentration is 10-30 g / L, and the culture is ended to obtain the citric acid fermentation broth.

[0012] Further, in step (1), the concentration is evaporative concentration at a temperature of 70-90℃; the volume ratio of the concentrate to the condensate is 1:1-5.

[0013] Further, in step (2), the microorganisms include at least one of Bacillus subtilis CCTCC No. M 2011443, Bacillus tegira CCTCC NO: M 2014004, Bacillus licheniformis CCTCC No. M 2016202, Bacillus coagulans CCTCC No. M 2015273, Bacillus cereus CCTCC NO: M 2016530, Bacillus argentea CGMCC 1.15821, Bacillus belye CGMCC NO. 5808, Saccharomyces cerevisiae, and Lactobacillus plantarum.

[0014] Further, in step (2), the pretreatment specifically involves: adjusting the pH of the concentrate to 5.5-7.5, adding activated carbon, heating and stirring, removing the filter residue through plate and frame filtration to obtain the filtrate, adding enzymes for saccharification reaction, and finally adding nutrients. After high-temperature sterilization, the filtrate is used for subsequent microbial culture.

[0015] Further, the amount of activated carbon added relative to the concentrate is 2-10 g / L; the heating and stirring temperature is 50-60℃, the rotation speed is 100-200 r / min, and the time is 15-30 min; the enzymes include pullulanase and saccharifying enzyme, the amount of pullulanase added relative to the concentrate is 100-500 U / L, and the amount of saccharifying enzyme added relative to the concentrate is 200-1000 U / L; the saccharification reaction temperature is 50-60℃, and the time is 15-45 min; the microbial culture temperature is 30-37℃, the rotation speed is 200-300 r / min, and the time is 30-37 h.

[0016] Furthermore, the nutrients include a carbon source, a nitrogen source, inorganic salts, and adenine nucleoside triphosphate; the carbon source includes at least one of glucose, sucrose, molasses, and starch; the nitrogen source includes at least one of ammonium sulfate, corn steep liquor, and peptone; and the inorganic salts include magnesium sulfate heptahydrate, dipotassium hydrogen phosphate, and sodium chloride.

[0017] Further, the carbon source is preferably glucose, and the nitrogen source is preferably corn steep liquor; the amount of glucose added relative to the concentrate is 1-5 g / L, the amount of corn steep liquor added relative to the concentrate is 1-5 g / L, the amount of magnesium sulfate heptahydrate added relative to the concentrate is 0.2-1 g / L, the amount of dipotassium hydrogen phosphate added relative to the concentrate is 0.5-3 g / L, the amount of sodium chloride added relative to the concentrate is 1-7.5 g / L, and the amount of adenine nucleoside triphosphate added relative to the concentrate is 2-10 mg / L.

[0018] Further, in step (3), the centrifugation speed is 5000-8000 r / min; the volume ratio of the concentrated bacterial solution to the tail liquid is 1:4-9; the protective agent includes starch and calcium sulfate, the amount of starch added relative to the concentrated bacterial solution is 5-50 g / L, and the amount of calcium sulfate added relative to the concentrated bacterial solution is 2-20 g / L; the carrier is diatomaceous earth, and the amount of diatomaceous earth added relative to the concentrated bacterial solution is 2-20 g / L.

[0019] Furthermore, in step (3), the inlet air temperature of the spray dryer is 35-170℃ and the outlet air temperature is 25-95℃.

[0020] The beneficial technical effects of this invention are as follows:

[0021] This invention addresses the problem that the residual liquid from citric acid production contains inhibitory substances that are detrimental to microbial growth. By optimizing the pH and combining it with activated carbon adsorption, the inhibitory effect of the residual liquid from citric acid production on microbial growth can be effectively eliminated.

[0022] This invention addresses the problem that residual polysaccharides in citric acid production effluent are difficult to fully degrade and utilize. By combining pullulanase and saccharifying enzyme for enzymatic hydrolysis and optimizing the conditions of pullulanase and saccharifying enzyme, the utilization rate of residual polysaccharides in citric acid production effluent is improved.

[0023] This invention addresses the problem of low concentrations of certain nutrients in the residue of citric acid production. By adding nutrients such as carbon sources, nitrogen sources, and inorganic salts, and optimizing the amount of each nutrient added, high-concentration microbial culture can be achieved. In particular, the addition of adenine triphosphate (ATP) significantly increases the growth vitality of microorganisms.

[0024] Since different microorganisms have different requirements for nutrients and culture conditions, this invention optimizes the types of microorganisms suitable for culturing citric acid production residue by screening microbial species. Among them, Bacillus subtilis, Bacillus tekirae, Bacillus licheniformis, Bacillus coagulans, Bacillus cereus, Bacillus argentea, Bacillus berberis, Saccharomyces cerevisiae, and Lactobacillus plantarum can all be used for culturing citric acid production residue. The best microorganisms for culturing are Bacillus subtilis, Bacillus tekirae, and Bacillus argentea.

[0025] This invention avoids the problem of a significant decrease in the number of viable bacteria during the preparation of microbial agents by adding protective agents and carriers and optimizing the amount of protective agents and carriers added, while ensuring that the viability of the bacteria is not affected. In addition, this invention further optimizes the spray drying conditions and optimizes the optimal inlet and outlet air temperatures during the spray drying process of different bacterial strains, thereby ensuring the concentration and viability of the prepared microbial agents.

[0026] This invention utilizes the residue from the citric acid production extraction stage for microbial culture and preparation of microbial inoculants. The condensate generated during the preparation of the microbial inoculants is reused as raw material for citric acid production, forming a green process for the cyclical co-production of citric acid and microbial inoculants. This technology, through internal microbial fermentation, achieves high-value and resource-based utilization of nutrients in the residue; it avoids the need for large-scale wastewater treatment plants requiring significant land area and equipment investment, saving land and investment, and preventing nutrient waste. Simultaneously, it achieves water resource recycling, significantly reducing water consumption and wastewater discharge. The citric acid-microbial inoculant co-production process described in this invention creates significant economic and social benefits for enterprises. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the process for the co-production of citric acid and microbial inoculants according to the present invention. Detailed Implementation

[0028] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0029] All raw materials and reagents used in the following examples are commercially available products;

[0030] The Aspergillus niger in the following examples was obtained from the China Industrial Microbial Culture Collection Center (CICC), with accession number CICC 40021.

[0031] The *Bacillus subtilis*, *Bacillus tekira*, *Bacillus tekira*, *Bacillus licheniformis*, *Bacillus licheniformis*, *Bacillus coagulans*, *Bacillus cereus ... NO.5808; Saccharomyces cerevisiae was purchased from Angel Yeast Co., Ltd.; Lactobacillus plantarum was purchased from Jiangsu Dayou Biotechnology Development Co., Ltd.

[0032] In the following examples, the amounts of activated carbon, pullulanase, glucoamylase, glucose, corn steep liquor, magnesium sulfate heptahydrate, dipotassium hydrogen phosphate, sodium chloride, and ATP added all refer to the amounts added relative to the concentrated solution.

[0033] In the examples below, the amounts of starch, calcium sulfate, and diatomaceous earth added all refer to the amounts added relative to the concentrated bacterial solution.

[0034] In the following examples, total sugar and reducing sugar were determined by Fehling's titration method; nitrogen source was determined by the Kjeldahl method; citric acid was determined by titration with 0.1429 mol / L NaOH; and spores were counted using a hemocytometer. Unless otherwise specified, commonly used equipment and processes in the art were employed.

[0035] Example 1

[0036] A process for the co-production of citric acid and microbial inoculant, the process comprising the following steps:

[0037] (1) Mix 1000 kg of corn flour with 3000 L of cooling water to form a slurry. Adjust the pH of the slurry to 6.0 with Ca(OH)2. Add α-high-temperature amylase at a dosage of 20 U / g corn flour. After spray liquefaction, the qualified corn liquefaction solution is obtained after the iodine test turns light brown. Filter 70% of the corn liquefaction solution through a plate and frame filter to remove the filter residue, and obtain corn sugar solution. Prepare a seed culture medium (total sugar 100 g / L, C / N ratio 20) with ammonium sulfate. Prepare a fermentation culture medium (total sugar 175 g / L, C / N ratio 60) with corn liquefaction solution, corn sugar solution and ammonium sulfate. Inoculate Aspergillus niger spores into the seed culture medium, with a final spore concentration of 5 × 10⁻⁶. 5 The seed culture was cultured at 37℃, 0.2 vvm airflow, 0.06 MPa pressure, and 200 rpm for 20 h to obtain a mature seed culture. The mature seed culture was then transferred to fermentation medium at a 15% volume inoculation ratio. Fermentation conditions were: 37℃, 0.1 vvm airflow, 0.06 MPa pressure, and 200 rpm stirring. The culture was terminated when the reducing sugar concentration dropped to 10 g / L, yielding the citric acid fermentation broth.

[0038] (2) The citric acid fermentation broth was subjected to plate and frame filtration to separate mycelium and citric acid solution. The citric acid solution was neutralized with calcium carbonate to a final pH of 5.0. Then, it was filtered to obtain calcium citrate and raffinate. Calcium citrate was used in subsequent extraction processes to obtain citric acid. 1200L of raffinate was placed in an evaporation and concentration device and concentrated at 90℃ to obtain 200L of concentrate and 1000L of condensate.

[0039] (3) The condensate was used for mixing corn flour in the production of citric acid; sodium hydroxide was added to the concentrate until the pH was 7.5, and then 10 g / L of activated carbon was added. The mixture was stirred at 60°C and 200 rpm for 30 min. The residue was removed by plate and frame filtration to obtain the filtrate. Then 500 U / L pullulanase and 1000 U / L saccharifying enzyme were added, and the saccharification reaction was carried out at 60°C for 45 min. Finally, 5 g / L glucose, 5 g / L corn steep liquor, 1 g / L magnesium sulfate heptahydrate, 3 g / L dipotassium hydrogen phosphate, 7.5 g / L sodium chloride, and 10 mg / L ATP were added. After sterilization at 121°C for 15 min, Bacillus subtilis was inoculated and cultured at 37°C and 200 rpm. When the spore rate was greater than 95%, the culture was stopped to obtain Bacillus subtilis bacterial culture.

[0040] (4) Then, 200L of Bacillus subtilis bacterial solution was centrifuged at 5000rpm to obtain 40L of concentrated Bacillus subtilis bacterial solution and 160L of tail liquid. The tail liquid and the residue were mixed and concentrated. 5g / L of starch, 20g / L of calcium sulfate and 20g / L of diatomaceous earth were added to the concentrated bacterial solution as a carrier. The solution was dried under spray drying conditions with an inlet air temperature of 170℃ and an outlet air temperature of 95℃ to obtain Bacillus subtilis bacterial agent.

[0041] Example 2

[0042] A process for the co-production of citric acid and microbial inoculant, the process comprising the following steps:

[0043] (1) Mix 1000 kg of corn flour with 3000 L of cooling water to form a slurry. Adjust the pH of the slurry to 6.0 with Ca(OH)2. Add α-high-temperature amylase at a dosage of 20 U / g corn flour. After spray liquefaction, the qualified corn liquefaction solution is obtained after the iodine test turns light brown. Filter 70% of the corn liquefaction solution through a plate and frame filter to remove the filter residue, and obtain corn sugar solution. Prepare a seed culture medium (total sugar 100 g / L, C / N ratio 20) with ammonium sulfate. Prepare a fermentation culture medium (total sugar 175 g / L, C / N ratio 60) with corn liquefaction solution, corn sugar solution and ammonium sulfate. Inoculate Aspergillus niger spores into the seed culture medium, with a final spore concentration of 5 × 10⁻⁶. 5 The seed culture was cultured at 37℃, 0.2 vvm airflow, 0.06 MPa pressure, and 200 rpm for 26 h to obtain a mature seed culture. The mature seed culture was then transferred to fermentation medium at a 10% volume inoculation ratio. The fermentation conditions were: 37℃, 0.1 vvm airflow, 0.06 MPa pressure, and 200 rpm stirring. The culture was terminated when the reducing sugar concentration dropped to 15 g / L, yielding the citric acid fermentation broth.

[0044] (2) The citric acid fermentation broth was subjected to plate and frame filtration to separate mycelium and citric acid solution. The citric acid solution was neutralized with calcium carbonate to a final pH of 5.0. Then, it was filtered to obtain calcium citrate and raffinate. Calcium citrate was used in subsequent extraction processes to obtain citric acid. 1200L of raffinate was placed in an evaporation and concentration device and concentrated at 80℃ to obtain 300L of concentrate and 900L of condensate.

[0045] (3) The condensate was used for mixing corn flour in the production of citric acid; sodium hydroxide was added to the concentrate to make the pH 6.5, and then 5 g / L of activated carbon was added. The mixture was stirred at 55°C and 150 rpm for 20 min. The residue was removed by plate and frame filtration to obtain the filtrate. Then 300 U / L pullulanase and 500 U / L saccharifying enzyme were added, and the saccharification reaction was carried out at 55°C for 30 min. Finally, 2.5 g / L glucose, 2.5 g / L corn steep liquor, 0.5 g / L magnesium sulfate heptahydrate, 1.5 g / L dipotassium hydrogen phosphate, 5 g / L sodium chloride, and 5 mg / L ATP were added. After sterilization at 121°C for 15 min, Bacillus tekirae was inoculated and cultured at 34°C and 250 rpm. When the spore rate was greater than 95%, the culture was stopped to obtain Bacillus tekirae bacterial culture.

[0046] (4) Then, 300L of Bacillus tekirae bacterial solution was centrifuged at 6500rpm to obtain 50L of concentrated Bacillus tekirae bacterial solution and 250L of tail liquid. The tail liquid and the residue were mixed and concentrated. 20g / L starch, 10g / L calcium sulfate and 10g / L diatomaceous earth were added to the concentrated bacterial solution as carriers. The solution was dried under spray drying conditions with an inlet air temperature of 160℃ and an outlet air temperature of 85℃ to obtain Bacillus tekirae bacterial agent.

[0047] Example 3

[0048] A process for the co-production of citric acid and microbial inoculant, the process comprising the following steps:

[0049] (1) Mix 1000 kg of corn flour with 3000 L of cooling water to form a slurry. Adjust the pH of the slurry to 6.0 with Ca(OH)2. Add α-high-temperature amylase at a dosage of 20 U / g corn flour. After spray liquefaction, the qualified corn liquefaction solution is obtained after the iodine test turns light brown. Filter 70% of the corn liquefaction solution through a plate and frame filter to remove the filter residue, and obtain corn sugar solution. Prepare a seed culture medium (total sugar 100 g / L, C / N ratio 20) with ammonium sulfate. Prepare a fermentation culture medium (total sugar 175 g / L, C / N ratio 60) with corn liquefaction solution, corn sugar solution and ammonium sulfate. Inoculate Aspergillus niger spores into the seed culture medium, with a final spore concentration of 5 × 10⁻⁶. 5The seed culture was cultured at 37℃, 0.2 vvm airflow, 0.06 MPa pressure, and 200 rpm for 32 h to obtain a mature seed culture. The mature seed culture was then transferred to fermentation medium at a 7% volume inoculation ratio. Fermentation conditions were: 37℃, 0.1 vvm airflow, 0.06 MPa pressure, and 200 rpm stirring. The culture was terminated when the reducing sugar concentration dropped to 30 g / L, yielding the citric acid fermentation broth.

[0050] (2) The citric acid fermentation broth was subjected to plate and frame filtration to separate mycelium and citric acid solution. The citric acid solution was neutralized with calcium carbonate to a final pH of 5.0. Then, it was filtered to obtain calcium citrate and raffinate. Calcium citrate was used in subsequent extraction processes to obtain citric acid. 1200L of raffinate was placed in an evaporation and concentration device and concentrated at 70℃ to obtain 600L of concentrate and 600L of condensate.

[0051] (3) The condensate was used as a feed mix for citric acid production of corn flour; sodium hydroxide was added to the concentrate until the pH was 5.5, and then 2 g / L of activated carbon was added. The mixture was stirred at 50°C and 100 rpm for 15 min. The residue was removed by plate and frame filtration to obtain the filtrate. Then 100 U / L pullulanase and 200 U / L saccharifying enzyme were added, and the saccharification reaction was carried out at 50°C for 15 min. Finally, 1 g / L glucose, 1 g / L corn steep liquor, 0.2 g / L magnesium sulfate heptahydrate, 0.5 g / L dipotassium hydrogen phosphate, 1 g / L sodium chloride, and 2 mg / L ATP were added. After sterilization at 121°C for 15 min, Bacillus aureus was inoculated and cultured at 30°C and 300 rpm. When the spore rate was greater than 95%, the culture was stopped to obtain Bacillus aureus bacterial culture.

[0052] (4) Then, 600L of Bacillus tekirae bacterial solution was centrifuged at 8000rpm to obtain 60L of concentrated Bacillus aureus bacterial solution and 540L of tail liquid. The tail liquid and the residue were mixed and concentrated. 50g / L starch, 2g / L calcium sulfate and 2g / L diatomaceous earth were added to the concentrated bacterial solution as carriers. The solution was dried under spray drying conditions with an inlet air temperature of 150℃ and an outlet air temperature of 75℃ to obtain Bacillus aureus bacterial agent.

[0053] Example 4

[0054] The process method of Example 4 is basically the same as that of Example 2, except that the microorganism cultured is Bacillus licheniformis.

[0055] Example 5

[0056] The process method of Example 5 is basically the same as that of Example 2, except that the microorganism cultured is Bacillus coagulans.

[0057] Example 6

[0058] The process method of Example 6 is basically the same as that of Example 2, except that the microorganism cultured is Bacillus cereus.

[0059] Example 7

[0060] The process method of Example 7 is basically the same as that of Example 2, except that the microorganism cultured is Bacillus belye.

[0061] Example 8

[0062] The process method of Example 8 is basically the same as that of Example 2, except that the microorganism cultivated is Saccharomyces cerevisiae. In addition, the bacterial culture method in step (3) is: "When the bacterial concentration does not increase, stop the culture to obtain Saccharomyces cerevisiae bacterial culture." In step (4), the bacterial agent preparation method is: "Add 20g / L starch, 2g / L calcium sulfate and 2g / L diatomaceous earth as carriers to the bacterial culture, and dry it under spray drying conditions with an air inlet temperature of 35℃ and an air outlet temperature of 25℃ to obtain Saccharomyces cerevisiae bacterial agent."

[0063] Example 9

[0064] The process method of Example 9 is basically the same as that of Example 2, except that the microorganism cultured is *Lactobacillus plantarum*, and the bacterial culture method in step (3) is: "When the bacterial concentration does not increase, stop the culture to obtain *Lactobacillus plantarum* bacterial culture." The bacterial agent preparation method in step (4) is: "Add 20 g / L starch, 2 g / L calcium sulfate and 2 g / L diatomaceous earth as carriers to the bacterial culture, and dry it under spray drying conditions with an inlet air temperature of 45°C and an outlet air temperature of 30°C to obtain *Lactobacillus plantarum* bacterial agent."

[0065] Comparative Example 1

[0066] The process method of Comparative Example 1 is basically the same as that of Example 2, except that the citric acid production residue is treated by anaerobic and aerobic biological methods and then discharged into the municipal sewage treatment plant after meeting the requirements.

[0067] Comparative Example 2

[0068] The process method in Comparative Example 2 is basically the same as that in Example 2, except that step (3) is adjusted as follows:

[0069] The condensate was used as a feed mix for citric acid production of corn flour; after the concentrate was sterilized at 121℃ for 15 min, it was inoculated with Bacillus tekirae and cultured at 34℃ and 250 rpm. When the spore rate was greater than 95%, the culture was stopped to obtain Bacillus tekirae bacterial solution.

[0070] Comparative Example 3

[0071] The process method in Comparative Example 3 is basically the same as that in Example 2, except that step (3) is adjusted as follows:

[0072] The condensate was used as a feed mix for citric acid production of corn flour. Sodium hydroxide was added to the concentrate to bring the pH to 6.5, and then 5 g / L of activated carbon was added. The mixture was stirred at 55°C and 150 rpm for 20 min. The residue was removed by plate and frame filtration to obtain the filtrate. After sterilization at 121°C for 15 min, Bacillus tekirae was inoculated and cultured at 34°C and 250 rpm. The culture was stopped when the spore rate was greater than 95%, and Bacillus tekirae bacterial culture was obtained.

[0073] Comparative Example 4

[0074] The process method in Comparative Example 4 is basically the same as that in Example 2, except that step (3) is adjusted as follows:

[0075] The condensate was used as a feed mix for citric acid production of corn flour. Sodium hydroxide was added to the concentrate to bring the pH to 6.5, and then 1 g / L of activated carbon was added. The mixture was stirred at 55°C and 150 rpm for 20 min. The residue was removed by plate and frame filtration to obtain the filtrate. After sterilization at 121°C for 15 min, Bacillus tekirae was inoculated and cultured at 34°C and 250 rpm. The culture was stopped when the spore rate was greater than 95%, and Bacillus tekirae bacterial culture was obtained.

[0076] Comparative Example 5

[0077] The process method in Comparative Example 5 is basically the same as that in Example 2, except that step (3) is adjusted as follows:

[0078] The condensate was used as a feed mix for citric acid production of corn flour. Sodium hydroxide was added to the concentrate to bring the pH to 6.5, and then 5 g / L of activated carbon was added. The mixture was stirred at 55°C and 150 rpm for 20 min. The residue was removed by plate and frame filtration to obtain the filtrate. Then, 300 U / L pullulanase and 500 U / L saccharifying enzyme were added, and the saccharification reaction was carried out at 55°C for 30 min. After sterilization at 121°C for 15 min, Bacillus tekirae was inoculated and cultured at 34°C and 250 rpm. The culture was stopped when the spore rate was greater than 95%, and Bacillus tekirae bacterial culture was obtained.

[0079] Comparative Example 6

[0080] The process method in Comparative Example 6 is basically the same as that in Example 2, except that step (3) is adjusted as follows:

[0081] The condensate was used as a feed mix for citric acid production of corn flour. Sodium hydroxide was added to the concentrate to bring the pH to 6.5, followed by 5 g / L of activated carbon. The mixture was stirred at 55°C and 150 rpm for 20 min. The residue was removed by plate and frame filtration to obtain the filtrate. Then, 300 U / L pullulanase and 500 U / L saccharifying enzyme were added, and the saccharification reaction was carried out at 55°C for 30 min. Finally, 2.5 g / L glucose, 0.5 g / L magnesium sulfate heptahydrate, 1.5 g / L dipotassium hydrogen phosphate, 5 g / L sodium chloride, and 5 mg / L ATP were added. After sterilization at 121°C for 15 min, Bacillus tekirae was inoculated and cultured at 34°C and 250 rpm. The culture was stopped when the spore rate was greater than 95%, yielding Bacillus tekirae bacterial culture.

[0082] Comparative Example 7

[0083] The process method in Comparative Example 7 is basically the same as that in Example 2, except that step (3) is adjusted as follows:

[0084] The condensate was used as a feed mix for citric acid production of corn flour. Sodium hydroxide was added to the concentrate to bring the pH to 6.5, followed by 5 g / L of activated carbon. The mixture was stirred at 55°C and 150 rpm for 20 min, and the residue was removed by plate and frame filtration to obtain the filtrate. Then, 300 U / L pullulanase and 500 U / L saccharifying enzyme were added, and the saccharification reaction was carried out at 55°C for 30 min. Finally, 2.5 g / L glucose, 2.5 g / L corn steep liquor, 0.5 g / L magnesium sulfate heptahydrate, 1.5 g / L dipotassium hydrogen phosphate, and 5 g / L sodium chloride were added. After sterilization at 121°C for 15 min, Bacillus tekirae was inoculated and cultured at 34°C and 250 rpm. The culture was stopped when the spore rate was greater than 95%, and Bacillus tekirae bacterial culture was obtained.

[0085] Comparative Example 8

[0086] The process method in Comparative Example 8 is basically the same as that in Example 2, except that step (3) is adjusted as follows:

[0087] The condensate was used as a feed mix for citric acid production of corn flour. Sodium hydroxide was added to the concentrate to bring the pH to 6.5, followed by 5 g / L of activated carbon. The mixture was stirred at 55°C and 150 rpm for 20 min. The residue was removed by plate and frame filtration to obtain the filtrate. 300 U / L pullulanase and 500 U / L saccharifying enzyme were then added, and the saccharification reaction was carried out at 55°C for 30 min. Finally, 7 g / L glucose, 7 g / L corn steep liquor, 1.5 g / L magnesium sulfate heptahydrate, 4 g / L dipotassium hydrogen phosphate, 8 g / L sodium chloride, and 12 mg / L ATP were added. After sterilization at 121°C for 15 min, Bacillus tekirae was inoculated and cultured at 34°C and 250 rpm. The culture was stopped when the spore count was greater than 95%, yielding Bacillus tekirae bacterial culture.

[0088] Comparative Example 9

[0089] The process method in Comparative Example 9 is basically the same as that in Example 2, except that step (4) is adjusted as follows:

[0090] Then, 300L of Bacillus tekirae bacterial solution was centrifuged at 6500rpm to obtain 50L of concentrated Bacillus tekirae bacterial solution and 250L of tail liquid. The tail liquid and the residue were mixed and concentrated. The concentrated bacterial solution was dried under spray drying conditions with an inlet air temperature of 160℃ and an outlet air temperature of 85℃ to obtain Bacillus tekirae bacterial agent.

[0091] Comparative Example 10

[0092] The process method in Comparative Example 10 is basically the same as that in Example 2, except that steps (1) and (2) are adjusted as follows:

[0093] (1) Mix 1000 kg of corn flour with 3000 L of cooling water to form a slurry. Adjust the pH of the slurry to 6.0 with Ca(OH)2. Add α-high-temperature amylase at a dosage of 20 U / g corn flour. After spray liquefaction, the qualified corn liquefaction solution is obtained after the iodine test turns light brown. Filter 70% of the corn liquefaction solution through a plate and frame filter to remove the filter residue and obtain corn sugar solution. Prepare a seed culture medium (total sugar 100 g / L, C / N ratio 20) with corn liquefaction solution and ammonium sulfate. Prepare a solution (total sugar 175 g / L, C / N ratio 60) with corn liquefaction solution, corn sugar solution and ammonium sulfate. Inoculate Aspergillus niger spores into the seed culture medium to a final spore concentration of 5 × 10⁻⁶. 5 The seed culture was cultured at 37℃, 0.2 vvm airflow, 0.06 MPa pressure, and 200 rpm for 26 h to obtain a mature seed culture. The mature seed culture was then transferred to fermentation medium at a 10% volume inoculation ratio. Fermentation conditions were: 37℃, 0.1 vvm airflow, 0.06 MPa pressure, and 200 rpm stirring. The culture was terminated when the reducing sugar concentration dropped to 5 g / L, yielding the citric acid fermentation broth.

[0094] (2) The citric acid fermentation broth was subjected to plate and frame filtration to separate mycelium and citric acid solution. The citric acid solution was neutralized with calcium carbonate to a final pH of 5.0, and then filtered to obtain calcium citrate and raffinate. The calcium citrate was then used in the subsequent extraction process to obtain citric acid. 1200L of raffinate was placed in an evaporation and concentration device and concentrated at 80°C to obtain 100L of concentrate and 1100L of condensate (the concentration of reducing sugar in the concentrate was controlled to be the same as in Example 2).

[0095] Test Example 1

[0096] Examples 1-9 and Comparative Examples 1-2 of the present invention were used to detect total sugar, citric acid concentration, residual reducing sugar, fermentation cycle, fermentation intensity, corn consumption per unit area, and water consumption per unit area in citric acid production. The cycle from which inoculated microorganisms were cultured to a spore rate greater than 95% or the cell concentration stopped growing, residual total sugar, viable bacteria concentration in fermentation broth, and viable bacteria concentration in microbial inoculum were detected in the production of microbial agents. The test results are shown in Table 1.

[0097] Table 1

[0098]

[0099]

[0100] As shown in Table 1, the residual liquid from Comparative Example 1 was not used for the production of microbial inoculants. After passing anaerobic and aerobic biological treatment, it was discharged into the municipal wastewater treatment plant, with a fermentation water consumption as high as 4.85 m³. 3 / t citric acid.

[0101] Comparative Example 2 did not require pretreatment of the residue and was used directly for the production of Bacillus tektii inoculum, reducing fermentation water consumption to 1.1 m³. 3 While the raffinate contained citric acid, residual substances in the raffinate inhibited microbial growth, resulting in a prolonged culture lag period of up to 45 hours. Residual sugars in the raffinate were difficult to fully degrade, with the total residual sugar remaining as high as 7.8 g / L after microbial culture. Furthermore, the concentrations of some nutrients were too low, preventing high-concentration culture; the viable cell concentration in the fermentation broth was only 1.5 billion CFU / mL, while the viable cell concentration in the spray-dried bacterial powder was 42 billion CFU / g. These results indicate that pretreatment is necessary to eliminate the negative impacts and achieve efficient utilization of the raffinate.

[0102] Examples 1-7 show that the pretreated leaching liquid was used to produce microbial inoculants, with significantly improved technical effects compared to Comparative Example 2. The most prominent achievements were the elimination of inhibitory effects, a shortened lag period, and a cycle time reduced to 30-32 hours; improved utilization of residual sugar in the leaching liquid, with total residual sugar decreasing to 2.1-2.8 g / L after microbial culture; and increased cell concentration through nutrient addition, resulting in a viable cell concentration of 5.3-6.5 billion CFU / mL in the fermentation broth and 150-180 billion CFU / g in the spray-dried inoculum. Compared to Comparative Example 1, the most significant improvement was a reduction in fermentation water consumption to 0.52-1.45 m³ / mL. 3 / t citric acid, realizing the resource utilization of residual liquid and reducing wastewater discharge.

[0103] In Examples 8-9, the raffinate was pretreated and used for the production of Saccharomyces cerevisiae and Lactobacillus plantarum inoculants. The culture cycle and residual sugar were effectively controlled. The viable cell concentrations of the inoculants after spray drying were 38 billion / g and 65 billion / g, respectively.

[0104] The results show that the method of the present invention pretreats the citric acid residue, eliminates negative impacts, and allows it to be used in the production of microbial agents, realizing the resource utilization of the residue and water resource recycling. The method of the present invention has an important role in promoting the technological advancement of the citric acid industry.

[0105] Test Example 2

[0106] Examples 2 and 2-8 of the present invention were used to detect total sugar, citric acid concentration, residual reducing sugar, fermentation cycle, fermentation intensity, corn consumption per unit area, and water consumption per unit area in citric acid production. The cycle of inoculating microorganisms to a spore rate greater than 95%, residual total sugar, viable bacteria concentration in fermentation broth, and viable bacteria concentration in microbial inoculant production were also detected. The results are shown in Table 2.

[0107] Table 2

[0108]

[0109]

[0110] As shown in Table 2, no treatment measures were taken in Comparative Example 2. The residual liquid was concentrated and directly used for Bacillus tekirae culture. The test results showed that the microbial fermentation cycle was long, the residual total sugar was high, and the viable cell concentration was low.

[0111] In Comparative Example 3, the raffinate was concentrated, and the pH was adjusted to 6.5 with sodium hydroxide. Then, 5 g / L of activated carbon was added, and the mixture was stirred at 55°C for 20 min. The residue was then removed by plate and frame filtration. After sterilization at 121°C for 15 min, the raffinate was used for Bacillus tekirae culture. Compared with Comparative Example 2, the fermentation cycle was significantly shortened, indicating that pH adjustment combined with activated carbon adsorption can effectively remove inhibitors, resulting in improved fermentation results.

[0112] In Comparative Example 4, although the pH was adjusted and activated carbon was added for adsorption after the raffinate was concentrated, the inhibitory effect was not eliminated, the lag period was not significantly shortened, and the fermentation cycle still reached 43 hours. This indicates that the pH and activated carbon dosage must be adjusted to an appropriate range to achieve better results.

[0113] In Comparative Example 5, the filtrate was concentrated, and the pH was adjusted to 6.5 with sodium hydroxide. Then, 5 g / L of activated carbon was added, and the mixture was stirred at 55°C for 20 min. The residue was then removed by plate and frame filtration. Subsequently, 300 U / L pullulanase and 500 U / L saccharifying enzyme were added, and the reaction was carried out at 55°C for 30 min. After sterilization at 121°C for 15 min, the mixture was used for Bacillus tekirae culture. Compared with Comparative Example 3, the total residual sugar in Comparative Example 5 was significantly reduced, and the fermentation results were further improved, indicating that the combined enzymatic hydrolysis of the two enzymes can improve the utilization rate of residual sugar in the citric acid filtrate.

[0114] Compared to Comparative Example 5, the fermentation broth of Example 2 underwent pretreatment after concentration of the raffinate, followed by the addition of nutrients. This resulted in a further increase in the viable cell concentration of the fermentation broth. The addition of nutrients promoted cell growth and achieved high-concentration culture. However, a comparison of Comparative Examples 6-8 with Example 2 showed that the composition and concentration of nutrients significantly affected cell growth. In Comparative Example 6, no corn steep liquor was added, and the viable cell concentration of the fermentation broth decreased from 6.4 billion / mL to 3.7 billion / mL. After spray drying, the viable cell concentration of the bacterial powder decreased from 180 billion / g to 84 billion / g. In Comparative Example 7, no ATP was added, resulting in decreased fermentation activity and a longer fermentation period of 36 hours. The viable cell concentration of the fermentation broth decreased to 4.2 billion / mL, and after spray drying, the viable cell concentration of the bacterial powder decreased to 98 billion / g. In contrast, although a large amount of nutrients were added to Comparative Example 8, the concentration exceeded the appropriate range, leading to an imbalance in the nutrient ratio. This resulted in a fermentation cycle extended to 35 hours, a total residual sugar increase to 3.4 g / L, a decrease in the viable cell concentration of the fermentation broth to 4.3 billion / mL, and a further decrease in the viable cell concentration of the spray-dried bacterial powder to 106 billion / g. Extensive experimental research determined the appropriate nutrient composition and concentration range.

[0115] The results show that pH adjustment combined with activated carbon adsorption can eliminate inhibition; dual-enzyme combined enzymatic hydrolysis can improve the utilization rate of residual sugars; and the addition of nutrients can promote cell growth and achieve high-concentration culture. However, these conditions need to be controlled within appropriate ranges to completely eliminate negative effects and achieve efficient recycling of the residual liquid.

[0116] Test Example 3

[0117] Examples 2 and 9-10 of the present invention were used to detect total sugar, citric acid concentration, residual reducing sugar, fermentation cycle, fermentation intensity, corn consumption per unit area, and water consumption per unit area in citric acid production. The cycle of inoculating microorganisms to a spore rate greater than 95%, residual total sugar, viable bacteria concentration in fermentation broth, and viable bacteria concentration in microbial inoculant production were also detected. The results are shown in Table 1.

[0118] Table 3

[0119]

[0120] As shown in Table 3, compared with Example 2, the bacterial culture obtained in Comparative Example 9 also achieved a high concentration of viable bacteria. However, without the addition of excipients as a protective agent, the bacterial cell activity was significantly lost during direct spray drying, and the concentration of viable bacteria in the obtained bacterial powder decreased significantly. This indicates that a protective agent needs to be added during the spraying process and the above conditions need to be controlled within an appropriate range.

[0121] Compared to Example 2, the main difference in Comparative Example 10 is that the residual reducing sugar at the endpoint of citric acid fermentation decreased from 15 g / L to 5 g / L. The results showed that the evaporation and concentration factor of the raffinate needed to be increased to 12 times, increasing energy consumption. Furthermore, due to the decrease in available sugars and other nutrients, the unusable residual sugar after microbial culture increased from 2.1 g / L to 5.6 g / L, while the viable cell concentration in the fermentation broth decreased from 6.4 billion / mL to 4.6 billion / mL. This indicates that the endpoint of citric acid fermentation needs to be controlled within a suitable range to ensure sufficient nutrients for microbial agent production. Considering the balance between citric acid production and the benefits of microbial agent production, controlling the residual reducing sugar at 15 g / L is optimal.

[0122] The above description is merely 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 variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. A process for the co-production of citric acid and microbial inoculant, characterized in that, The process includes the following steps: (1) The citric acid fermentation broth was subjected to plate and frame filtration to separate mycelium and citric acid broth. The citric acid broth was neutralized with calcium carbonate and filtered to obtain calcium citrate and raffinate. Calcium citrate was then used in the subsequent extraction process to obtain citric acid. The raffinate was then used in the evaporator for concentration to obtain concentrate and condensate. (2) The condensate was used as the mixing water for citric acid production. The concentrate was pretreated and then used to cultivate microorganisms to obtain microbial inoculum. (3) Then the microbial liquid is centrifuged to obtain concentrated liquid and tail liquid. The tail liquid is mixed with the residue liquid for concentration treatment. After adding a protectant and a carrier to the concentrated liquid, it is spray-dried to obtain microbial agent. In step (2), the microorganism is at least one of Bacillus subtilis CCTCC No. M 2011443, Bacillus tegira CCTCC NO: M 2014004, Bacillus licheniformis CCTCC No. M 2016202, Bacillus coagulans CCTCC No. M 2015273, Bacillus cereus CCTCC NO: M 2016530, Bacillus argentea CGMCC 1.15821, Bacillus belyss CGMCC NO. 5808, Saccharomyces cerevisiae, and Lactobacillus plantarum. In step (2), the pretreatment specifically involves: adjusting the pH of the concentrate to 5.5-7.5, adding activated carbon, heating and stirring, removing the filter residue through plate and frame filtration to obtain the filtrate, adding enzymes for saccharification reaction, adding nutrients, and then sterilizing at high temperature for subsequent microbial culture. The activated carbon is added at a rate of 2-10 g / L relative to the concentrate; the enzymes are pullulanase and saccharifying enzyme, with pullulanase added at a rate of 100-500 U / L relative to the concentrate and saccharifying enzyme added at a rate of 200-1000 U / L relative to the concentrate; the saccharification reaction is carried out at a temperature of 50-60 ℃ for a time of 15-45 min; The nutrients consist of a carbon source, a nitrogen source, inorganic salts, and adenine nucleoside triphosphate; the carbon source is at least one of glucose, sucrose, molasses, and starch; the nitrogen source is at least one of ammonium sulfate, corn steep liquor, and peptone; and the inorganic salts consist of magnesium sulfate heptahydrate, dipotassium hydrogen phosphate, and sodium chloride. The amount of glucose added relative to the concentrate is 1-5 g / L, the amount of corn steep liquor added relative to the concentrate is 1-5 g / L, the amount of magnesium sulfate heptahydrate added relative to the concentrate is 0.2-1 g / L, the amount of dipotassium hydrogen phosphate added relative to the concentrate is 0.5-3 g / L, the amount of sodium chloride added relative to the concentrate is 1-7.5 g / L, and the amount of adenine nucleoside triphosphate added relative to the concentrate is 2-10 mg / L.

2. The process method according to claim 1, characterized in that, In step (1), the method for preparing the citric acid fermentation broth is as follows: Aspergillus niger spores are inoculated into the seed culture medium and cultured for 20-32 h to obtain mature seed broth; The mature seed culture is then transferred to the fermentation medium at a volume inoculation ratio of 7-15%. The culture is stopped when the reducing sugar concentration is 10-30 g / L to obtain citric acid fermentation broth.

3. The process method according to claim 1, characterized in that, In step (1), the concentration is evaporative concentration at a temperature of 70-90 ℃; the volume ratio of the concentrate to the condensate is 1:1-5.

4. The process method according to claim 1, characterized in that, The heating and stirring temperature is 50-60 ℃, the rotation speed is 100-200 r / min, and the time is 15-30 min; the microbial culture temperature is 30-37 ℃, the rotation speed is 200-300 r / min, and the culture is stopped when the spore rate is greater than 95% or the cell concentration does not increase.

5. The process method according to claim 1, characterized in that, In step (3), the centrifugation speed is 5000-8000 r / min; the volume ratio of the concentrated bacterial solution to the tail liquid is 1:4-9; the protective agent is starch and calcium sulfate, the amount of starch added relative to the concentrated bacterial solution is 5-50 g / L, and the amount of calcium sulfate added relative to the concentrated bacterial solution is 2-20 g / L; the carrier is diatomaceous earth, and the amount of diatomaceous earth added relative to the concentrated bacterial solution is 2-20 g / L.

6. The process method according to claim 1, characterized in that, In step (3), the inlet air temperature of the spray dryer is 35-170 ℃ and the outlet air temperature is 25-95 ℃.

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